CHARACTERIZATION OF GILT’S OVARIES AND ATTEMPTS
TO ACHIEVE A SUCCESSFUL IN VITRO MATURATION OFOOCYTES FROM SMALL AND MEDIUM FOLLICLES

September, 2016

Pilar FERRÉ PUJOLGRADUATE SCHOOL OF
ENVIRONMENTAL AND LIFE SCIENCE(Doctor’s Course)
OKAYAMA UNIVERSITY

Dedication

To both my parents, for giving me life.

IITable of contents

Table of contents ……..…………………………………………………………......……..

III

Abstract ……………………..………………………………………………...………...…

VI

Declaration ……………………..……………………………………...………...…...……

IX

Acknowledgements ………………...………………………………………………..…….

X

Publications arising from this thesis …….……………………………………………..….

XII

Conference proceedings ……………………..…………………………………………….

XIII

List of figures …………………………………...……..……...………………………...…

XIV

List of tables ………………………………………………..…………………………...…

XVI

Abbreviations ……………………………………………………..……...…………...…...

XVII

CHAPTER 1: LITERATURE REVIEW ……………..………....……………………...…

1

1.1 Preface ………………………………………………………………………………

1

1.2 The pig as a research model ………………………………………………………...

1

1.3 Factors influencing reproduction performance in pigs ……………………………..

1

1.3.1 Extrinsic factors …………………………...…………………………………...

2

1.3.2 Intrinsic factors ……………………..………………………………………….

2

1.4 The porcine ovary …………………………………………………………………..

3

1.5 Problematic about the differences in the ovarian samples for experimentation ……

3

1.6 Oocyte maturation in vivo …………………………………………………………..

3

1.6.1 Oogenesis in vivo ………………………………………………………………

4

1.6.2 Folliculogenesis in vivo ………………………………………………………..

5

1.6.2.1 Activation of primordial follicles ………………………………………..

5

1.6.2.2 Pre-antral and early antral stages ………………………………………...

5

1.6.2.3 Follicular antral stage …………………………………………………….

6

1.7 Oocyte maturation in vitro ………………………………………………………….

7

1.7.1 Nuclear maturation …………………………………………………………….

8

1.7.2 Cytoplasmic maturation ………………………………………………………..

9

1.8 Problematic of the IVM protocols ……………………………………………...…..

10

1.9 Conclusion …………………………………...……………………………………..

10

1.10 OBJECTIVES AND HYPOTHESES ….………………………………………….

11

CHAPTER 2: GENERAL MATERIALS AND METHODS ………...…………………...

13

2.1 Chemicals and culture media ……………………………….………………………

13

2.1.1 Ovary transport and COCs collection mediums ……………………………….

13

2.1.2 In vitro maturation medium ……………………………………………………

13

2.1.3 Parthenogenesis and early embryo culture mediums …………………………..

13

III

2.2 Animals ………………………………………………………….………………….

14

2.3 Ovary collection and COCs selection ………………………………………………

14

2.4 Routine protocol for in vitro maturation of COCs ………………………………….

16

2.5 Parthenogenetic activation of the oocytes …………………………………………..

16

2.6 Early embryo culture …………...…………………………………………………...

17

CHAPTER 3: Characterization of gilt’s ovaries with different patterns in the follicularmorphology and the meiotic competence of the oocytes .………………………………....

18

3.1 Introduction …………………………………………………………………………

18

3.2 Materials and methods ……………………………………………………………...

19

3.2.1 Ovary collection ……………………………………………………………….

19

3.2.2 Experiment 1: Ovarian weight, surface patterns and follicular populationassessment …………………………………………………………………………...

19

3.2.3 Experiment 2: Oocyte in vitro maturation ……………....…………………….

20

3.2.4 Statistical analysis ……………………………………………………………..

20

3.3 Results ………………………………………………………………………………

21

3.3.1 Ovarian weight and follicular surface patterns ………………..……………….

21

3.3.2 Follicular population ……………………………………...……………............

23

3.3.3 Oocyte maturation in vitro ……………………………………………………..

23

3.4 Discussion …………………………………………………………………………..

26

3.5 Conclusion …………………...……………………………………………………..

27

CHAPTER 4: Effect of removing cumulus cells from porcine cumulus-oocyte complexes
derived from small and medium follicles during in vitro maturation on the apoptotic
status and meiotic progression of the oocytes .……………………………….……………

28

4.1 Introduction …………………………………..………………...…………………...

28

4.2 Materials and methods ……………………………………….……………………..

29

4.2.1 Preparation of COCs …………………………………..……………………….

29

4.2.2 In vitro maturation and decumulation of the oocytes ………….…………...….

29

4.2.3 Fluorescence assays for the apoptotic status, viability and meiotic progressionof the oocytes ……………...………………………………………..………………..

30

4.2.4 Statistical analysis ……………….……………………………………...……...

32

4.3 Results ……………..……………………………..…………………………………

32

4.3.1 Apoptotic status, viability and nuclear stage of oocytes before IVM ………….

32

4.3.2 Viability, nuclear stage of the oocytes after 44 h of IVM and MII spindle
quality……………………………………………………………………………..…..

32

4.4 Discussion …………………………………………………………………………..

38

4.5 Conclusion …………………………………………………...……………………..

39

IV

CHAPTER 5: Effect of removing cumulus cells from porcine COCs derived from small
and medium follicles during IVM in the presence or absence of GDF9 or VEGF on the
meiotic and developmental competences of the oocytes .….………………...……………

40

5.1 Introduction …………………………………………………………………………

40

5.2 Materials and methods ……………………………………………………………...

41

5.2.1 Ovary transport and COCs collection ………………………………………….

41

5.2.2 Experiment 1: Effect of the removal of cumulus cells during IVM on oocyte
nuclear maturation ……………………………………………………………………

42

5.2.3 Experiment 2: Effect of the supplementation of GDF9 on meiotic progressionof oocytes …………………………………………………………………………….

42

5.2.4 Experiment 3: Effect of the supplementation of VEGF on oocyte IVM ………

43

5.2.5 Experiment 4: Developmental competence of oocytes denuded during IVM ....

43

5.2.6 Statistical analysis ……………………………………………………………...

44

5.3 Results ………………………………………………………………………………

44

5.3.1 Experiment 1: Effect of the removal of cumulus cells on oocyte IVM …….….

44

5.3.2 Experiment 2 and 3: Effect of exogenous supplementation GDF9 or VEGF on
oocyte IVM …………………………………………………………………………..

45

5.3.3 Experiment 4: Oocyte developmental capacity and embryo quality of nonsupplemented oocytes denuded before, during and after IVM ………………………

49

5.4 Discussion …………………………………………………………………………..

51

5.5 Conclusion ……………………………………………………………...…………..

52

CHAPTER 6: GENERAL DISCUSSION ………………………………….………...…...

53

6.1 General conclusions ………………………………………………………………..

55

CHAPTER 7: SUMMARY ………………………………………………………………..

56

BIBLIOGRAPHY …...………………………………………………………….…………

58

Appendices ………………………………………………………………………...………

73

V

Abstract

In the developed countries, pigs are usually slaughtered for pork production at 5-6 months
old when those gilts have still not reached puberty. Porcine oocytes are easily prepared, as
cumulus-oocyte complexes (COCs), from ovaries of slaughtered females at local abattoirs.
Although there are, at least, three different types of ovaries; smooth surfaced ovaries (thefollicles do not protrude through the ovarian cortex thus presenting a smooth appearance),
bubbled-like surface ovaries (the follicles protruded strongly through all the ovarian surface)and mixed surface ovaries (the ovaries showed a mixed pattern with some smooth appearance
areas and some with protruding follicles), it is not clear whether the morphology of the ovaries
affects the meiotic competence of the oocytes in each ovary type. For in vitro production of
mammalian embryos, furthermore, usually COCs are collected from middle-sized (3-6 mm indiameter; MF) or larger follicles of the ovaries, due to a higher developmental competence ofthe oocytes as compared with those from small-sized follicles (less than 3 mm in diameter; SF).
Since larger numbers of SF exist in the ovary, if we can develop a new IVM system forachieving higher developmental competence of oocytes from SF, we may be able to further
improve the efficiency to utilize female gametes available in the ovaries for their use inreproductive technologies. Through three experiments described below, therefore, themorphological differences of ovaries and the conditions for in-vitro maturation (IVM) were
examined for oocytes from SF and MF to achieve a more efficient meiotic and developmental
competence.
1. Characterization of prepubertal gilt ovaries with different patterns in the follicularmorphology on the surface and the meiotic competence of the oocytes.
Oocytes from middle-size follicles have been commonly utilized in domestic animals
for in vitro production of embryos, but their maturation and developmental competencecapacities depend on extrinsic and intrinsic factors, which are unknown in ovaries obtained fromslaughterhouses. And so, in this study we evaluated if ovaries from prepubertal gilts have anymorphological differences according to their follicular populations. For this reason, ovaries
were classified into three categories; ovaries with smooth (SSO), bubbled (BSO) or mixed(uneven) surface (MSO), weighted, and the number of small (SF; < 3 mm), medium (MF; 3-6mm) and large (> 6 mm) follicles present on their surface were counted. Cumulus-oocyte
complexes from SF and MF of SSO or MSO were cultured in vitro to assess their meiotic
competence capacity. Based on the follicular numbers, a significantly higher number of SF wasobserved in MSO than SSO and BSO (138.8 vs. 116.6 and 94.4, respectively) and the number ofVIMF per ovary was significantly higher in BSO and MSO than SSO (18.44 and 14.18 vs. 11.04,
respectively). When the meiotic competence of oocytes was assessed, MSO showed asignificantly higher percentage of metaphase-II stage oocytes than SSO in SF (55.6% vs. 50.0%,
respectively) and MF (80.2% vs. 75.6%, respectively). In conclusion, we propose that MSO can
suppose a good source of oocytes for improving the efficiency of the actual reproductive
technologies.
2. Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived fromsmall and medium follicles during in vitro maturation on the apoptotic status and meiotic
progression of the oocytes.
The present study was undertaken to examine the apoptotic status and meiotic
progression of oocytes from small (SF; 0.5-2 mm in diameter) and medium follicles (MF; 3-6mm in diameter) when the oocytes were denuded before, during and after in-vitro maturation(IVM). Cumulus-oocyte complexes (COCs) from SF or MF of prepubertal gilt ovaries were
cultured for IVM, in the presence of 1 mM dibutyryl cAMP, 10 IU/ml eCG and 10 IU/ml hCG
for 20 h and then in the absence of these supplements in the same medium for another 24 h.
Before or 20 h after the start of IVM culture, some of the oocytes were isolated from COCs andcontinued the culture for IVM. At the end of IVM, the apoptotic status and meiotic progressionof the oocytes were compared with oocytes cultured in the presence of cumulus cells (CCs) byAnnexin-V/PI assay and DAPI staining. At the start of IVM, there was not a significant
difference in the viability between oocytes from SF and MF, but the incidence of oocytes at theGV0 stage was higher in the specimens from SF than MF, and that of oocytes at the GVI stage
was the opposite. Apoptotic status of the oocytes was only affected by the time when theoocytes were denuded. The percentage of intact oocytes was significantly higher when CCs
were removed at 20 and 44 h after the start of IVM in both, oocytes from SF and MF. The
incidence of early and late apoptotic oocytes was significantly higher when the CCs were
removed before IVM in both, SF and MF. The incidence of mature oocytes was significantly
affected by both, the origin of COCs and decumulation timing. Although the percentage ofmature oocytes was higher in MF, maturation rates were significantly higher when oocytes were
denuded at 20 h, as compared with 0 and 44 h after the start of IVM. However, the percentage
of mature oocytes with a morphologically normal spindle was significantly higher when oocytes
were denuded at 44 h, rather than 20 h of IVM. In conclusion, removing CCs at 20 h after thestart of IVM seems to promote meiotic progression of the oocytes to the metaphase-II stage
even when the COCs were collected from SF, but factor(s) from or communication with CCs
during IVM may be needed to obtain a morphologically normal spindle in mature oocytes.

VII3. Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived fromsmall and medium follicles during IVM in the presence or absence of GDF9 or VEGF on
the meiotic and developmental competences of the oocytes.
Strategies to induce meiotic resumption of oocytes could be reducing the cAMP levels
by interruption of communication between oocyte and cumulus cells, or by supplementation of
the IVM medium with exogenous hormones and growth factors. Vascular endothelial growth
factor (VEGF) and growth-differentiation factor 9 (GDF9) have been proved to improve themeiotic competence of oocytes. Firstly, the effect of timing of removing cumulus cells during
IVM on meiotic and developmental competences of oocytes was assessed, and it was also
examined if the addition of GDF9 or VEGF could further improve IVM rates of oocytes
denuded during maturation culture. In all experiments, it was observed that SF oocytes had
lower maturation, cleavage and blastocyst rates than those from MF. Denuding oocytes before
the start of IVM had detrimental effects on all the parameters studied, whereas culturing theoocytes as COCs during the first 10 h was enough to induce meiotic resumption in the samelevels as the controls. When intact COCs were denuded 20 h after the start of IVM, maturation
rates of the oocytes from SF (64.1%) and MF (82.5%) were significantly higher than SF(51.2%) and MF controls (76.0%), whereas developmental competence to the blastocyst stageof those oocytes was similar with SF and MF controls, respectively. When an exogenous growth
factor, VEGF or GDF9, was supplemented to the IVM medium, any significant effects were notobserved in oocytes denuded 20 h after the start of IVM. For this, we concluded that oocytes
from MF have a higher quality than those from SF and that denuding the oocytes 20 h after thestart of IVM could be a good method to promote the meiotic resumption of oocytes, without any
reduction in the developmental competence.
Results from current experiments demonstrate that using COCs from MSO or byremoving cumulus cells of COCs 20 h after the start of IVM can promote the meiotic
resumption of oocytes, without any reduction in the developmental competence. This
information may contribute to improve the efficiency to prepare female resources for in-vitro
embryo production.

VIII

Declaration

This dissertation contains no materials which have been accepted for the award of any other
degree or diploma in any other tertiary institution and to the best of my knowledge and belief,
contains no materials previously published or written by another person, except for thereferences that have been included in this text.

Signature.

Date.

IX

Acknowledgements

My deepest and foremost thanks are for my mentor and supervisor, Doctor Hiroaki
Funahashi, for all these years I have been able to spend in his laboratory, the guidance he hasoffered me and the patience, the understanding and all the kindness he showed towards me
during the entire journey for the completion of my PhD.
I would also like to thank Doctor Takuya Wakai, who even if he arrived when myproject had already started, still offered his help and guidance until the end. I also thank-you forsurfing through the University webpage and helping me find all the application forms anddocuments I needed all the time.
I must also thank the local slaughterhouse, the Okayama Prefectural Meat for donating
the porcine ovaries I needed for this project and to all the staff members who work in there. I
specially convey my appreciation to the nice old man (whose name I never knew) for always
passing me the ovaries without throwing them to my face. I totally appreciated it, really. I also
want to thank all the laboratory members who, independently of the weather, brought theovaries to the laboratory every morning without fail during these four years.
I also thank Mr. Khanh, from our laboratory “Vietnamese team”, for teaching me how
to perform oocyte parthenogenesis and for the help I got every time I would perform theseexperiments; you saved me lots of time and trouble!
I also must thank Anna Sánchez, one of the few Spanish in the University for those talks
and discussions we held in any restaurant, cafeteria or whichever place we ended up meeting. I
totally enjoyed them; even if we were supposedly meeting for just a coffee and when we would
check the time it would already be night time. It was fun to have someone from a different
specialty to comment the protocols and results of my experiments, because you always offered afresh view.
To my best friend Leandro, the guy who I would not have met if I had not come to
Japan. I am glad I met you on my first day in Okayama. Thank-you for being one of the few
guys I could speak to in one of my native languages. You were the only one who totally
understood my daily frustrations in this country, and for being the one who introduced me to myactual boyfriend. You are my Argentinian brother!
To my boyfriend, Jun, I am thankful that our paths crossed in Japan. Thanks for being
with me and for encouraging and supporting me always, even in those days I wanted toXrenounce and throw everything away. Even if during this last part of my PhD journey you havenot been physically here with me, you are still one of the greatest pillars that support me.
I also cannot forget to express my thanks to my parents, who always supported me in all
the decisions I took until now and to whom I am dedicating this thesis. Without you it would
have been impossible.
I finally would like to thank everybody who was also important for the realization of mythesis, as well as expressing my apologies to all those whom I could not mention personally one
by one.

XI

Publications arising from this thesis

1. Ferré P, Bui TMT, Wakai T, Funahashi H (2016). Effect of removing cumulus cells from
porcine cumulus-oocyte complexes derived from small and medium follicles during in vitro
maturation on the apoptotic status and meiotic progression of the oocytes. Theriogenology. [In
Press] doi: 10.1016/j.theriogenology.2016.05.024

XII

Conference proceedings

1. Ferré P and Funahashi H. Effect of cumulus cell removal during in vitro maturation of
porcine cumulus-oocyte complexes on the apoptotic status and meiotic progression of theoocytes. Poster was presented at the 41st International Embryo Transfer Society meeting in
Versailles, France (2015).
2. Ferré P, Bui TTM, Wakai T and Funahashi H. Effect of cumulus cell removal at different
timings during IVM on nuclear maturation of porcine oocytes. Poster was presented at the 108th
Meeting of the Society for Reproduction and Development in Miyazaki, Japan (2015).
3. Ferré P, Bui TTM, Tran MT, Wakai T and Funahashi H. Characterization of prepubertal gilt
ovaries with different patterns in the follicular morphology on the surface and the meiotic
competence of the oocytes. Poster was presented at the 5th International Conference on
Sustainable Animal Agriculture for Developing Countries in Pattaya, Thailand (2015).
4. Ferré P, Bui TTM, Tran MT, Wakai T and Funahashi H. Effect of addition of follicular fluidor growth differentiation factor-9 on in vitro maturation of porcine oocytes denuded 20 h after
the start of in vitro maturation. Poster was presented at the 42 nd International Embryo Transfer
Society meeting in Louisville, USA (2016).
5. Ferré P, Wakai T and Funahashi H. Effect of cumulus cell removal during in-vitro
maturation on the meiotic competence of oocytes derived from small and medium follicles.
Poster was presented at the 18th International Congress on Animal Reproduction in Tours,
France (2016). Poster was also presented at the 11th Research and exchange meeting of highschool students and graduate students, Okayama University, Japan (2016).

XIII

List of figures
CHAPTER 1.
Figure 1.1

Mammalian oogenesis and folliculogenesis stages from the developmentof the primordial germ cells (PGCs) to the ovulation of a mature oocytefrom a Graafian follicle……………………………………………………

4

CHAPTER 2.
Figure 2.1

Classification of ovaries according to the morphology of their cortex ...… 15

Figure 2.2

Ovaries with the presence of follicles with different diameters on theirsurface …………………………………………………………………….

Figure 2.3

15

Different types of cumulus-oocyte complexes according to the quality ofthe surrounding cumulus cells ……………………………………………

16

CHAPTER 3.
Figure 3.1

Three categories of porcine ovaries according to their surface
morphology …..………………………..…………………………...

Figure 3.2

Oocyte nuclear chromatin status after 44 h of IVM culture when stainedwith DAPI ………………………………………………………...………

Figure 3.3

19

21

Percentages of GVBD in oocytes derived from SF and MF of different
morphologies of ovaries (SSO and MSO) after IVM ……....……..……...

24

CHAPTER 4.
Figure 4.1

Apoptotic status of the oocytes stained with Annexin V/PI …………..….

Figure 4.2

Oocyte nuclear status including germinal vesicle stages in oocytes before

Figure 4.3

30

IVM when stained with DAPI ….………………………………………...

31

Nuclear morphologies of MII stage oocytes when stained with DAPI …..

31

CHAPTER 5.
Figure 5.1

Nuclear status of oocytes after 44 h of IVM when stained with orcein …..

Figure 5.2

Oocyte GVBD rates after 44 h of IVM culture when CCs were removed
at different times of the IVM culture ……………………..………………

Figure 5.3

46

Oocyte nuclear maturation rates of SF and MF denuded at 20 h of IVM
and supplemented or not with VEGF …………………………………….

Figure 5.6

45

Oocyte nuclear maturation rates of SF and MF denuded at 20 h of IVM
and supplemented or not with GDF9 ……………………………………..

Figure 5.5

44

Rates of mature oocytes after 44 h of IVM culture when CCs were
removed at different times of the IVM culture …………………………...

Figure 5.4

42

46

Nuclear maturation rates of SF oocytes denuded at 20 h of IVM culture
when supplemented or not with GDF9 when compared with the control...

XIV

47

Figure 5.7

Nuclear maturation rates of SF oocytes denuded at 20 h of IVM culture
when supplemented or not with VEGF when compared with the control...

Figure 5.8

Nuclear maturation rates of MF oocytes denuded at 20 h of IVM culture
when supplemented or not with GDF9 when compared with the control...

Figure 5.947

48

Nuclear maturation rates of MF oocytes denuded at 20 h of IVM culture
when supplemented or not with VEGF when compared with the control...

XV

48

List of tables
CHAPTER 3.
Table 3.1

Table 3.2

Table 3.3

Weight of prepubertal porcine ovaries according to their surface
morphological characteristics …………………………..…………………… 22
Number of follicles present in prepubertal porcine ovaries with different
morphologies ……………………………………………………………… 22
Nuclear stage of oocytes from SF and MF of smooth (SSO) and mixed
surface ovaries (MSO) after 44 h of IVM culture ……………...................... 25

CHAPTER 4.
Table 4.1 Apoptotic status of oocytes just after collection of the COCs from SF andMF………………..………………………………………………………….. 32
Table 4.2 Nuclear stage of live oocytes just after collection of the COCs from SF and
MF ……….………………………………………………………………….. 34
Table 4.3 Effect of removal of cumulus cells from COCs derived from SF and MF
before or during IVM on the apoptotic status of oocytes at the end of IVM ....

35

Table 4.4 Effect of removal of CCs from COCs derived from SF and MF before andduring IVM of COCs on meiotic competence of the oocytes 44 h after thestart of IVM ………….………………………………………………………. 36
Table 4.5 Effect of removal of CCs during IVM on the normal morphology ofmetaphase spindle in the mature oocytes ……………………………………. 37
CHAPTER 5.
Table 5.1

Maturation of SF and MF oocytes by polar body selection when CCs are
removed at 0, 20 and 44 h of IVM …………………………………………….

49

Table 5.2 Embryo developmental capacity of SF and MF oocytes denuded at 0, 20 and
44 h of IVM after parthenogenetic activation ……..………………………….

XVI

50

Abbreviations%

Percentage°C

degree(s) Celsiusµg

microgram(s)µlmicroliter(s)µmmicrometer(s)µM

micromole(s)µsec

microsecond(s)ACalternating currentAIanaphase I

BSA

bovine serum albumin

cAMP

cyclic adenosine monophosphateCCs

cumulus cells

COCs

cumulus-oocyte complexes

Da

DaltonDAPI

4’,6-Diamidino-2-phenylindole

dbcAMP

dibutyryl cyclic adenosine monophosphateDCdirect current

Deg

degenerated oocyte

DMSO

dimethyl sulfoxideDOdenuded oocyte

eCG

equine chorionic gonadotropin

EDTA

ethylenediaminetetraacetic acidFF

follicular fluidggram(s)GDF9growth differentiation factor-9

GV

germinal vesicle

GV0

germinal vesicle stage 0

GVI

germinal vesicle stage I

GVII

germinal vesicle stage II

GVIII-IVgerminal vesicle stage III-IV

GVBD

germinal vesicle breakdown

hCG

human chorionic gonadotropin

HEPES

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidIVC

in vitro culture

IVF

in vitro fertilization
XVII

IVM

in vitro maturation

LF

large follicle

M

Molar

MF

medium folliclemg

milligram(s)MImetaphase I

MII

metaphase IImMmilimolar

mM199

modified medium 199

mPOM

modified porcine oocyte mediumng

nanogram(s)nMnanomolarPI

propidium iodide

PMI

prometaphase I

PVA

polyvinyl alcoholSFsmall follicle

TI

telophase I

TL-HEPESHEPES buffered Tyrode Lactate solution

VEGF

vascular endothelial growth factor

ZP

zona pellucida

XVIII

CHAPTER 1: GENERAL INTRODUCTION

1.1 Preface
The history of oocyte maturation in-vitro started long ago, with the pioneer studies ofEvans (1931) and of Pincus and Enzmann (1935). In the reproductive field, in-vitro maturation(IVM) consists in the culture of reproductive cells (most commonly oocytes or germinal stem
cells) outside the body in controlled conditions which allow them to grow and develop in an
optimal manner. Actually, reproductive technologies are important for the maintenance of thegenetic diversity in many farm and endangered species, and they are also basic for theapplication of other technologies such as the creation of transgenic animal models, xenografting
and the creation of bioreactors.
The success of the in-vitro technologies is influenced by many factors in which the ones
with most importance would be the source of samples and the quality of reproductive cells, so it
is of great interest to research new methods that will allow us to obtain great numbers of highquality gametes. For this, it is important to understand the factors that affect the reproductive
performance of animals, since it will affect the quality of gametes and also we must understandthe pathways for their growth and maturation in vivo, so we can improve and develop new
systems to culture oocytes in vitro in a more efficient way.
1.2 The pig as a research model
The domestic pigs are closely related to humans in terms of anatomy, genetics andphysiology (Hart et al., 2007; Dyce et al., 1987; Swindle et al., 2012) making them into a goodmodel for the study of many infectious and metabolic disorders or illnesses as an alternative tothe rodent models (Meurens et al., 2012). Pigs can also be used for the creation transgenic
animals that can be used as bioreactors for producing proteins of pharmacologic interest (Park etal., 2006; Lee et al., 2009) and they are also important for the creation and development of new
medical therapies, such as xenotransplants (Cozzi and White, 1995; Levy, 2000) which have
had relative success (Reinholt et al., 1998; Deacon et al., 1997) giving hope into improving
human life quality. For being able to apply these innovative technologies, reproductive in vitro
techniques are of crucial importance, since they constitute the first step for their successfulachievement.
1.3 Factors influencing reproduction performance in pigs

1

Animal reproductive performance and efficiency is affected by many elements that will
influence the quality of the gametes and they can be classified into two main groups: extrinsic
factors; which depend on the environment and the management conditions and intrinsic factors;
those which depend on the individual (Rodríguez FPC., 2005).
1.3.1 Extrinsic factors
The intense selection of the growth and productivity characters for the domestic pig
breeding has developed a continuous polyestric female (Love et al., 1981; Falceto, 1992), butthere are still influences of seasons (and photoperiod) on prolificacy in both, male and female
pigs (Greer, 1983). In males, in the summer, heat stress affects negatively to spermatogenesis(Wettemann et al., 1979). During this period of stress, the semen quality parameters, such asconcentration of sperm and total number of sperm in the ejaculate lower (Wettemann et al.,
1976, 1979) and the proportion of morphologically abnormal spermatozoides increases(Cameron and Blackshaw, 1980). In females, sows can present heat stress anoestrus during thesummer or in elevated ambient temperatures (Einarsson et al., 2008) and mating the animals inthese conditions can lead to lower conception rates and smaller litters (Edwards et al., 1968;
Nelson et al., 1970).
Zak et al., (1997) detected that sows with delayed heat had low levels of insulin, so
nutrition can be used as a tool for improving fertility. It has been reported that providing gilts
and sows with abundant feed after mating can alleviate these negative effects caused byseasonality (Peltoniemi and Virolainen, 2006). Supplementation of exogenous insulin in thecyclic gilt increases ovulation and stimulates LH secretion (Cox et al., 1987) which can reverse
the negative effects of feed restriction by affecting the brain glucose levels (Almeida et al.,
2001; Ubeda, 2003).
1.3.2 Intrinsic factors
There are differences among animal species, for example, cows (monoovulatory
species) have lower ovulation rates than pigs or sheep (polyovulatory species) (Kumar Pramod
et al., 2013). Differences in fertility can also be detected between different breeds of the same
species, as Miller et al. (1998) showed that Meishan sows have higher ovulation rates thanLarge White hybrids (27.7 vs 17.6 corpora lutea) probably due to differences in follicle
recruitment and atresia. Finally, there can also be differences among individuals, since many
reproductive anomalies and illnesses are heritable, producing malformations in males andfemales that can lead to gonad inactivity such as having ovotestes or ovarian hypoplasy (Baker
JR., 1925; Falceto et al., 1990).

2

1.4 The porcine ovary
The ovary is the main organ at a reproductive level; they are found in pairs, but theirplacement and size varies among species. They are responsible for the supply of germ cells(oocytes) and the production of steroidogenic hormones from circulating cholesterol for the
production of estrogen and progesterone that control many aspects of the female development
and physiology (Edson et al., 2009).
The reproductive system in pigs is situated dorsal to the intestines in the pelvic cavity.
Macroscopically, the ovaries from prepubertal gilts have a blackberry-like shape with abundant
pink follicles of different diameters with less than 6 mm in diameter. In these young females,
there are continuous waves of follicular growth and atresia that never overcome the intermediate
follicular size, so the ovaries from these young gilts do not possess corpora albicans (Falceto,1987). In post-pubertal females presenting anestrous, the ovaries look flattened and smooth withfew numbers of follicles of variable size on the surface with less than 6 mm in diameter that will
regress by atresia and not ovulate, but, unlike the ovaries of prepubertal gilts, these ovaries
possess corpora albicans, indicating that these females reached puberty even if their estrous was
not detected (Falceto et al., 2004).
1.5 Problematic about the differences in the ovarian samples for experimentation.
We indicated that the differences in the reproductive performance of the pigs usedcommercially for the production of meat can be due to numerous factors such as breed, age,season, nutrition and housing conditions that can result in differences on the quality of oocytes
and sperms.
Since the ovaries are highly-available and because they constitute an unwanted sub
product of the meat industry, most of the actual studies in reproductive biotechnologies using
the pig as a model are performed with ovaries obtained from animals daily abated for meatproduction. Among these animals, we can find prepubertal gilts, pubertal gilts or gilts that spend
a long time in anestrous. As we said, anestrous is a symptom of ovarian inactivity, which is a
cause of infertility and sterility in the swine (Falceto et al., 2004). For this, the samples will befrom a variable origin, making it difficult to find a method for standardizing samples for making
the IVM protocols more efficient.
1.6 Oocyte maturation in vivo

3

The mammalian oocytes develop and mature inside the ovarian follicles. The growthand development of these follicles requires a series of coordinated events that induce
morphological and functional changes within the follicle that lead to cell differentiation andsubsequent oocyte growth and development (Palma et al., 2012).
1.6.1 Oogenesis in vivo
In the mammalian species, the process of oogenesis is initiated early during the fetal
development and it ends when the female becomes sexually mature and happens synchronicallywith the folliculogenesis process. The oogenesis begins with the formation of the primordial
germ cells (PGCs) during the gastrulation process. These oocyte precursors have motility andcan respond to chemotactic stimuli making them able to migrate from the site of origin in theembryonic epiblast through the genital ridge until they arrive to the undifferentiated but
developing gonad. During and after their migration, the PGCs proliferate by mitoses and, when
they become surrounded by somatic cells, are called oogonia (Picton et al., 1998; Palma et al.,2012).
In a determined moment, the oogonia will enter the meiosis process and will becomearrested at the diplotene stage of the first meiotic prophase (dictyate stage) surrounded by asingle layer of flat pre-granulosa cells forming the primordial follicles. The oocytes will remain
at this stage until the female reaches puberty, when a surge of LH will induce the resumption ofmeiosis and the ovulation of the oocytes arrested at the MII stage (Huang et al., 2010; Zuccotti
et al., 2011).

Fig. 1.1: Mammalian oogenesis and folliculogenesis stages from the development of the primordial germ
cells (PGCs) to the ovulation of a mature oocyte from a Graaffian follicle. Follicular diameter,
chromosomal (n) and DNA content (C) of the oocyte are shown in the scheme.
(modified from Picton et al., 1998).

4

1.6.2 Folliculogenesis in vivo
An ovarian follicle is a multicellular structure consisting in a meiotically arrested oocyte
surrounded by one or several layers of somatic cells, pre-granulosa or granulosa cells. Pregranulosa cells are the primary cell type in the ovary and are cells that provide the physicalsupport and the microenvironment required for the developing oocyte. These granulosa cells
establish strong connections with the oocyte (intermediate junctions and gap junctions) which
ensure a bi-directional communication pathway for the delivery of signals necessary for the
production of hormones and the growth of the oocytes (Nicholson et al., 1997; Huang et al.,2010; Palma et al., 2012).
In the pig, follicular growth from the primary to nearly pre-ovulatory status (also called
Graafian follicle) takes more than three months, and the process can be divided into two distinct
phases:-A slow growth phase, which is independent on gonadotropin stimulation. During thisphase, pre-antral follicles grow by increasing the number of granulosa cells and the sizeof the oocyte.

-

A rapid growth phase, which is dependent on gonadotropin stimulation. The size of thefollicle augments due to a dramatic increase in the proliferation of granulosa cells andthe formation of the antrum occurs (Morbeck et al., 1992).
1.6.2.1 Activation of primordial follicles
Oocytes can control follicular formation through the activity of transcription factor ingerm cell α (TNFα), which regulates the initial organization of primordial follicles andmodulates the survival of the germ cells (Zuccotti et al., 2011; Sánchez et al., 2012). Numerous
factors have been demonstrated to arrest or induce the recruitment of primordial follicles in anexperimental way, but the exact role of these factors is not yet fully understood, but they can beclassified according to their effect on the follicular development as inhibiting or activating
factors. (Skinner, 2005; Zuccotti et al., 2011; Palma et al., 2012).
1.6.2.2 Pre-antral and early antral stages
When a primordial follicle leaves the resting pool, the pre-granulosa cells secrete kitligand (KL) that will interact with the oocyte promoting its growth. During this growth phase,the oocyte will secrete glycoproteins which will condense around it to form a translucent layer,
the zona pellucida (ZP; Gupta et al., 2012). When the ZP synthesis starts, the granulosa cells
will become cuboid and they will extend processes through the newly synthetized structure to
the ooplasm where they will form gap junctions allowing a bi-directional communication
5

between the oocyte and the cumulus cells (Nicholson et al., 1997; Sugiura et al., 2005; Gilchrist
et al., 2008; Palma et al., 2012).
Gap junctions present between oocyte and surrounding cumulus cells will allow the lastones to support oocyte development and to play an essential role during its growth and its
metabolism by exchanging molecules of less than 1kDa, inorganic ions and electrical impulses(Kidder and Mhawi, 2002). The cumulus cells will also provide the oocyte with aminoacids andsubstrates derived from glycolysis, such as pyruvate, that will be used for nutrition and energy
production (Biggers JD, 1967), thanks to the oocyte secretion of paracrine factors that will
stimulate the glycolytic activity of cumulus cells by promoting the expression of genes encoding
glycolytic enzymes (Sugiura et al., 2005; Zuccotti et al., 2011; Palma et al., 2012). During thistime of oocyte growth, the stromal cells outside the basement membrane will become organized
into the theca cell layers by the presence of soluble factors secreted by the granulosa cells(Young and McNeilly, 2010).
The regulation of the follicular cell proliferation during the recruitment is due to thecooperative effect of the oocyte secreted factors from the TGF-β superfamily growthdifferentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15). The first has adominant effect during the early phases, while the activity of the second is performed during
more advanced phases (Hunter et al., 2005). The correct growth of the follicle is balanced
trough the regulation of the KL expression, factor that co-ordinates the growth of the oocyte andthe proliferation of the granulosa cells. Follicle growth is inhibited by GDF-9 and is activatedby BMP-15. BMP-15 also inactivates itself by the KL expression in a negative feedback loop
(Hunter et al., 2005; Zuccotti et al., 2011). Growth differentiation factor-9, BMP-15 and also
BMP-6 act through receptors to activate the intracellular signal transducer molecules (SMAD)
cascade by binding to a type-I receptor (ALK) or a type II receptor, that leads to ALK
phosphorylation of numerous SMAD molecules necessary for inducing the gene transcription
pathway (Gilchrist et al., 2008).
1.6.2.3 Follicular antral stage
The antral stage of follicular development is characterized by its dependence on
gonadotrophins (FSH and LH) secreted cyclically by the pituitary gland, and since the oocyte
lacks the receptors, their action on oocyte development will be exerted via cumulus cells(Schoevers et al., 2007; Zuccotti et al., 2011).
When a follicle reaches to 300 µm in diameter, the oocyte has acquired its final size andit is in this moment when the follicle undergoes pre-antral to antral transition. In this phase, thegranulosa cells will continue to proliferate, and when the follicle reaches a diameter of 400 µm,
6follicular fluid will accumulate and it will end up coalescing to form a single follicular antrum(Morbeck et al., 1992; Huang et al., 2010). With the formation of the antral cavity, thegranulosa cells differentiate into two anatomically and functionally distinct lineages: the mural
granulosa cells that line the follicle wall and have principally a steroidogenic role, and thecumulus cells, which form an intimate association with the oocyte that will gradually acquire
meiotic and developmental competence (Sugiura et al., 2005). This differentiation is due to the
suppressive effect that the oocyte exerts on the protein expression in cumulus cells, maintaining
their phenotype by down-regulating several mural granulosa cell markers such as: LHCGR,
CYP11A1 and CD34 (Gilchrist et al., 2008; Paradis et al., 2010; Zuccotti et al., 2011).
The response of cumulus cells to the gonadotropin surge is mediated by a highconcentration of FSH that accompanies the LH surge together with the action of local paracrine
effectors such as epidermal growth factor-like ligands from the granulosa cells, interleukin-1
from the thecal compartment and oocyte-derived signals. In the mural granulosa cells, LH binds
to its receptor and stimulates the adenyl cyclase (AC) causing an increase in the intracellularconcentration of cAMP, which activates the protein kinase A (PKA) and the downstream
extracellular-signal-regulated kinases 1 and 2 (ERK1, ERK2 or MAPK3/1) signaling cascade.
Subsequently, the ERK1/2 pathway induces the down-regulation of genes related to folliculardevelopment and the up-regulation of the ovulation-related genes, concretely those required for
cumulus expansion. The products of these genes, will act in an autocrine/juxtacrine way within
the mural granulosa cells or they will diffuse to activate the cumulus cells (Conti et al., 2005;
Shimada et al., 2006; Procházka et al., 2011). The ERK1/2 pathway also mediates theimmediate effect of LH on gap junctional closure in granulosa cells, stopping the somatic influx
of cAMP to the oocyte, which will lead to a drop of the ooplasm level of cAMP allowing
meiotic resumption and germinal vesicle breakdown (GVBD; Sánchez et al., 2012).
Finally, the process of follicular development will be completed with the creation of arupture pore in the apical follicle wall and the ovulation of a MII arrested oocyte surrounded bya surrounding mass of expanded cumulus cells (Akison et al., 2012).
1.7 Oocyte maturation in vitro
In vitro maturation is an important reproductive biotechnology that allows the
production of large numbers of viable oocytes capable to be fertilized and generate embryoswith the ability of developing to term and it can also reduce the generation intervals andincrease the quality of the offspring obtained by this process. This technology allows to use
many of the oocytes present in the ovaries that otherwise would generally be lost due to atretic
processes in the ovarian follicles and by allowing the use of ovaries derived from7

slaughterhouses that otherwise would be wasted. These oocytes can be used in basic research
and also for advanced animal husbandry and that is basic to the application of otherbiotechnological applications such as cloning (Kim et al., 2005) or the production of transgenic
animals to be used as xenotransplants or bioreactors (Cozzy and White, 1995; Lee et al., 2009).
A very important point for the IVM technology is to be able to succeed into obtaining
oocytes that can achieve both, nuclear and cytoplasmic maturation. Since many years ago, it is
known that mammalian oocytes can resume meiosis when they are liberated from the follicularenvironment (Pincus and Enzmann, 1935), and that they can be matured in vitro in defined
mediums supplemented with exogenous agents like bovine serum (Edwards, 1965). Currently,
the mechanisms responsible for oocyte maturation are still not completely understood causing
the actual IVM systems to be unable to mimic the in-vivo conditions of follicular environment
before and during culture.
1.7.1 Nuclear maturation
In oocytes, nuclear maturation involves the condensation and redistribution ofchromatins during meiosis.
In pigs, the oocytes have a high variability in the GV morphologies after collection(Nagai et al., 1997) and also during culture (Funahashi et al., 1997a). This heterogeneity is
responsible for observable differences in the meiotic and developmental competences of theoocytes collected for IVM purposes (Mcgaughey and Polge, 1971) and might also be
responsible for the oocytes to induce meiotic resumption on different times, thus causing
variations in their maturation at the end of the culture. It is known that at the end of IVM culture,
not all oocytes are able to complete their nuclear maturation. Some oocytes remain at the GV or
MI stages, whereas others simply degenerate through apoptosis (Miao et al., 2009). These
differences not only affect oocyte maturation but also their developmental ability and can becaused by numerous factors such as coming from oocytes and follicles with a different size intheir diameters (Szybek, 1972; Motlik and Fulka, 1986; Eppig et al., 1994; Grabowska et al.,2016), stress caused by the usage of inadequate culture media (Laurinick et al., 1994; Fischer
Russell et al., 2006) or by differences in the age of the donor (Revel et al., 1996; O’Brien et al.,
1996; Marchal et al., 2001; Khatun et al., 2011).
A way of partly solving the previously stated problems is by a temporary inhibition of
the meiotic maturation during the first part of the IVM culture, fact that also increases oocyte
developmental competence (Funahashi et al., 1997b; Hashimoto et al., 2002) because thisprocess does not inhibit the protein synthesis during the GV stages (Marchal et al., 2001). The
inhibition of the GVBD can be achieved with different methods: the inhibition of the maturation8

promoting factor (MPF) activity (Mermillod et al., 2000; Kulbeka et al., 2000) or with the
supplementation of agents capable of incrementing the intracellular levels of cAMP such as
dibutyryl cyclic AMP (dbcAMP), a cAMP analogue that is permeable to cell membranes andcan also increase the activity of the PKA (Kim et al., 2008). It has been demonstrated that theaddition of this molecule during the first part of IVM, can inhibit oocyte GVBD, has asynchronization effect on the nuclear stages of pig oocytes (Funahashi et al., 1997b) and also
has a positive effect on promoting MI to MII transition thus enhancing the meiotic potential of
the oocytes (Somfai et al., 2003). Furthermore, it has been suggested that the lower
developmental capacity of oocytes from prepubertal gilts might be due to their incapacity ofaccumulating cAMP in their cytoplasm during IVM culture (Bagg et al., 2006).
1.7.2 Cytoplasmic maturation
The cytoplasmic maturation of an oocyte, also known as oocyte capacitation, occurs
during the oocyte growth phase and it comprises a series of events that imply ultrastructural andmolecular changes occurring in the cytoplasm of the oocytes (Hyttel et al., 1997).
It is believed that the storage of a determined amount of transcripts or proteins isrequired for the oocytes to acquire higher meiotic and developmental competence. Pig oocytes
are known to require a phase of protein synthesis before the GVBD occurs for a normal
progression of meiosis (Fulka et al., 1986), and that after 12-16 h after the LH peak takes place,a new synthesis period begins, being necessary for breaking the nuclear envelope (Kulbeka et al.,1988).
Ultrastructural changes during the period of cytoplasmic maturation can suppose therelocation of organelles such as cortical granules or mitochondria. In the oocyte, the cortical
granules develop from the Golgi apparatus and, when the oocyte has finished its maturation they
get positioned just under the oolema for, after fertilization, undergo exocytosis and release theircontents into the perivitelline space for blocking polyspermy (Liu, 2011). An irregular
distribution of the oocyte cortical granules might be responsible for a high incidence ofpolyspermy after IVF in vitro (Cran and Cheng, 1986).
Mitochondria relocation seems to be important for a normal development in many
species. In immature oocytes, their shape is round or ovoid, possess few cristae and can containvacuoles (Motta et al., 2000), and they gradually will become more elongated and the number oftransverse cristae will increase (Hyttel and Niemann, 1990). Mitochondria are distributed
throughout all the cytoplasm in mature bovine oocytes, but they can be not present in thecortical region (Van Blerkom, 1990; Plante and King, 1994). In oocytes at the GV stage,
mitochondria are situated mostly in the cortex but during maturation they are relocated9depending on the nutrients present in the medium (Krisher and Bavister, 1997). If themitochondria remain in the cortex, it is a sign of low developmental competence. The
functionality of this redistribution is not understood, but it is speculated that they might supply
energy to the nucleus or reduce the concentrations of oxygen, thus acting as an antioxidant agent(Bavister and Squirrell, 2000).
1.8 Problematic of the IVM protocols
The oocytes obtained in the ovarian follicles present heterogenic characteristics that willaffect to their maturation and developmental competence capacities. Furthermore, themechanisms implicated in the synchronous nuclear and cytoplasmic maturation of oocytes invivo are not completely understood, fact that causes difficulties in the development of good
methods for maturing oocytes in vitro. Protocols based in the inhibition of an early meiotic
resumption have been developed to improve the quality and number of mature oocytes, but even
if the actual maturation protocols allow similar maturation rates of oocytes derived from both
stimulated large and unstimulated middle or small follicles, the blastocyst rates of the later ones
are still much lower, fact that reduces significantly the effectivity of the reproductive
technologies.
Strategies to characterize the oocyte quality by morphologic and biochemical criteria
have recently gotten attention with the objective of improving oocyte meiotic potential anddevelopmental competence.
1.9 Conclusion
The pig is a farm species with a high research interest for its use in numerous
reproductive technologies. Many factors unknown to researchers affect to the quality of theoocytes from ovaries of slaughtered pigs for pork production, and the in vivo mechanisms
implied in the synchronic nuclear and cytoplasmic maturation of oocytes is not totally
understood due to the complexity of the follicular environment and the pathways implicated.
Technique for IVM has been developed to allow the maturation of oocytes fromdifferent sources, but as the oocyte samples are highly heterogenic, current technologies arelimited to obtain large proportions of mature oocytes with a good quality, especially if they arederived from small follicles. It is of great importance to develop new methods for improving theefficiency of the actual IVM technologies by obtaining large numbers of highly competent
oocytes.

10

1. 10 OBJECTIVES AND HYPOTHESES

After having analyzed the actual status of porcine in vitro maturation protocols, two main
objectives were proposed for this doctoral thesis.
-

Firstly, to find an ovarian intrinsic factor that may be used for the improvement on theefficiency of the actual in vitro maturation protocols.

-

Secondly, to find a method for improving the in vitro maturation protocols for theobtainment of high-quality mature oocytes from small and medium-sized ovarianfollicles.

For the achievement of these main objectives, the following working hypotheses wereprepared:
1. Reproductive efficiency in farm animals, such as pigs, depends on two main factors;
intrinsic and extrinsic. As most of ovaries used for experimentation purposes are
obtained from slaughtered animals, extrinsic factors which influence reproduction suchas nutrition, the environment and the sanitary conditions are factors which are unknown
and cannot be controlled by the experimenter. To be able to select a criterion that is
intrinsic on the sample, (which means that must be a characteristic which has to beobserved on the sample, in this case the ovaries from slaughtered gilts), the morphology
of ovarian surface was selected as the main criterion that will allow to select highquality cumulus-oocyte complexes with the objective of improving the efficiency of
culture protocols for IVM.
For this, firstly ovaries were divided, according to their surface characteristics, into 3
groups: smooth-surfaced ovaries (SSO), bubbled-like surfaced oocytes (BSO) andmixed-surface ovaries (MSO), and their weight and the number of small, medium andbig follicles present on the surface were assessed for each type. Afterwards, groups of
COCs from small and medium follicles of SSO and MSO types were in vitro cultured
and their nuclear status was subsequently observed under a microscope to detect if theefficiency of the maturation culture can be improved by the criterion.

2. In the actual protocols used for the IVM of oocytes, these cells are usually matured in
the presence of meiotic inhibitors that act in a reversible manner. After co-incubating
the oocytes with the inhibitor for some time, they are afterwards transferred to a
medium without the inhibitor supplements to complete their maturation. During theculture, cumulus cells surrounding oocytes decrease their communication as the time11

passes, diminishing the transport of inhibiting substances such as cAMP and cGMP tothe ooplasm. That is the main reason why it was hypothesized that a way to improve
the nuclear maturation of oocytes can be performed by diminishing the intracellularlevels of these inhibiting molecules such as cAMP by removing the cumulus cells
during IVM.
For this, it was firstly investigated the oocyte nuclear and apoptotic status of oocytes
from small and medium follicles before IVM. The second experiment was to assess thematuration rates and the apoptotic status of oocytes after IVM when they were denuded
after the first part of IVM (after 20 h) when compared with the controls (before IVM,
after 0 h; and at the end of the culture, after 44 h). It was also examined the quality of
meiotic spindle of mature oocytes by observing the morphology under a fluorescencemicroscope.

3. The third part of this study was centered on the maturation rates and thedevelopmental competence capacity of oocytes from small and medium follicles whencumulus cells were removed before, during and after IVM. It is known that whengrowth factors are added to the in vitro maturation culture media can increase nuclear
and developmental competence in oocytes. This time it was assessed if maturation
rates could be further improved when cumulus cells were removed after 20 h,when the culture media were supplemented with two different growth factors, growthdifferentiation factor 9 (GDF9) or vascular endothelial growth factor (VEGF).
For this objective, firstly it was examined the maturation rates of oocytes from SF and
MF when denuded at 0, 20, 32 and 44 h of IVM, and chose the timing considered as the
optimum for maturation according to the results. After that, the maturation media were
supplemented with exogenous GDF9 or VEGF at concentrations which had been
proven to increase maturation rates in standard IVM conditions (100 ng/mL for GDF9
and 200 ng/mL for VEGF) and analyzed nuclear maturation of the oocytes after
treatments. Finally, as an observable maturation increase was not obtained, oocytes
denuded at 0, 20 and 44 h were parthenogenetically activated to assess theirdevelopmental competence ability and the blastocyst quality.

12CHAPTER 2: GENERAL MATERIALS AND METHODS

2.1 Chemicals and culture media.
The reagents NaCl, NaOH, MgCl2·6H2O, CaCl2·2H2O, KH2PO4, gentamicin-sulphate
and paraffin liquid were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Mannitol,
potassium chloride, NaH2PO4·2H2O and MgSO4·7H2O were purchased from Ishizu
Pharmaceutical Co., Ltd. (Osaka, Japan). Equine chorionic gonadotropin (eCG; Serotropin) andhuman chorionic gonadotropin (hCG; Gonatropin) were purchased from ASKA PharmaceuticalCo., Ltd. (Tokyo, Japan). Unless specified, all the other reagents were obtained from SigmaAldrich Japan K. K. (Tokyo, Japan).
2.1.1 Ovary transport and COCs collection mediums.
The ovaries were transported in the laboratory in a 0.9% (w/v) saline solution
supplemented with 75 mg/L of potassium penicillin G and 50 mg/L of streptomycin-sulphate at
25 °C (Funahashi, 2005). The medium used for the collection and the washing of the COCs was
modified TL-HEPES-PVA medium composed of 114 mM NaCl, 3.2 mM KCl, 2.0 mM
NaHCO3, 0.34 mM KH2PO4, 10.0 mM sodium lactate, 0.5 mM MgCl2·6H2O, 2.0 mM
CaCl2·2H2O, 10.0 mM HEPES, 0.2 mM sodium pyruvate, 12.0 mM sorbitol, 0.1% (w/v)
polyvinylalcohol, 25 µg/mL gentamycin and 65 µg/mL potassium penicillin G (Funahashi et al.,1997).
2.1.2 In vitro maturation medium.
The medium used for IVM culture was a BSA-free chemically defined medium, porcine
oocyte medium which was composed of (Research Institute for the Functional Peptides,
Yamagata, Japan), this medium was supplemented with 50 μM β-mercaptoethanol (mPOM;
Akaki et al., 2009), and it has been shown to support successful development to the blastocyst
stage following IVF. This IVM medium has been showed to support successful development tothe blastocyst stage following IVF (Yoshioka et al., 2008) and piglet production (Akaki et al.,2009).
2.1.3 Parthenogenesis and early embryo culture mediums.
The mediums used for the parthenogenetic activation of the oocytes were an
Electroporation solution composed of 0.25 M mannitol, 0.5 mM HEPES, 100 μM CaCl2·2H2O,
100 μm MgCl2·6H2O and 0.01% (w/v) polyvinylalcohol (PVA) at a pH of 7.2 (Patel et al.,2014).
13

The other medium used just after the parthenogenetic activation of the oocytes was abasic fertilization medium, Medium-199 with Earle’s salts (Gibco, Grand Island, NY) modified
with 3.05 mM D-glucose, 2.92 mM hemi-calcium lactate, 0.91 mM sodium pyruvate, 12 mM
sorbitol, 75µg/L potassium penicillin G and 25 µg/L gentamicin at a pH of 7.4 (mM199;
Funahashi and Day, 1993).
The medium used for early embryo culture was porcine zygote medium (PZM) which
consisted in 108 mM NaCl, 10 mM KCl, 0.35 mM KH2PO4, 0.4 mM MgSO4·7H2O, 25 mM
NaHCO3, 0.2 mM sodium pyruvate, 2 mM L(+)-lactic acid, 2 mM L-glutamine, 5 mM
hypotaurine, 10 μg/mL gentamycin, 2% (v/v) BME amino acids solution, 1% (v/v) MEM nonessential amino acids solution and 3% (w/v) polyvinylalcohol.
All the mediums (except the Electroporation solution) were equilibrated overnight underparaffin oil at 39°C in a wet atmosphere of 5% CO2 in air before their usage. The
Electroporation solution was simply pre-warmed at 39°C overnight.
2.2 Animals
The animals used for obtaining the ovaries were prepubertal gilts which proceeded fromfarms in the Okayama prefecture which were sacrificed when their total corporal live weight
was of approximately 110 kg in a local slaughterhouse (Okayama Prefectural Meat). The
samples were obtained over four years and, as the pig is a continuous polyoestric species, theanimals were continuously in different stages of the estrous cycle at the moment of the sacrifice.
2.3 Ovary collection and COCs selection
Ovaries from prepubertal gilts were collected at a local abattoir and placed in a 0.9%(w/v) NaCl solution supplemented with 75 mg/L of potassium penicillin G and 50 mg/L ofstreptomycin-sulphate. They were transported to the laboratory within 2 h at 25°C. On arrival,
the ovaries were washed twice in new pre-warmed transport solution and they were classified
according to their external morphology into two main groups: early follicular phase and latefollicular phase ovaries.
-

Early follicular phase ovaries included those ovaries which did not show signals of
having hemorrhagic bodies, corpora lutea and/or corpora albicans visible on thesurface.

-

The ovaries classified as late follicular phase, were those with the presence ofhemorrhagic bodies, corpora lutea and/or corpora albicans visible on their surface.

14

The late follicular phase ovaries were discarded, and they were not used for any
experiment in this study. Ovaries which showed the presence of illnesses as the ones with thepresence of cysts were also discarded for all the experiments (Fig 1.1).

Fig. 1.1: Classification of ovaries according to the morphology of their cortex.
A. Early follicular phase ovary. The cortex looks pink without signs of having had an estrous
cycle. B. Late follicular phase ovary, we can appreciate 3 corpora lutea (black arrowheads) on thesurface. C. Ovary with a cyst on the surface (red circle).

Figure 1.2: Ovaries with the presence of follicles with different diameters on their surface.
A: Ovary with big follicles (black circles) and medium follicles (black arrowheads) on the cortex.
B: Ovary with medium (black arrowheads) and small follicles (red arrowheads) present.

COCs were obtained from the follicles present in the surface of the ovaries. They wereclassified as small follicles (SF) if they had a diameter of < 3 mm, as medium follicles (MF) if
their diameters were between 3-6 mm and as large follicles if their diameters were > 6 mm (Fig
1.2). For our in vitro maturation experiments, COCs were aspirated only from SF and MF byusing a disposable 10 mL plastic syringe attached to an 18-gauge needle, separately. The pooled
follicular contents were then placed in a 50 mL centrifuge tube and washed 3 times with TLHEPES-PVA to remove any remaining follicular fluid and endogenous hormonal activity.
COCs were selected only if the oocytes had a uniform ooplasm and were surrounded completely
15

by at least 3 layers of clear and compact cumulus cells. Those oocytes incompletely surroundedby CCs or those COCs in which the cumulus cells were already expanded were not used for our
experiments (Fig 1.3).

Figure 1.3: Different types of COCs according to the quality of the surrounding cumulus
cells. A: Good quality COCs oocytes have a uniform dark cytoplasm and are surrounded bynumerous layers of a clear compact cumulus cell mass. B: Low quality COCs, oocytes present
areas without cumulus cells (white arrow head), and some oocytes have granulated cytoplasm(red arrow head). C: Oocytes surrounded by an expanded cumulus cell mass.
2.4 Routine protocol for in vitro maturation of COCs
Groups of approximately 50 COCs were cultured in a well of a 4-well dish (Nunclon
Delta Surface, Thermo Scientific) with 500 µL mPOM supplemented with 1 mM dibutyryl
cyclic AMP (dbcAMP), 10 IU/mL equine chorionic gonadotropin (eCG) and 10 IU/mL humanchorionic gonadotropin (hCG) for 20 h at 39°C and 5% CO2 in a wet atmosphere. After that, theoocytes were washed 3 times in pre-warmed mPOM and changed into a new dish with fresh
pre-warmed medium without the hormone supplements and dbcAMP to continue maturation foranother 24 h at 39°C and 5% CO2 in air.
2.5 Parthenogenetic activation of the oocytes after in vitro maturation.
After oocyte IVM, the oocytes from the experimental groups were denuded of cumulus
cells with a solution of 0.1% (w/v) hyaluronidase in TL-HEPES by pipetting with the help of a
glass narrow-bore pipette. Then the oocytes were examined under a confocal microscope andthey were only selected for parthenogenetic activation if they showed signs of nuclear
maturation by the detection of the first polar body (1st PB) extruded to the perivitelline space.
After selection, the mature oocytes were washed three times with 2 ml ofElectroporation solution and the equilibrated oocytes were placed in a drop of electroporation
medium between the two parallel wire electrodes of an electroporation chamber connected to a
BTX electro cell manipulator ECM 2001M (BTX, San Diego, CA, USA) and a single DC pulse16

of 120V for 30 µsec was given to the oocytes for their activation. Next, the oocytes were
washed 3 times in mM199 supplemented with 0.4% BSA and 5 µM cytochalasin B at a pH of
7.8 under paraffin oil and they were kept in a drop of the same medium for 4 h at 39°C and 5%
CO2 in air. Finally, the oocytes were washed 3 times thoroughly with PZM medium andtransferred to a drop of the same medium for embryo culture.
2.6 Early embryo culture
The oocytes were cultured for a total of 5 days in a drop of PZM under paraffin oil at
39°C and 5% of CO2 in a wet atmosphere. Cleavage and blastocyst formation rates wereobserved at day 2 and day 5 after the start of culture. Non-cleaved oocytes were removed fromthe culture on day 2; and on day 5, the blastocysts present in the culture were fixed for at least
24 h in a drop of 4% PFA at 4°C, washed in a 1x PBS solution supplemented with 0.1% (w/v)
PVA to avoid attachment to the dish, stained with Vectashield™ mounting medium with 4’,6Diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA) and finallyobserved under a fluorescence microscope for counting the number of cells in the blastocysts.

17

CHAPTER 3: Characterization of gilt’s ovaries with different patterns in the follicularmorphology and the meiotic competence of the oocytes.

3.1 Introduction
In many farm species, it is of general knowledge that the reproductive efficiency isaffected by extrinsic factors (those that depend on the environment and the management) andintrinsic factors (those depending on the animal) (Rodríguez FPC, 2005). Amongst the extrinsic
ones we can find environmental factors such as heat stress (Usui and Koketsu, 2005); nutritional
factors (Peltoniemi and Virolainen, 2006); and those derived from the manipulation and housing
conditions of the animals (Caton et al., 1986). In the factors that are considered intrinsic, we candistinguish factors such as the breed (Yen et al., 1987) and the age (Faillace et al, 1994) which
affect greatly to the animal fertility.
Prepubertal animals suppose a potential important stock of ovarian follicles and oocytes,
because their ovaries contain higher numbers of growing follicles than in pubertal ones
(Reynaud et al., 2002). Gilts generally reach puberty between 150 and 220 days of age (Soede etal., 2011) but they are usually slaughtered before its onset, so their ovaries, which contain highnumbers of developing follicles, can constitute a good source of oocytes for reproductive
biotechnologies such as in vitro maturation (IVM), fertilization (IVF) and embryo production.
In the ovaries, the population of follicles shows non-homogenic characteristics. There
are not only differences on their diameter sizes, but also in their hormonal, lipid and protein
contents (Fahimiya et al., 2010). Amongst this heterogenic population, oocytes destined to IVM
are mainly classified according to the size of the follicle of origin in small (SF; less than 3 mm
in diameter) and medium follicles (MF; 3-6 mm in diameter). It has been observed that oocytes
from MF are capable to achieve higher degrees of nuclear maturation and have improved
developmental competence than those derived from SF in the pig (Marchal et al., 2002; Kohata.
et al., 2013).
A way of optimizing reproduction protocols for reproductive biotechnologies in pigs
could be performed by obtaining elevated numbers of high quality oocytes derived from MF in
ovaries from prepubertal slaughtered animals. As the extrinsic and intrinsic factors of the abatedanimals are usually unknown to the investigators, the aim of the present study was to evaluate if
we could find any differences in the numbers of the follicular populations of gilt ovaries whenclassified according to the morphology of their surface. We also intended to assess if any

18differences in the maturation capacity of oocytes obtained from SF and MF of ovaries withdifferent morphologies could be detected.
3.2 Materials and methods
3.2.1 Ovary collection
Ovaries were randomly collected from prepubertal gilts of approximately 110 kg of live
body weight at a local abattoir, immediately separated from the reproductive tract and placed ina 0.9% (w/v) saline solution supplemented with 75 mg/L of potassium penicillin G and 50 mg/Lof streptomycin sulphate and transported to the laboratory within 1 hour according to ourlaboratory routine protocol (Funahashi, 2005). On arrival, the ovaries were washed twice andonly those ovaries which had no presence of corpora lutea on their surface were selected for use
in this study.
3.2.2 Experiment 1: Ovarian weight, surface patterns and follicular populationassessment
A total of 250 ovaries were trimmed from excess mesovaric tissue, blotted with a paper
towel and individually weighted on an electronic digital scale (H120, Sartorius, Germany).
Accordingtomorphology,
classified

thesurfaceovarieswere

Figure

3.1:

Three

three

types

categories

ofporcineinto

according to thoseovaries according to theirpreviously

described (Grasso et al., 1988);

surface morphology.

briefly; smooth surfaced ovaries

A. Smooth surfaced ovary(SSO) if the follicles did not(SSO), the ovarian cortex

protrudepresents a smooth appearance

throughtheovarianwithout follicles protruding

cortex thus presenting a smooththroughappearance, bubbled-like surface

it.

B.

Bubbledsurface ovary (BSO), theovaries (BSO) if the follicles

cortex is totally covered withprotruded strongly through all thefollicles strongly protruding

ovarian surface, or mixed surface

through it. C. Mixed surface

ovaries (MSO) if the ovaries

ovary (MSO), the cortex

showed a mixed pattern with somepresents some areas withsmooth appearance areas and some

protruding

and

non-

with protruding follicles (see Fig.

protruding

follicles.

Scale

3.1).

bars represent 1 cm in all the

After

that,

follicular

pictures.

19

populations on the ovarian cortex were classified according to their diameter size in: smallfollicles (SF; < 3 mm in diameter), medium follicles (MF; 3 to 6 mm in diameter) and large
follicles (LF; > 6 mm in diameter). To avoid counting a follicle more than once, following thecount of a follicle, its contents were aspirated with a disposable syringe. When no follicles werevisible on the ovarian surface, the ovaries were strongly pressed with a paper towel for
removing the remaining follicular fluid and weighed for a second time to know the remaining
ovarian weight. The difference between the ovarian weight with and without follicles represents
the follicular fluid weight, as shown in a previous study (Edey et al., 1972).
3.2.3 Experiment 2: Oocyte maturation in vitro
For this experiment, we selected 30 SSO and MSO each time. Due to the low numbersof BSO specimens in the sample, we were unable to include this type of ovary in our
experiment. Cumulus-oocyte complexes (COCs) were aspirated only from SF and MF
separately. After washing 3 times with TL-HEPES-PVA, only groups of 50 COCs with auniform ooplasm and completely surrounded by a compact cumulus cell mass of at least 3
layers were cultured in a well of a 4-well dish (Nunclon Delta Surface, Thermo Scientific) with
500 µL mPOM supplemented with 1 mM dibutyryl cyclic AMP (dbcAMP), 10 IU/mL equinechorionic gonadotropin (eCG) and 10 IU/mL human chorionic gonadotropin (hCG) for 20 h andthen without eCG, hCG and dbcAMP for another 24 h at 39°C and 5% CO2 in a wet atmosphere(Funahashi et al., 1997).
After the IVM, cumulus cells were completely removed by pipetting with 0.1% (w/v)
hyaluronidase in TL-HEPES-PVA, the oocytes were fixed in a solution of 4 % (w/v)
paraformaldehyde in PBS for 20 min at room temperature, washed with PBS and mounted on
glass slides with Vectashield™ mounting medium with 4’,6-Diamidino-2-phenylindole (DAPI;
Vector Laboratories Inc., Burlingame, CA, USA) to stain the nucleus. Meiotic status of theoocytes (see Fig. 3.2) was examined under a fluorescence microscope (Nikon Eclipse 80i,
Tokyo, Japan).
3.2.4 Statistical analysis
All statistical analyses were performed with the IBM SPSS Statistics 21.0 for Windows
software. Statistical analyses with 250 ovaries (Exp. 1) and 5 replicated trials (Exp. 2) wereconducted with a one-way and two-way ANOVA tests, respectively. If the P value was inferior
to 0.05 in ANOVA, a Tukey post-hoc test was performed. The data of the ovarian follicularnumbers were subjected to an analysis with a Kruskal-Wallis test. Independently of the test used
for the analysis, all the data in Exp. 1 are expressed as means ± SD and P < 0.05 was considered20

to be statistically significant in all the cases. Before the statistical analysis, all the percentage
data for the IVM experiment were subjected to arc-sine transformation if there were values> 90% or < 10%. All data were posteriorly transformed back into percentages for the tables andfigures and are expressed as means ± SEM.

Figure 3.2: Nuclear morphologies of oocytes after 44 h of IVM culture.
A: GV stage oocyte. B: PM-I stage oocyte. C: M-I stage oocyte. D: A-T stage oocyte. E:
M-II stage oocyte. F: Degenerated oocyte.
3.3 Results
3.3.1 Ovarian weight and ovarian surface patterns
A total of 250 ovaries were analyzed and classified into 3 groups according to thepatterns of the follicles on their surface. Totally 114 (45.6%) ovaries were SSO, 120 (48.0%)
were MSO and only 16 (6.4%) were BSO, being this last one the scarcest type of ovary. The
mean ovarian weight of the sample was 3.728 g, and after follicle aspiration, the remaining
ovarian weight was of only 2.072 g. When the ovary weight was assessed, MSO had asignificantly higher mean weight as compared with the SSO and BSO (Table 3.1; 3.944 g vs.
3.586 g and 3.126 g, respectively), whereas the weight after follicular aspiration was
significantly lower in BSO than SSO and MSO (Table 3.1; 1.452 g vs. 2.043 g and 2.182 g
respectively).

21

Table 3.1: Weight of prepubertal porcine ovaries according to their surface morphologicalcharacteristics
Weight (g; Mean ± SD) of
Ovary type

Number (%)
Ovary

Ovary after aspiration

Follicular fluidSSO

114 (45.6 %)3.586 ± 1.167a

2.043 ± 0.823a

1.543 ± 0.050a

MSO

120 (48.0 %)3.944 ± 0.965b

2.182 ± 0.69a

1.762 ± 0.412b

BSO

16 (6.4 %)3.126 ± 1.257a

1.452 ± 0.66b

1.674 ± 0.166ab

250 (100 %)3.728 ± 1.101

2.072 ± 0.77

1.657 ± 0.032

Total

SSO: smooth surfaced ovary, MSO: mixed surfaced ovary, BSO: bubbled surfaced ovary.
a,b

Different superscript letters indicate significant differences amongst the values present in thesame column (P < 0.05).

Table 3.2: Number of follicles present in prepubertal porcine ovaries with different
morphologies.
No. ofTotal no. of

ovariesfollicles/ovary

examined(mean ± SD)

SSO

114

MSO

Ovary
type

Number (mean) of follicles (% ± SD)
SF

MF

LF

128.4a ± 58.0

116.6b (87.2 ± 12.5)

11.0b (12.0 ± 12.6)

0.8 (0.8 ± 2.1)

120

153.6b ± 58.7

138.8a (88.2 ± 9.7)

14.2a (11.1 ± 9.6)

0.6 (0.8 ± 2.7)

BSO

16

113.6a ± 72.6

94.4b (73.5 ± 19.8)

18.4a (24.7 ± 17.6)

0.8 (1.8 ± 4.5)

Total

250

139.6 ± 60.7

125.8 (86.8 ± 12.3)

13.0 (12.4 ± 12.1)

0.7 (0.8 ± 2.6)

SF, small follicles with less than 3 mm in diameter; MF, middle follicles with 3-6 mm indiameter; LF, large follicles with a diameter larger than 6 mm; SSO, smooth surfaced ovary;
MSO, mixed surfaced ovary; BSO, bubbled surfaced ovary.
a,b

Different superscript letters indicate significant differences amongst the values present in thesame column (P < 0.05).

22

3.3.2 Follicular population
As shown in Table 3.2, the mean number of follicles per ovary was 139.6. When weanalyzed these data according to the ovarian morphology type, the total number of follicles was
significantly higher in MSO than in SSO and BSO (153.6 vs. 128.4 and 113.6 respectively).
MSO also had a significantly higher number of SF per ovary than the SSO and BSO (138.81 vs116.58 and 94.44, respectively). The MSO and BSO types had a significantly higher number of
MF per ovary, although the percentage of MF was lower in MSO than that of BSO (11.1% vs.
24.7%), because MSO was also the type which possessed the most follicles.
3.3.3 Oocyte maturation in vitro
The two-way ANOVA analysis (Figure 3.2) showed that there were significant
differences in the germinal-vesicle break down (GVBD) capacity between SSO and MSO
oocytes, especially in those derived from SF. Independently of the ovary type, oocytes from MF
always had a significantly higher rate of GVBD than those from SF. The meiotic stages ofoocytes after IVM are shown in table 3. The analysis revealed that there were significant
differences in the percentages of oocytes at the GV and M-II stages between the ovarian types
and the follicular size was found to have a significant effect on all the oocyte nuclear stages.
When the type of ovary was taken into account, oocytes presented significant differences onlyin the incidence of GV and M-II nuclear configurations. The incidence of oocytes from MF and
SF at the GV stage was significantly higher in SSO than in MSO (16.4% and 4.1% vs. 7.7% and2.1% respectively). The analysis revealed also a significant effect of the ovarian type on the MII oocyte achievement, being slightly higher in MSO than SSO in both, SF and MF (55.6% and80.2% vs. 50.0% and 75.6% respectively). When the nuclear stages according to the follicular
type were analyzed, oocytes at the GV stage always showed a significantly higher number whenthose were derived from SF (SSO: 7.7% vs. MSO: 16.4%) as compared with the MF derived
ones (SSO: 4.1% vs. MSO: 2.1%). Significantly higher percentages of oocytes at the M-I stage
and degenerated configurations were observed in oocytes from SF than those from MF, whichin contrast, had significantly higher rates of oocytes at the M-II stage (SSO: 50.0% MSO:
55.6% vs. SSO: 75.6% MSO: 80.2%, respectively).

23

Figure 3.3: Percentages of GVBD oocytes derived from SF and MF of different
morphologies of ovaries (SSO vs. MSO).

a,b

Different superscripts indicate significant differences (P<0.05) between oocytes from SF

and MF. A,B Different superscripts indicate significant differences (P<0.05) between oocytes
collected from SSO and MSO.

24

Table 3.3: Nuclear stage of oocytes from SF and MF of smooth (SSO) and mixed surface ovaries (MSO) after 44 h of IVM culture.
Total No.

Ovaryof oocytes

type

MSO

examined

GV

ProM-I

FFollicular type
M-I

A-I/T-I

M-II

Deg

SF

248

19 (7.7 ± 0.7)Ba

4 (1.6 ± 0.8)55 (22.2 ± 3.2)a4 (1.6 ± 0.8)138 (55.6 ± 3.2)Ab

28 (11.3 ± 0.5)a

MF

248

5 (2.1 ± 1.1)Bb

1 (0.4 ± 0.4)

35 (14.1 ± 1.6)b7 (2.8 ± 0.8)

199 (80.2 ± 1.8)Aa

1 (0.4 ± 0.4)b

SF

250

44 (16.4 ± 0.8)Aa

4 (1.6 ± 1.0)

43 (16.8 ± 1.5)a9 (2.8 ± 0.8)117 (50.0 ± 1.5)Bb

31 (12.0 ± 3.2)a

MF

246

10 (4.1 ± 1.5)Ab

2 (0.8 ± 0.5)

31 (12.6 ± 2.4)b13 (5.3 ± 0.5)

186 (75.6 ± 2.5)Ba

4 (1.6 ± 0.8)b

SSO

AB, ab

Number (% ± SEM) of oocytes at the stage of:Different superscripts indicate significant differences among the values in the same column (P < 0.05).

Result of 2-way ANOVA.
GV

ProM-I

M-I

A-I/T-I

M-II

Deg

MSO vs SSO< 0.01

-

0.179

-

< 0.05

0.368

SF vs MF

< 0.001-< 0.05-< 0.001< 0.001

Interaction

0.361

-

0.549

-

0.835

0.339

25

3.4 Discussion
Ovaries from prepubertal gilts were classified into 3 types according to their cortex
morphology characteristics. Our result of macroscopic classification was consistent with datacollected from 105 days-old gilts in a previous report (Dufour et al., 1985). In the current
experiment, porcine ovaries were collected from gilts slaughtered when they reached a corporalweight of approximately 110 kg, which is a common time for pork production in Japan. For
reaching this weight, these gilts could be estimated around 165 days of age (Kusec et al., 2008),
which approximately coincides with the time of puberty (Oxender et al., 1979). Here wedemonstrated that SSO and MSO are majority of the morphology of ovaries in these gilts, andthat the ratios were similar in both.

In the present study, we also observed the mean weight offollicular fluid (1.657 + 0.032 g) accounted for about 44% of the weight of the ovary (3.728 +
1.101 g), whereas the weight was significantly lower than a previous report (Clark et al., 1982),
probably because we aspirated follicular fluid from follicles only located in the cortex. In thisexperiment, we could characterize the morphology of porcine ovaries as COC-resource forembryo production in vitro.
In the present study, the mean number of follicles was 139.6 + 60.7, and the value wassimilar to a previous study (Dufour et al., 1993) with Meishan gilts near puberty. This resultshows that there are many follicles on the surface of ovaries. In addition, classification of theovarian morphology allowed us to find that total number of follicles in MSO was significantly
larger than those in SSO and BSO. The number of SF in MSOs was significantly larger thanthose of SSO and BSO, whereas the number of MF in MSO was larger than that of SSO, MSO
and BSO and no differences could be detected in the numbers of LF. These results demonstrate
the superiority of MSO in the number of SF and MF to SSO. Although Grasso et al. (1988)
showed that the number of SF was lower in BSO than SSO and MSO, our result showed asimilar trend, but not significant. Therefore, the ovarian cortex morphology seems tosignificantly affect the numbers of SF and MF in their cortex, and hence it may be a goodcharacteristic to take into account for improving the efficiency of COC collection for IVM
protocols.
Oocyte meiotic competence is known to be acquired gradually during the follicular
growth, until it reaches the early antral stages (Armstrong et al., 2001). In the pig, follicles with
0.5 mm in diameter have a fully differentiated antrum, but the oocytes have not yet reached their
final size. These small oocytes have very limited ability to initiate nuclear IVM (Motlik et al.,1984) because growing oocytes are unable of undergoing GVBD thus being meiotically
incompetent (Szybek et al., 1972; Combelles et al., 2002). In the present study, we found thatthe meiotic competence of oocytes was significantly higher when the COCs were collected from26

MSO than SSO, whereas the competence was significantly higher when the COCs were derived
from MF than SF in both MSO and SSO. This result shows not only that MSO has a larger
number of MF and SF as compared with SSO but also that the oocytes in MSO have a higher
meiotic competence. According to a previous study (Dufour et al., 1985), the number offollicles with 2.05-3.56 mm in diameter was larger in MSO than SSO, whereas the number of
those with 1.13-2.00 mm in diameter was smaller. Furthermore, a recent study by Töpfer et al.
(2016) has demonstrated that those follicles with a diameter superior than 4 mm have better
nuclear and cytoplasmic maturation capacities than those oocytes with a smaller diameter. In thepresent study, the follicles were considered to be MF if the diameter of follicles was 3-6 mm
and SF if that was less than 3 mm. It is possible that the difference in the meiotic competence
observed in this study, would be because the mean diameters in each criterion were smaller in
SSO than MSO.
3.5 Conclusion
In conclusion, the total number of follicles (MF plus SF) was larger in MSO than SSO
and BSO and the meiotic competence of oocytes was also higher when the COCs were collectedfrom MSO than SSO. We propose that MSO can constitute a good source of oocytes for the
improvement of the efficiency of the actual in vitro reproductive technologies. Further studies
on the developmental competence of the oocytes and on oocyte viability and apoptosis shouldbe performed to have a better insight on this matter.

27

CHAPTER 4: Effect of removing cumulus cells from porcine cumulus-oocyte complexes
derived from small and medium follicles during in vitro maturation on the apoptotic
status and meiotic progression of the oocytes.

4.1 Introduction
Usually pigs for pork production have been slaughtered before the onset of puberty indeveloped countries. The ovaries are a common resource of follicles and oocytes for their use inreproductive biotechnologies (Day and Funahashi, 1996; Funahashi and Day, 1997) and manystudies have shown that oocytes in the cumulus-oocyte complexes (COCs) obtained from
prepubertal gilts have significantly lower meiotic and cytoplasmic competence than those fromcycling sows. For in vitro embryo production, routinely COCs from medium follicles (MF) with
3-6 mm in diameter of prepubertal gilt’s ovaries are used, because oocytes from MF have arelatively higher rate of meiotic competence as compared with those from small follicles (SF;with less than 2 mm in diameter), though the number of MF in the ovary is significantly lower
than that of SF (Bolamba et al., 1991; Knox, 2005; Marchal et al., 2002; Kohata et al., 2013).
The oocytes recovered from antral follicles are surrounded by multiple layers of
cumulus cells (CCs) which are in deep communication with the oocyte via cytoplasmic
projections that go through the zona pellucida and contact with the oolema via gap junctions
forming a structure known as COC (Albertini et al., 2007). It has been well known that COCs
not only secrete paracrine factors but they also interact through the gap junctions (passing
molecules only smaller than 1 kDa, (Kidder and Mhawi, 2002) in a bi-directional manner,which is essential for oocyte nutrition, growth and regulation of meiotic progression (Hunter,
2000; Sanchez-Lazo et al., 2014; Gilchrist et al., 2004). Also, CCs contribute partially tomaintain the oocyte into meiotic arrest by the production and transfer of cAMP via gapjunctions, which accumulates into the ooplasm (Mao et al., 2013). The communication throughthe gap-junctions between the oocyte and the CCs decreases progressively during IVM (Ozawa
et al., 2008; Bagg et al., 2009). This disconnection between the cells seems to be responsible formeiotic resumption in fully grown oocytes, due to a reduction in the intracellular cAMP level(Thomas et al., 2004; Sela-Abramovich et al., 2006), which will activate CDC2/CDK1 and
MAP kinases (Shimada et al., 2002; 2006), and consequently permit the oocytes to achievematuration to the metaphase-II stage in vitro (Edry et al., 2006; Norris et al., 2009) and in vivo
(Sela-Abramovich et al., 2006). In the pig, this communication between the oocyte and the CCs
during the first 4 hours of IVM appears to be most important when the COCs were collectedfrom MF (Sasseville et al., 2009).

28

However, there are no studies about if removing CCs or breaking off thecommunication between oocyte and CCs affects the nuclear maturation of oocytes derived fromSF. Therefore, the aims of the present study were to analyze the viability and meiotic ability of
porcine oocytes from SF and MF when they were denuded before, during or after IVM.
4.2 Materials and methods
4.2.1 Preparation of COCs
Ovaries without any evidences of corpora lutea were collected at a local abattoir andplaced in a 0.9% (w/v) NaCl solution supplemented with 75 mg/L of potassium penicillin G and
50 mg/L of streptomycin-sulphate. They were transported to the laboratory within 2 h at 25°C.
Cumulus-oocyte complexes were aspirated from SF (0.5-2 mm in diameter) and MF (3-6 mm indiameter) located on the ovarian surface using a disposable 10-mL syringe with an 18-gauge
needle and collected into a 50-mL centrifuge tube. The pooled follicular contents were washed 3times with modified TL-HEPES-PVA medium at room temperature to remove any traces offollicular fluid and thus any endogenous gonadotropic activity and growth factor activity.
Thereafter, only COCs from SF and MF were selected if they had a uniform ooplasm and acompact cumulus cell mass of at least 3 layers of CCs.
4.2.2 In vitro maturation and decumulation of the oocytes.
Cumulus-oocyte complexes derived from SF and MF were randomly distributed into
groups (30 to 35 COCs per group). One group from each, SF and MF, was used to analyze theapoptotic status and nuclear stage of the oocytes before IVM. The oocytes were denuded with
TL-HEPES-PVA containing a 0.1% (w/v) hyaluronidase by using a glass narrow-bore pipette,
and washed 3 times with TL-HEPES-PVA and then used for fluorescence assays.
The remaining groups of COCs from SF and MF were cultured separately in 300 µL of
mPOM supplemented with 1mM dibutyryl cyclic AMP (dbcAMP), 10 IU/mL eCG and 10
IU/mL hCG for 20 h at 39°C in an atmosphere of 5% CO2 in air. The COCs were then washed 3times in equilibrated mPOM without the dbcAMP and gonadotropins, transferred to 300 µL of
the same medium and continued culture for an additional 24 h at 39°C in an atmosphere of 5%
CO2 in air. Before (0 h) and after the start 20 h of IVM, the oocytes from SF and MF COCs
were denuded as described above and continued the IVM culture after washing three times with
mPOM. At the end of IVM culture (totally 44 h from the start), the remaining oocytes were alsodenuded, washed 3 times and then used for the fluorescence assays.

29

4.2.3 Fluorescence assays for the apoptotic status, viability and meiotic progressionof oocytes
Denuded oocytes were processed for an apoptotic/viability assay before (0 h) and afterIVM for totally 44 h of culture, according to the manufacturer’s instructions by using the Alexa
Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Molecular Probes, Eugene, USA). Briefly,
after washing 3 times with cold PBS, the denuded oocytes were incubated in a 100 µL drop of
1x Annexin-binding buffer, in which 5 µL of Annexin V conjugate and 2 µL of 100 µg/mL
propidium iodide were added, at room temperature for 15 min. After washing twice with 1x
Annexin-binding buffer, the oocyte specimens were mounted on a glass slide with
Vectashield™ mounting medium with 4’,6-Diamidino-2-phenylindole (DAPI; Vector
Laboratories Inc., Burlingame, CA, USA). The slide was immediately observed under afluorescence microscope with excitation (450-490 nm) and emission (520 nm) filters (Nikon
eclipse 80i, Tokyo, Japan).
For the apoptosis/viability assessment, oocytes were considered as normal if they
showed no fluorescence, in a state of early apoptosis if they showed a partial green fluorescencein the oolema, in a state of late apoptosis if they showed a strong green fluorescence whichsurrounded the oolema forming a complete fluorescent circle and dead if they had a positive redsignal of propidium iodide in the chromatin or in the cytoplasm, respectively (see Fig. 4.1).

Figure 4.1: Apoptotic status of the oocytes stained with Annexin V/PI.
A: Normal live oocyte, there is no fluorescence in the cell. B: Early apoptotic oocyte withthe membrane partially stained green (white arrowhead). C: Late apoptotic oocyte, all themembrane is strongly stained in green. D: Dead oocyte. The cytoplasm is stained in red.

For the assessment of meiotic progression, the oocytes were classified by themorphology of DAPI fluorescence. For the oocytes analyzed before IVM culture, themorphology of germinal vesicle was determined according to the modified criteria of previous
reports (Motlik and Fulka, 1976; Funahashi et al., 1997). Briefly, GV0 stage is that characterized
by the presence of a nucleolus surrounded by filamentous chromatin which occupies all the
nuclear space, the GVI stage is that with the presence of a nucleolus surrounded by chromatin in30

the form of a ring or a horseshoe, the GVII stage is the one with the presence of a nucleolus as aring or horseshoe and a few well stained clumps located near it, the GVIII stage is characterized

Figure 4.2: Nuclear morphologies of GV stage oocytes.
A: GV0 stage oocyte. B: GVI stage oocyte. C: GVII stage oocyte. D: GVIII-IV stage oocyte. E:
PM-I stage oocyte. F: M-I stage oocyte.
by the presence of a nucleolus surrounded by an irregular network of filamentous bivalents andclumps in the whole area of the GV, and the GVIV stage is characterized by the presence offilamentous bivalents and no nucleolus or by the presence of diakinesis chromatin (See figure4.2). After IVM culture, mature oocytes at the metaphase-II stage were classified into twogroups according to the spindle morphology. Oocytes were considered as having a normal
spindle if the revolver configuration could be seen, and as having an abnormal configuration if
the spindle DNA appeared highly compacted or fragmented (see Fig. 4.3).

Figure 4.3: Nuclear morphologies of MII stage oocytes.
A: Normal M-II stage oocyte, the revolver configuration can be seen clearly. B-C:
31
Abnormal configurations of the MII spindle. Chromosomes
are disorganized (B) or highly
compacted (C).

4.2.4 Statistical Analysis
Data obtained from 5 replicated trials were processed by one-way or two-way ANOVA
using StatView 4.0 statistical software (Abacus Concepts, Inc., Berkeley, CA). All the data wereexpressed as mean ± SEM. Findings were considered to be significantly different at P<0.05, andwhen there was a significant effect, values were compared with a Tukey’s multiple comparisonstest.
4.3 Results
4.3.1 Apoptotic status, viability and nuclear stage of oocytes before IVM
As shown in Table 4.1, there were no significant differences in the apoptotic status andviability between oocytes derived from SF and MF just after collection from ovaries. However,when the nuclear stage of the oocytes was examined (Table 4.2), the incidence of oocytes at theGV0 stage was significantly higher in those from SF (24.8 ± 2.4%) than MF (3.3 ± 1.4%),
whereas the incidence of GVI stage oocytes was the opposite, being significantly higher in MF(57.8 ± 5.9%) than SF (22.7 ± 4.4%). No significant differences were observed in any of theother nuclear stages between the MF and SF oocytes.
Table 4.1: Apoptotic status of oocytes just after collection of the COCs from SF and MF.

Origin of
COCs

No. ofoocytes
examined

No. (% ± SEM) of oocytes at the status of:No. (% ± SEM)

Normal

Early apoptotic

Late apoptotic of dead oocytes

SF

198

78 (39.4 ± 3.8)

99 (49.8 ± 5.5)

10 (5.2 ± 3.0)

11 (5.6 ± 2.6)

MF

191

81 (42.6 ± 6.3)

103 (53.6 ± 7.4)

5 (2.8 ± 1.8)

2 (1.0 ± 0.6)

Experimental data were obtained from trials replicated 5 times.
4.3.2 Viability, nuclear stage of the oocytes after 44 h of IVM and MII spindle
quality.
When the apoptotic status of the oocytes was assessed, the status was not different
between oocytes from SF and MF, but it was affected by the time when the oocytes were
denuded (Table 4.3). The percentage of intact oocytes was significantly higher when CCs were
removed after 20 h (39.7 ± 2.8% in SF oocytes and 45.4 ± 3.1% in MF oocytes) or 44 h (39.3 ±
3.9% in SF oocytes and 36.8 ± 3.3% in MF oocytes) after the start of IVM, as compared withwhen the CCs were removed 0 h after the start of IVM (17.7 ± 3.8% in SF oocytes and 22.2 ±
2.3% in MF oocytes). Denuding the oocytes 20 h after the start of IVM did not affect the32

incidence of the oocytes at early apoptotic stage (57.4 ± 1.9% in SF oocytes and 52.8 ± 2.6% in
MF oocytes) as compared with controls (when oocytes were denuded 44 h after the start of IVM;
57.2 ± 4.0% in SF oocytes and 62.6 ± 3.4% in MF oocytes), whereas denuding the oocytes atthe start of IVM significantly increased the incidence of early apoptotic oocytes in the same
origin of COCs (74.3 ± 5.4% in SF oocytes and 69.4 ± 3.4% in MF oocytes). The percentage oflate apoptotic oocytes was also significantly higher when oocytes were denuded before IVM(7.4 ± 2.5% in SF oocytes and 6.7 ± 3.3% in MF oocytes), as compared with when the oocytes
were denuded 20 h (2.9 ± 1.3% in SF oocytes and 1.8 ± 0.8% in MF oocytes) and 44 h after thestart of IVM (3.5 ± 1.7% in SF oocytes and 0.6 ± 0.6% in MF oocytes).
When the nuclear stages of oocytes were examined 44 h after the start of IVM, theincidence of oocytes at the metaphase-II stage was significantly affected by both, the origin of
the COCs and by the time when the oocytes were isolated from the COCs (Table 4.4). In both,oocytes derived from SF and MF, denuding the oocytes 20 h after the start of IVM significantly
increased the percentage of mature oocytes 44 h after the start of IVM (65.4 ± 2.3% in SF
oocytes and 83.1 ± 1.4% in MF oocytes), whereas denuding 0 h after the start of IVM
significantly decreased their incidence (27.9 ± 3.6% in SF oocytes and 32.3 ± 2.5% in MF
oocytes), as compared with control oocytes which were denuded at the end of IVM for 44 h
(41.0 ± 2.1% in SF oocytes and 68.5 ± 1.9% in MF oocytes). As shown in Table 4.5, however,the percentage of oocytes with a morphologically normal spindle at the metaphase-II stage wassignificantly higher in those derived from MF (91.5 ± 1.8%) than SF (70.7 ± 5.4%) when theCCs were not removed until the end of IVM. In both SF and MF oocytes, the percentage wassignificantly lower when oocytes were denuded 20 h after the start of IVM (66.8 ± 3.9% and73.0 ± 3.0%, respectively). Regardless of the type of follicle the oocytes were derived from, SF
and MF, the incidence of oocytes with a normal morphological spindle at the metaphase-II stage
was lowest when the oocytes were denuded just before the start of IVM culture.

33

Table 4.2: Nuclear stage of live oocytes just after collection of the COCs from SF and MF.

Origin of
COCs

No. (% ± SEM) of live oocytes at each nuclear stage before IVM

No. ofoocytes
examined

GV0

GVI

GVII

GVIII-IV

ProM-I

Deg.

SF

187

46 (24.8 ± 2.4)a43 (22.7 ± 4.4)a24 (12.9 ± 2.3)

72 (38.5 ± 4.1)-

2 (1.1 ± 1.1)

MF

189

6 (3.3 ± 1.4)b

109 (57.8 ± 5.9)b

18 (9.6 ± 1.5)

55 (28.7 ± 6.0)

1 (0.5 ± 0.5)

-

a, b

Different superscript letters indicate significant differences between the values in the same column (P < 0.05).

Experimental data were obtained from trials replicated 5 times.

34

Table 4.3: Effect of removal of cumulus cells from COCs derived from SF and MF before or during IVM on the apoptotic status of oocytes at the end of IVM

Origin of
COCs

SF

MF

Time (h) whenCCs wereremovedNo. (% ± SEM) of oocytes in the status of:

No. of oocytes
examined

No. (% ± SEM) of

Normal

Early apoptotic

Late apoptotic

dead oocytes0174

31 (17.7 ± 3.8)a129 (74.3±5.4)a13 (7.4 ± 2.5)a1 (0.6 ± 0.6)

20

171

68 (39.7 ± 2.8)b98 (57.4 ± 1.9)bc

5 (2.9 ± 1.3)b

-

44

173

68 (39.3 ± 3.9)b99 (57.2 ± 4.0)bc

6 (3.5 ± 1.7)b

-

0

170

38 (22.2 ± 2.3)a118 (69.4 ± 3.4)ab11 (6.7 ± 3.3)a1 (0.7 ± 0.7)

20

165

75 (45.4 ± 3.1)b87 (52.8 ± 2.6)c

3 (1.8 ± 0.8)b

-

44

168

62 (36.8 ± 3.3)b105 (62.6 ± 3.4)abc

1 (0.6 ± 0.6)b-

Origin of COCs

0.34

0.64

0.33

-

Time when CCs were removed<0.001<0.001<0.05-

Significant effect(P-values)

a,b,c

Different superscript letters indicate significant differences between the values in the same column (p < 0.05).

Experimental data were from trials replicated 5 times.

35

Table 4.4: Effect of removal of CCs from COCs derived from SF and MF before and during IVM of COCs on meiotic competence of the oocytes 44 h after
the start of IVM.
Time (h) when

No. ofNo. (% ± SEM) of oocytes at the meiotic stage of:

Origin of
CCs wereoocytesremovedexamined

GV

ProM-I

M-I

A-I/T-I

M-II

Deg.

0

173

82 (47.3 ± 3.9)a8 (4.7 ± 1.2)

9 (5.2 ± 1.4)ab8 (4.5 ± 2.8)

48 (27.9 ± 3.6)a17 (9.9 ± 2.4)a

20

171

38 (22.2 ± 2.8)b4 (2.34 ± 1.5)

4 (2.3 ± 0.6)a6 (3.5 ± 1.1)

112 (65.4 ± 2.3)b7 (4.1 ± 1.5)a

44

173

61 (35.2 ± 1.3)c

5 (2.9 ± 1.3)

21 (12.2 ± 2.4)b7 (4.1 ± 1.5)

71 (41.0 ± 2.1)c

8 (4.6 ± 1.1)a0

169

75 (44.7 ± 3.1)a10 (5.9 ± 1.5)

12 (6.8 ± 2.6)ab

7 (4.2 ± 1.2)

55 (32.3 ± 2.5)a8 (4.7 ± 1.1)a

20

165

17 (10.3 ± 1.1)d

1 (0.6 ± 0.6)

3 (1.8 ± 1.2)a5 (3.0 ± 0.1)

137 (83.1 ± 1.4)d

2 (1.2 ± 0.7)b

44

167

18 (10.7 ± 1.1)d6 (3.5 ± 1.1)

19 (11.4 ± 2.3)b6 (3.7 ± 1.5)

115 (68.5 ± 1.9)b

2 (1.1 ± 0.7)b

Origin of COCs

< 0.001

0.970

0.938

0.756

< 0.001< 0.05Time when CCs were removed< 0.001

0.184< 0.001

0.795< 0.001< 0.005

COCs

SF

MF

Result of 2-way ANOVA (P-values)a, b, c, dDifferent superscript letters indicate significant differences between the values in the same column (p < 0.05).
Experimental data were obtained from trials replicated 5 times.

36

Table 4.5: Effect of removal of CCs during IVM on the normal morphology of metaphasespindle in the mature oocytes.

Origin of
COCs

Time (h) when

No. of mature

No. (% + SEM) of oocytes with aoocytes examinedmorphologically normal MII spindle

048

32 (56.6 ± 2.4)a

20

112

75 (66.8 ± 3.9)b44

71

50 (70.7 ± 5.4)c055

31 (56.0 ± 3.8)a

20

137

100 (73.0 ± 3.0)c

44

115

105 (91.5 ± 1.8)d

CCs wereremovedSF

MF

Result of 2-way ANOVA

a, b, c, dOrigin of COCs

< 0.05Time when CCs were removed< 0.001

Different superscript letters indicate significant differences between the values in the same

column (p < 0.05).
Experimental data were obtained from trials replicated 5 times.

37

4.4 Discussion
In the present study we found that, at the beginning of IVM, the apoptotic status of
COCs was not different between those derived from SF and MF, whereas the percentage ofoocytes at the GV0 stage was significantly higher in those derived from SF than MF, as
described previously (Funahashi et al., 1997). It has been reported that the meiotic anddevelopmental oocyte competences are significantly lower when the COCs were collected fromSF, as compared with MF (Marchal et al., 2002; Kohata et al., 2013). Our results suggest thatthe lower competence of the oocytes derived from SF could be due to the differences in themorphological GV stages, but not in the apoptotic status at the start of IVM.
We also found in this study that the time when CCs were removed from COCs during
IVM significantly affected the apoptotic status of the oocytes at the end of IVM, but the originof the COCs did not affect it. In the present study, although we did not observe any reductionsin the incidence of live oocytes by denuding during IVM, which is different from resultspresented recently (Lin et al., 2015), removing CCs before the start of IVM significantly
decreased the incidence of intact oocytes and increased the incidence of early and late apoptotic
ones. Cumulus cells seem to act as a barrier, which protects the oocytes from in vitro induced
lipotoxic effects, such as reactive oxygen species formation, caspase 3 activation, andmitochondria deterioration (Lolicato et al., 2015). Denuding cumulus cells has been known toseverely damage oocytes, leading to their apoptosis or degeneration in rabbits (Lu et al., 2010).
On the other hand, in the present study, any differences in the state of apoptosis of oocytes were
not observed when the CCs were removed 20 h and 44 h after the start of the culture. The
current results, therefore, demonstrate that denuding oocytes at the first half of IVM (20 h after
the start of IVM), at least, can maintain the oocytes intact in relation to apoptosis.
In the present study, the percentage of oocytes matured to the metaphase-II stage wassignificantly affected not only by the origins of the COCs but also by the time when CCs were
removed from the COCs. Although the maturation rate was lower in oocytes derived from SF
than MF, we found that the denudation of oocytes at 20 h after the start of IVM significantly
increased the maturation rate of the oocytes from both, SF and MF. Bidirectional
communication between the oocytes and the surrounding somatic cells is required for the
maintenance of the meiotic arrest in mammalian oocytes (Wigglesworth et al., 2013). It has
been known that the C-type natriuretic peptide – natriuretic peptide receptor 2 signaling
pathway is involved in meiotic arrest and cumulus oophorous formation in mice (Zhang et al.,2010; Kiyosu et al., 2012) and porcine oocytes (Santiquet et al., 2014). Oocyte-derived
paracrine factor (Hiradate et al., 2014) and estradiol (Zhang et al., 2011) seem to participate in
the regulation and expression of natriuretic peptide receptor 2 in cumulus cells. The present38results demonstrate that disruption of the communication between CCs and oocyte 20 h after thestart of IVM is effective even in COCs derived from SF to induce the resumption of meiosis
arrest and maturation to the metaphase-II stage.
However, the percentage of oocytes having a normal metaphase-II spindle was affected
not only by the COCs precedence but also the time when the surrounding CCs were removed
from the COCs. The percentage was significantly higher in oocytes from MF than SF and also
in oocytes denuded after the IVM culture. A recent paper showed that the percentages of mature
oocytes having a normal spindle, as well as the blastocyst formation rate, were significantly
lower in oocytes denuded 22 h than 44 h after the start of IVM (Lin et al., 2015). The
metaphase-II spindle assembly has been reported to be improved by elevated intra-ooplasmic
ATP levels following either COC preincubation or ATP microinjection into the oocytes prior to
IVM (Xu et al., 2014).
4.5 Conclusion
The current results obtained in this study suggest that disruption of the communication
between CCs and the oocyte 20 h after the start of IVM may have a detrimental effect on theintra-ooplasmic content of ATP. Our results also suggest that secretion(s) from CCs and/or
communication through the gap-junctions between CCs and the oocyte are required to obtain amorphologically normal spindle after IVM. Further studies will be required to elucidate if thefactor carried into the oocyte from CCs and/or an increased ooplasmic ATP content somehow
can improve the spindle quality of the oocytes derived from SF.

39

CHAPTER 5: Effect of removing cumulus cells from porcine COCs derived from small
and medium follicles during IVM in the presence or absence of GDF9 or VEGF on the
meiotic and developmental competences of the oocytes.

5.1 Introduction
Porcine ovaries used for experimentation purposes are usually procured fromcommercial slaughterhouses resulting in the obtainment of a sample with highly heterogenic
characteristics that will affect oocyte quality (Ding and Foxcroft, 1992). Many strategies havebeen developed to improve oocyte maturation, fertilization and embryo development during thepast years (Zhang et al, 2012). Some of these strategies for improving oocyte maturation consist
in using factors present in the follicular environment for somehow mimic the in-vivo conditionsof the follicle. Some factors secreted from oocytes and CCs have been commonly used as
supplements in the in vitro maturation mediums.
Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor-β(TGF-β) superfamily. In follicles, it is mainly expressed in the oocyte, and the expression isnecessary for female fertility (Juengel and McNatty, 2005; Hanrahan et al., 2004). When thisfactor is added to granulosa or cumulus cells in vitro, it can mimic oocyte control on theiractions by the activation of the SMAD cascade pathways that regulate proliferation, cumulus
expansion and apoptosis (Gilchrist et al., 2004a; 2004b). The IVM treatment of COCs with
GDF9, improves their capacity to develop to the blastocyst stage and their embryo quality(Hussein et al., 2006; Yeo et al., 2008).
Vascular endothelial growth factor (VEGF) was originally identified as a tumorsecreted protein, known for its ability to increase vascular permeability which results in theextravasation of proteins and other molecules (Dvorak et al., 1995). VEGF regulates
angiogenesis in the developing ovarian follicles (Tempel et al., 2000; Barboni et al., 2000) and
can also interact with the receptors present on the surface of the follicular somatic cells acting asa mitogenic factor in developing follicles (Bruno et al., 2009; Greenaway et al., 2004). The
expression and production of this protein are both upregulated as the follicle develops (Shimizu
et al., 2003; Greenaway et al., 2004). Supplementation of VEGF protein in the oocyte IVM
medium has been reported to have a stimulating effect by improving the maturation anddevelopmental competence capacity in the pig (Kere et al., 2014).

40

Oocytes are usually matured in vitro in the form of cumulus-oocyte complexes (COCs).
These structures consist in an oocyte surrounded by several layers of cumulus cells that
communicate with the central oocyte via gap junctions (Albertini et al., 2001) and can transfer
small molecules and ions to the oocyte cytoplasm through them (Huang and Wells, 2010).
Amongst these molecules, the continuous flow of high concentrations of cyclic adenosine 3’-5’monophosphate (cAMP) into the oocyte cytoplasm is believed to maintain the oocyte meioticarrest and that a transient increase of the cytoplasmic cAMP levels is associated with theinitiation of meiotic resumption in the pig (Chaube SK, 2000). Several studies have shown thatthe interruption of the communication via gap junctions by using inhibitors (Mao et al., 2013) orjust by the removal of the cumulus cells during IVM culture in the presence of gonadotropins
can induce meiotic resumption of the oocytes (Sasseville et al., 2009).
The objective of this study was to evaluate the effect of CCs removal on different times
during IVM of prepubertal gilt oocytes from SF and MF and to evaluate their quality by theevaluation of their developmental competence capacity and blastocyst formation ability afterparthenogenetic activation. In addition, it was evaluated if the exogenous supplementation of
GDF9 or VEGF into the culture medium was able to further improve the nuclear maturation of
the denuded oocytes.
5.2 Materials and methods
5.2.1 Ovary transport and COCs collection.
Oocytes were obtained from prepubertal gilts at a local slaughterhouse and brought into
the laboratory into a 0.9% (w/v) saline solution supplemented with 75 mg/L of potassium
penicillin G and 50 mg/L of streptomycin-sulphate within 2 h at 25°C (Funahashi, 2005).
Only ovaries without any visible corpora lutea were used for this experiment. Cumulusoocyte complexes (COCs) were aspirated from small follicles (SF; < 3 mm in diameter) andmedium follicles (MF; 3-6 mm in diameter) located on the ovarian surface using a disposable
18-gauge needle and collected into a 50 mL centrifuge tube separately. The pooled follicular
contents were washed 3 times with modified TL-HEPES-PVA medium at room temperature,and only COCs with a uniform ooplasm and with at least 3 layers of clear and compact cumulus
cells were selected for each type.
5.2.2 Experiment 1: Effect of the removal of cumulus cells during in vitro
maturation on oocyte nuclear maturation.

41

SF and MF COCs were randomly distributed into groups (30-35 COCs per group) andcultured separately in a 300µL drop of mPOM supplemented with 1 mM dibutyryl cyclic AMP(dbcAMP), 10 IU/mL eCG and 10 IU/mL hCG under paraffin oil for 20 h at 39 °C in an
atmosphere of 5% CO2 in air. Then, the COCs were washed 3 times in equilibrated mPOM
without the dbcAMP and the gonadotropins and transferred into a new dish with a 300 µL dropof the same medium and continued culture for another 24 h in the same conditions. At 0, 10, 20,
32 and 44 h of IVM the CCs were removed with a 0.1% (w/v) hyaluronidase solution and a
glass narrow-bore pipette and, after washing 3 times in equilibrated mPOM solution, thedenuded oocytes continued to culture. At the end of IVM (after 44 h), denuded oocytes were
mounted on glass slides, fixed in 0.25% (v/v) acetic acid in ethanol solution for at least 72 h,
stained with 1% (w/v) orcein in acetic acid solution and the nuclear configurations were
evaluated under a phase-contrast microscope according to the criteria of Motlik and Fulka(1976; see Fig. 5.1).

Figure 5.1: Oocyte nuclear stages after 44 h of IVM when stained with orcein.
A: GV stage oocyte, the DNA is disorganized inside the nuclear envelope; B: PM-I stage oocyte,
the DNA looks highly compacted and there is no nuclear envelop; C: M-I stage oocyte, the DNA isorganized into a configuration revolver-like; D: A/T stage oocyte, the chromosomes are being
separated by the microtubules in opposite ways; E: MII stage oocyte, there is a meiotic spindle
smaller than the one in MI stage accompanied with a polar body.

5.2.3 Experiment 2: Effect of the supplementation of GDF9 on meiotic progressionof oocytes
Groups of approximately 30 COCs from SF and MF were cultured in a 300 µl drop ofmPOM (as described in experiment 1) with or without 100 ng/mL GDF9 (Biovision, Milpitas,
CA, USA) at 39 °C and 5% CO2 in air for 20 h in the presence of dbcAMP, eCG and hCG.
After the first period of IVM, CCs surrounding oocytes were removed as described previously
and the denuded oocytes continued culture for IVM with or without GDF9 in the absence of
dbcAMP and gonadotropins in the same medium for another 24 h. At the end of IVM culture,
all oocytes were denuded and the oocytes were fixed at room temperature for 20 min in asolution of 4% (w/v) PFA in PBS solution and meiotic progression of the oocytes was examined
after DAPI staining under a fluorescence microscope (Nikon Eclipse 80i, Tokyo, Japan).

42

5.2.4 Experiment 3: Effect of the supplementation of VEGF on oocyte in vitro
maturation
As in the previous experiments, groups of approximately 30 COCs from SF and MF
were cultured in a 300 µl drop of mPOM in the presence of dbcAMP, eCG and hCG for 20 h at
39 °C in a wet atmosphere of 5% CO2 in air. During this first half of the culture, the medium
was supplemented or not with a final concentration of 200 ng/mL of VEGF (V4512-5UG,
Sigma-Aldrich), concentration that has been proven to promote maturation of porcine oocytes
from SF in our laboratory (Bui et al., 2015). After the first half of the IVM protocol, oocytes
were denuded and they continued the culture in a new pre-warmed drop of mPOM without any
supplements. As in experiment 2, after the IVM period had finished; all the oocytes were
denuded, fixed and stained with DAPI for the evaluation of the nuclear status under afluorescence microscope.
5.2.5 Experiment 4: Developmental competence of oocytes denuded during IVM
Groups of 60 (30 COCs/dish) COCs were cultured for IVM in a 300 µL drop of mPOM
and the oocytes were denuded at 0, 20 and 44 h of IVM as explained in experiment 1. At theend of the IVM culture, after CCs were removed from the remaining COCs, the cumulus-freeoocytes were observed under a stereomicroscope for the presence of the first polar body (PB)
extruded in the perivitelline space and only those with PB were selected for the parthenogenetic
activation.
The selected oocytes were washed 3 times with electroporation solution (pH 7.2) andthen transferred in a drop of the same medium situated between the electrodes of an activationchamber. A single electrical pulse (DC: 1.2 kV/cm, 30 µs) was given to the oocytes for theiractivation by using a BTX Electro-Cell Manipulator 2001M (BTX, San Diego, CA, USA). Next,
the stimulated oocytes were washed three times in mM199 solution supplemented with 0.4%(w/v) BSA and 5 µmol/L cytochalasin B at 39 °C in an atmosphere of 5% CO2 in air for 4 h.
After that, the oocytes were washed three times in PZM and cultured in a drop of the same
medium covered with paraffin oil in the same ambient conditions for 5 days. Cleavage andblastocyst formation rates were observed at day 2 and day 5 after the start of culture,
respectively. Non-cleaved oocytes were removed from the culture on day 2; and on day 5, theblastocysts were fixed for at least 24 h in a drop of 4% PFA at 4°C, washed in a 1x PBS
solution supplemented with 0.1% (w/v) PVA to avoid attachment to the dish and stained with
Vectashield™ mounting medium with 4’,6-Diamidino-2-phenylindole (DAPI; Vector
Laboratories Inc., Burlingame, CA, USA) and observed under a fluorescence microscope for43

counting the blastocyst cell number. Embryos were considered to be pseudo-blastocysts they
had less than 20 cells.
5.2.6 Statistical analysis
Data obtained from at least 5 replicated trials were processed by one-way or two-way
ANOVA using IBM SPSS Statistics 21.0 software for Windows. Findings were considered tobe significantly different at P<0.05, and when there was a significant effect, values werecompared with a Tukey’s multiple comparisons test. Before performing the statistical analysis,
all percentage data in the experiments were subjected to arc-sine transformation if there werevalues > 90% or < 10%. All data were posteriorly transformed back into percentages for thetables and figures and are expressed as means ± SEM, except for the blastocyst cell count thatwas expressed as means ± SD.
5.3 Results
5.3.1 Experiment 1
The percentage of oocytes which achieved the germinal vesicle break down (GVBD),
was significantly lower when the oocytes from SF and MF were denuded just before the start ofIVM culture (51.6% and 73.6%, respectively) as compared with the other experimental groups(see Fig. 5.2).

Figure 5.2: Oocyte GVBD rates after 44 h of IVM culture.
The experiment was replicated 5 times.
A,B: Different superscript letters indicate significant differences among the size of the follicles(P<0.05).
a, b: Different letters indicate significant differences among the different times of decumulation(P<0.05).

44

Figure 5.3: Oocyte nuclear maturation rates after 44 h of IVM culture.
The experiment was replicated 5 times.
A,B: Different superscript letters indicate significant differences among the size of the follicles(P<0.05).
a, b: Different letters indicate significant differences among the different times of decumulation(P<0.05).

The percentage of oocytes reaching to the metaphase-II (MII) stage was always
significantly higher in those oocytes obtained from MF than in the SF ones (see Fig. 5.3 andsupplementary table 1 in the Annex) and in both, COCs from SF and MF, the maturation ratesof oocytes denuded at 10 h, 20 h and 32 h of IVM culture were similar with those which theCCs were removed after IVM. Even if there were no significant differences in the maturation
rates of these groups, as the group we denuded after 20 h had the highest IVM rates, we used
this timing for removing CCs in the following experiments.
5.3.2 Experiments 2 and 3
Regardless of the supplementation with GDF9 or VEGF, the maturation rates werealways significantly higher in the denuded oocytes from MF than SF for both experiments (seeFig. 5.4 and Fig. 5.5, and supplementary tables 2 and 3 in the Annex).
In both cases, removing CCs 20 h after the start of IVM improved meiotic resumption
rates in SF oocytes (Fig. 5.6; 59.2% vs 41.6% in GDF9 and Fig. 5.7; 61.3% vs 40.2% in VEGF),
whereas no differences were detected in the MF groups (see Fig. 5.8 and Fig. 5.9). Despite of
the origin of COCs (SF or MF), maturation rates of oocytes denuded 20 h after the start of IVM
were not affected by supplementation with GDF9 or VEGF during the first or/and second halfof IVM.

45

Figure 5.4: Oocyte nuclear maturation rates of SF and MF denuded at 20 h of IVM culture andsupplemented or not with GDF9
The experiment was replicated 5 times.
A,B: Different superscript letters indicate significant differences among the size of the follicles(P<0.05).

Figure 5.5: Oocyte nuclear maturation rates of SF and MF denuded at 20 h of IVM culture andsupplemented or not with VEGF
The experiment was replicated 5 times.
A,B: Different superscript letters indicate significant differences among the size of the follicles(P<0.05).

46

Figure 5.6: Nuclear maturation rates of SF oocytes denuded at 20 h of IVM culture andsupplemented or not with GDF9
The experiment was replicated 5 times.
a, b: Different superscript letters indicate significant differences among the time of CCs removal(P<0.05).

Figure 5.7: Nuclear maturation rates of SF oocytes denuded at 20 h of IVM culture andsupplemented or not with VEGF
The experiment was replicated 5 times.
a, b: Different superscript letters indicate significant differences among the time of CCs removal(P<0.05).

47

Figure 5.8: Nuclear maturation rates of SF oocytes denuded at 20 h of IVM culture andsupplemented or not with GDF9
The experiment was replicated 5 times.

Figure 5.9: Nuclear maturation rates of MF oocytes denuded at 20 h of IVM culture andsupplemented or not with VEGF
The experiment was replicated 5 times.

48

5.3.3 Experiment 4
Independently of the time of CCs removal, oocytes from MF had always significantly
higher rates of maturation (Table 5.1), cleavage and blastocyst rates (Table 5.2) than those fromSF, but there were no differences in the blastocyst cell number.
When oocytes were before the start of IVM, the oocytes caused significantly lower rates
of maturation in both groups, SF (37.6% vs 51.2%) and MF oocytes (50.8% vs 76%) and also
had negative effects on the blastocyst rates in both groups (2.7% vs 25.8% in SF; 27.3% vs
48.5% in MF). When CCs were removed 20 h after the start of IVM, it significantly promoted
nuclear maturation in both, SF (64.1% vs 51.2%) and MF oocytes (82.5% vs 76.0%) and there
were no significant differences on cleavage or blastocyst rates in both groups when comparedwith the controls.

Table 5.1: Maturation of SF and MF oocytes by polar body selection when CCs are removed at
0, 20 and 44 h of IVM.

Follicle
type

CCs

Num.

removaloocytestime (h)examined0180

92 (50.82 ± 4.59) Ac

20

183

151 (82.49 ± 4.18) Aa

44

188

143 (76.00 ± 3.35) Ab

0

186

70 (37.56 ± 1.81) Bc

20

181

116 (64.08 ± 2.60) Ba

44

182

93 (51.15 ± 1.92) Bb

MF

SF

Maturation rate (n (% ± SEM))

Result of 2-way ANOVA
Follicle type

< 0.001

CCs removal time

< 0.001

Interaction

0.225

A, B

: Different superscript letters indicate significant differences amongst oocytes from different

follicle size.
a, b, c: Different superscript letters indicate significant differences among the time when were

denuded.

49

Table 5.2: Embryo developmental capacity of SF and MF oocytes denuded at 0, 20 and 44 h of
IVM after parthenogenetic activation.
Embryo culture (Day 2)
Follicle
type

Embryo culture (day 5)

CCs

MatureremovaloocytesCleavage rate

Blastocyst rate

time (h)(n)(n (% ± SEM)(n (% ± SEM)

092

82 (88.78 ± 2.24) A

27 (27.34 ± 5.77) Ab

32.19 ± 8.42

151

139 (91.79 ± 2.35)

A

69 (44.88 ± 5.62)

Aa

32.75 ± 3.90

130 (90.92 ± 1.23)

A

69 (48.52 ± 4.53)

Aa

34.02 ± 2.63

57 (81.66 ± 4.28)

B

100 (85.92 ± 4.84)

B

74 (79.07 ± 7.00)

B

MF

20
44
0

SF

20
44

143
70
116
93

2 (2.68 ± 1.65)

Blastocyst cellnumber(X̅ ± SD)

Bb

25.00 ± 1.41

27 (23.34 ± 4.94)

Ba

31.88 ± 1.70

24 (25.79 ± 7.99)

Ba

32.01 ± 2.54

Result of 2-way ANOVA
Follicle type

< 0.05< 0.001

0.077

CCs removal time

0.410

< 0.001

0.169

Interaction

0.881

0.275

0.392

A, B

: Different superscript letters indicate significant differences amongst oocytes from different

follicles.
a, b: Different superscript letters indicate significant differences among the time when oocytes

were denuded.

50

5.4 Discussion
Mammalian oocytes that have reached their final size are arrested at the prophase of thefirst meiotic division indicated by the presence of a germinal vesicle (GV). Arresting oocytes at
the GV stage is maintained by inherent factors correlated with low activity levels of cell cycle
regulatory proteins (Yang et al., 2012) and also by the transference of cAMP from the CCs to
the oocyte cytoplasm via gap junctions (Dekel, 1988). In some species, meiosis can be resumed
spontaneously when oocytes are removed from the follicle and introduced in an adequate
culture medium (De Loos et al., 1994; Edwards, 1965), so it has been suggested that cAMP
derived from the follicle is the responsible for maintaining oocyte meiotic arrest (Tsafriri andChanning, 1975) by activating the cAMP dependent protein kinase A (PKA) (Conti et al., 2002).
Meiosis also can be resumed by a reduction in the oocyte gap-junctional coupling of its
surrounding somatic cells (Kalous et al., 1993; Larse WJ, 1986). In the pig, COCs maintain thisgap-junctional communication during the first 24-32 of culture, and even if the CCs expansion
starts after 16 h of IVM (Motlik and Fulka, 1986), the oocytes do not become disconnectedfrom them until they reach a stage between the MI and MII a result of the CCs expansion
process (Suzuki et al., 2000).
Our results indicate that oocytes from MF had higher maturation, cleavage andblastocyst rates than those from SF. Many studies also corroborate existing differences on theseparameters (Kohata et al., 2013; Bagg et al., 2007; Marchal et al., 2002). Oocyte developmental
competence is acquired during the process of folliculogenesis and, with the increasing size ofthe ovarian follicle, the oocytes will obtain a more adequate cytoplasmic maturation thanks totheir companion somatic cells (Gilchrist et al., 2008). Oocytes from SF must have a poorer
cytoplasmic maturation than those from MF, and, as follicles of less than 2 mm in diameter
depend on the ovarian endogenous activity for their growth (Driancourt et al., 1995).
Culturing denuded oocytes impaired meiotic resumption and had negative effects on thedevelopmental capacity of oocytes, especially in those from SF, whereas that when they were
cultured as a COC during the first part of IVM, their nuclear maturation rates improved
significantly. It has been widely reported that CCs provide the oocytes with nutrients and othermolecules such as ions or transcripts necessary for achieving a good cytoplasmic maturation(Eppig, 1991; Park et al., 2013), and an early detachment of the CCs might cause the lack forsome of these factors provided during the first hours of culture.
The pronounced improvement in the maturation rates of porcine oocytes from SF
denuded at 20 h might be related to a transient raise in the levels of cAMP during the first partof IVM by the addition of the homologous molecule dbcAMP. That, together with the CCs

51

removal and the culture of the denuded oocytes in a medium without the modulator, might havecaused a rapid decrease on the ooplasmic levels of cAMP. This diminution on the cAMP
concentration levels, together with the activity of the PD3A into further reducing the levels ofcAMP (Sun et al., 2015) and the phosphorylation of MAPK (Liang et al., 2007) caused theinitiation of meiotic resumption.
The exogenous addition of GDF9 or VEGF had no effect in promoting oocyte
maturation when supplemented during the first part of IVM culture when the CCs were
removed at 20 h, but high concentrations of VEGF have been proven to affect negatively tooocyte maturation and developmental capacity in the human due to COC hypoxia (MalamitsiPuchner et al., 2001). Many studies in cumulus-enclosed oocytes thorough IVM culture haveshown a positive effect in maturation rates and developmental competence when growth factors
were added into the maturation medium (Xia et al., 1994; Hussein et al., 2006; Singal andPrasad, 2009; Toori et al., 2014). The lack of response in our experiment indicates that the CCs
probably exert their beneficial effect during the second part of culture, and the positive effects
are not seen due to CCs removal. As oocyte maturation is correlated with the follicular stages invivo (Gilchrist et al., 2001), developing pre-culture conditions with hormones or growth factors
might be a good way for testing if the maturation rates when CCs are removed after the first halfof IVM can be improved further by letting the factors to exert their beneficial action before CCs
removal.
5.5 Conclusions
In conclusion, oocytes from MF have higher meiotic resumption and developmental
competence than those from SF probably because they have reached a higher phase of
maturation in vivo. Denuding oocytes 20 h after the start of IVM can be a good method forincreasing the efficiency of IVM protocols since it can improve oocyte meiotic resumption rates
without any reduction in the developmental competence of oocytes from SF. The addition of
100 ng/mL of GDF9 or 200 ng/mL of VEGF, were unable to further increase the percentages ofnuclear maturation in both types of oocytes. We suggest that culturing the COCs with growth
factors and/or hormones prior to IVM might help the oocytes by promoting maturation anddevelopmental competence when denuded after the first part of culture.

52

CHAPTER 6: GENERAL DISCUSSION
The ovarian samples used for experimenting purposes are usually obtained fromcommercial slaughterhouses, in there, young animals and those with the presence of anestrous
will be sacrificed, so the ovaries from slaughtered animals will have different characteristics
which will affect to the quality of the gametes. Furthermore, even if the available protocols for
IVM have improved greatly for the past years, the available protocols in the porcine species arestill deficient, especially when researchers try to mature oocytes from small follicles.
The first aim of this study was to find an ovarian factor that could be used for reducing
the variability of the samples and would help to improve the efficiency of oocyte IVM protocols.
Firstly, the samples were classified into 3 categories according to the characteristics of theirsurface, weighed the ovaries and counted the numbers of different-sized follicles present on
their surface. Cumulus-oocyte complexes from SF and MF of ovaries with a smooth or a mixed
surface were cultured in vitro to assess their meiotic competence capacity. The number of small
and medium follicles was higher in mixed surface ovaries than in the other ovarian types andthis type of ovaries also presented significantly higher maturation rates in oocytes from small
and medium follicles. This could be because the ovaries would be in the recruitment follicularphase, characterized by high proliferation rates of the ovarian follicles, which would also affectto the oocyte quality by having less numbers of atretic follicles, or by having higher oocyte
quality.
As researchers generally have a limited amount of samples available for experiments, amethod to improve maturation rates and the quality of oocytes from SF and MF was tried todevelop, independently of the type of ovarian morphology. It is known that the decrease of thelevels of cAMP in the ooplasm by cutting the communication between the oocyte and its
surrounding cumulus cells can lead to GVBD and to the meiotic resumption of oocytes. To test
removing cumulus cells can be used as a meiotic promotion factor, in the second experiment,
the apoptotic and nuclear status of oocytes from SF MF were examined after removing CCs
during IVM. Before IVM, the incidence of oocytes at the GV0 and GVI stage was different
according to the follicle of origin, being higher in oocytes from SF and MF respectively;
whereas no differences could be detected in the apoptotic status. Researchers have proposed that
GVI oocytes have started to receive maturation stimuli and are in a more advanced phase of
maturation than those arrested in the GV0. This fact could be one of the reasons why oocytes
from MF reach higher rates of maturation than the ones derived from SF. After culture, the time
when oocytes were denuded affected the oocyte apoptosis rates. The percentage of intact
oocytes was significantly higher when cumulus cells were removed at 20 and 44 h and theincidence of early and late apoptotic oocytes was significantly higher when we cultured
53

denuded oocytes, because CCs act as a barrier that protects the oocytes against reactive oxygen
species and in a final instance apoptosis. Maturation rates were affected by the size of thefollicle of origin and by the time the oocytes were denuded. By denuding the oocytes after 20 hof culture we improved their maturation rates, due to the activation of the meiotic resumption
process, but the percentages of oocytes with normal spindles were lower than the controls,meaning that there are probably differences in the energy contents when CCs are removed early.
The above concept led me to perform a third experiment for further investigating on thequality of the oocytes denuded at 20 h of IVM. Since VEGF and GDF9 have been proved to
improve maturation of oocytes when cultured in vitro, it was examined if oocyte maturation
rates could be further improved by the supplementation of these factors. Firstly it was assessed
if denuding oocytes 20 h after the start of IVM was optimal and, after confirmation,
developmental competence of the oocytes was examined. Afterwards, GDF9 or VEGF were
supplemented to elucidate if maturation rates could be further improved. SF oocytes had lower
maturation, cleavage and blastocyst rates than those from MF, and this coincides with current
previous findings in which oocytes from SF possess lower quality because they have not
become meiotically competent, and they are probably lacking in cytoplasmic maturation.
Oocytes denuded after 20 h of IVM, had higher rates of maturation by maintaining thedevelopmental competence in the same conditions as the controls. The exogenous addition of
growth factors in the medium did not significantly improve oocyte maturation of theexperimental groups, probably, because of the lack of cumulus cells during the second part of
the IVM, they could not exert their beneficial effect on the oocytes, so it is suggested that, tofurther improve maturation rates, the addition of hormones and growth factors should be
performed as a culture prior to the IVM protocol.
The work presented in this thesis shows that ovaries and oocytes from slaughteredanimals have heterogenic characteristics and the efficiency of maturation protocols can be
improved by selecting ovaries with a mixed surface that will provide elevated numbers ofoocytes with high nuclear maturation capacity. Or, when the samples are limited, denuding
oocytes after 20 h of IVM maintains their viability and promotes meiotic progression in both,SF and MF oocytes. Furthermore, even if the rates of abnormal spindles are higher in the treatedoocytes, parthenogenetic activation does not reduce their developmental competence.
General conclusions
In conclusions, removing the cumulus cells 20 h after the start of IVM allow theobtainment of higher numbers of good quality oocytes because of the improvement in54

maturation rates while maintaining the developmental competence of oocytes, especially inthose derived from SF.

55

CHAPTER 7: SUMMARY
Currently, female gametes are easily collected from slaughtered or live ovaries and usedfor various reproductive biotechnologies to improve the efficiency and value of mammalianembryos. Pigs have been utilized as a model in biomedical research, whereas the gametes andembryos are quite unique from other species. In the developed countries, although pigs areusually slaughtered for pork production at 5-6 months old when those gilts have neared or still
not reached to the puberty, ovarian morphologies have not been considered for the resource of
cumulus-oocyte complexes (COCs). Furthermore, the effect of interrupting communication
between oocyte and the surrounding cumulus cells has not been examined in porcine COCs
derived from small follicles (SF; < 3 mm in diameter).
In the present studies, firstly, gilt’s ovaries with different follicular morphologies werecharacterized and the meiotic competence of oocytes was compared among the criteria.
Porcine ovaries were able to be classified into three categories; ovaries with smooth (SSO),
bubbled (BSO) or mixed (uneven) surface (MSO). A significantly higher number of SF wasobserved in MSO than SSO and BSO and the number of middle follicles (MF; 3-6 mm indiameter) per ovary was significantly higher in BSO and MSO than SSO. Since MSO showeda significantly higher percentage of metaphase-II stage oocytes than SSO in SF and MF, it
was concluded that MSO could suppose a good source of oocytes for improving the efficiencyof the actual reproductive technologies.
In the second experiments, the apoptotic status and meiotic progression of oocytes from SF
and MF were examined when the oocytes were denuded during culture for in-vitro maturation(IVM). Apoptotic status of the oocytes was only affected by the time when the oocytes were
denuded (0, 20 and 44 h after the start of IVM). Although the percentage of mature oocytes
was higher in MF, maturation rates were significantly higher when oocytes were denuded at
20 h, as compared with 0 and 44 h after the start of IVM. Therefore, it was concluded thatremoving CCs at 20 h after the start of IVM seemed to promote meiotic progression of theoocytes to the metaphase-II stage even when the COCs were collected from SF.
In the last experiments, the effect of the timing of removing cumulus cells during IVM ondevelopmental competences of oocytes was assessed, and it was also examined if the addition
of GDF9 or VEGF could further improve IVM rates of oocytes denuded during maturation
culture. Regardless of the presence of GDF9 or VEGF, denuding oocytes 20 h after the start ofIVM did not affect their developmental competence to the blastocyst stage, concluding thatdenuding the oocytes 20 h after the start of IVM could be a good method to promote themeiotic resumption of oocytes, without any reduction in the developmental competence.

56

Results from the current experiments demonstrate that using COCs from MSO or byremoving the cumulus cells of COCs from SF and MF 20 h after the start of IVM can promote
the meiotic resumption of oocytes, without any reduction in the developmental competence.
This information may contribute to improve the efficiency to prepare female resources for invitro embryo production.

57

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APPENDICES

73

Supplementary table 1. Oocyte nuclear maturation and GVBD rates when CCs are removed during IVM in SF and MF oocytes.

Follicle Time of CCs
type

removal (h)N. ofOocyte nuclear stage after 44 h of IVM ((n (% ± SEM))

oocytes
examined

GV

PMI
Aa

MII

9 (5.9 ± 1.3)

42 (26.9 ± 3.2)

Deg
Bb

80 (51.6 ± 6.3) B b

59 (38.8 ± 6.3)

10

154

27 (17.6 ± 1.8) A b 18 (11.6 ± 2.5)

15 (9.8 ± 3.7) A b

6 (4.0 ± 1.6)

75 (48.5 ± 8.2) Ba

13 (8.6 ± 2.4) A 114 (73.8 ± 3.3) B a

20

143

26 (18.7 ± 4.2) A b

4 (2.8 ± 1.3) A b

5 (3.2 ± 1.4)

90 (62.0 ± 4.0) Ba

11 (7.7 ± 1.4) A 107 (73.6 ± 4.1) B a

32

149

15 (10.2 ± 1.9) A b 10 (6.6 ± 2.2) 18 (12.1 ± 2.2) A bc 9 (6.1 ± 2.4)

89 (59.5 ± 3.2) Ba

8 (5.6 ± 1.9) A

44

146

20 (13.8 ± 3.0) A b

7 (4.8 ± 2.1)

40 (27.4 ± 3.9) A ac 3 (2.2 ± 0.9)

65 (44.3 ± 4.1) Ba

11 (7.6 ± 2.6) A 115 (79.7 ± 5.4) B a0155

33 (21.2 ± 4.5) B a

3 (2.0 ± 1.3)

17 (10.9 ± 4.3) B bc 2 (1.3 ± 0.8) 92 (59.4 ± 5.2) A b

8 (5.2 ± 2.3) B

114 (73.6 ± 6.2) A b

10

146

14 (9.9 ± 5.2) B b

5 (3.4 ± 1.1)

3 (2.1 ± 0.9) B b

4 (2.6 ± 1.8) 112 (76.4 ± 4.4) A a

8 (5.6 ± 1.9) B

124 (84.5 ± 6.6) A a

20

144

9 (6.2 ± 2.8) B b

6 (4.2 ± 0.7)

7 (4.8 ± 2.5) B b

2 (1.4 ± 0.8) 114 (79.2 ± 5.0) A a

6 (4.3 ± 2.6) B

129 (89.5 ± 3.0) A a

32

150

8 (5.2 ± 2.4) B b

3 (2.0 ± 1.3)

13 (8.8 ± 2.6) B bc 2 (1.3 ± 1.3) 118 (78.6 ± 4.6) A a

6 (4.2 ± 2.1) B

136 (90.6 ± 1.9) A a

44

147

13 (8.9 ± 3.0) B b

4 (2.8 ± 1.4)

20 (13.6 ± 1.5) B ab

105 (71.3 ± 4.3) A a

5 (3.3 ± 1.1) B

130 (87.8 ± 3.1) A a

Follicle type

< 0.001-< 0.05-< 0.001< 0.05< 0.001

Time of CCs removal< 0.001-< 0.001-< 0.001

0.791< 0.001

Interaction

0.928

-

0.433

-

0.678

0.980

0.703

MF

0

16 (10.3 ± 3.5)

GVBD
A

154

8 (5.5 ± 1.7)

19 (12.3 ± 3.8)

A/T
A bc

0

SF

9 (5.9 ± 0.7)MI

126 (84.3 ± 3.3) B a

Result of 2-way ANOVA

A, B,Ca, b, c: Different superscript letters indicate significant differences amongst oocytes from different follicles.

: Different superscript letters indicate significant differences amongst oocytes decumulated on different times.

74Supplementary table 2. Effect of CCs removal time and GDF9 supplementation on the nuclear maturation of SF oocytes.
Follicle
type

SFTime ofCCs
removal

GDF9
1st part
IVM
-

GDF9
2nd part
IVM
-

-

+

20 h

44 h

Result of 2-way ANOVA
Time of CCs removal
GDF9 supplementation
Interactiona, b, c

Oocytes
Nuclear stage of oocytes after 44 h of IVM (n (% ± SEM))
examined
GV
PMI
MI
A/T
MII
(n)
151
28 (18.38 ± 3.34) 4 (0.65 ± 0.65) 13 (10.48 ± 2.11) 3 (2.07 ± 1.41) 101 (59.24 ± 5.83)a151

30 (19.7 ± 2.87)

3 (2 ± 1.33)

Deg
14 (9.18 ± 4)

12 (7.88 ± 1.64) 3 (2.02 ± 0.83)

88 (57.76 ± 5.05)a16 (10.64 ± 3.56)

a12 (7.74 ± 2,41)

+

-

153

23 (14.94 ± 3.2)

3 (1.96 ± 1.3) 16 (10.67 ± 1.91) 8 (5.21 ± 1.23)

90 (59.47 ± 4.84)

+

+

153

21 (13.67 ± 2.07) 3 (1.92 ± 1.29) 16 (10.38 ± 3.09) 5 (3.31 ± 1.06)

92 (60.16 ± 4.84)a15 (9.87 ± 4.76)

37 (24.56 ± 5.02) 1 (0.67 ± 0.67) 26 (17.52 ± 4.48) 4 (2.69 ± 1.25)b

20 (12.98 ± 3.4)

-

-

151

0.267
0.542
-

-

0.194
0.880
-

: Different superscript letters indicate significant differences amongst oocytes decumulated on different times.

75-

63 (41.58 ± 4.97)< 0.05
0.989
-

0.387
0.918
-

Supplementary table 3. Effect of CCs removal time and GDF9 supplementation on the nuclear maturation of MF oocytes

Follicle
type

Time of

GDF9

GDF9

CCs

1st part

2nd part

removal

IVM

IVM

-

Nuclear stage of oocytes after 44 h of IVM

Oocytes
examined

GV

PMI

MI

-

149

12 (8.03 ± 3.04)

1 (0.65 ± 0.65)

13 (8,78 ± 2.07)

7 (4.81 ± 2.10) 109 (73.06 ± 1.79)

6 (4.00 ± 1.64)

-

+

150

18 (12.16 ± 3.56)

015 (9.88 ± 3.35)

2 (1.29 ± 0.79) 109 (72.75 ± 2.92)

6 (3.92 ± 1.20)

+

-

149

19 (12.94 ± 4.01) 1 (0.74 ± 0.74)

13 (8.53 ± 5.97)

+

+

155

14 (9.01 ± 3.88)

1 (0.63 ± 0.63)

12 (7.72 ± 2.99)

-

-

151

14 (9.32 ± 1.66)

A/T

MII

Deg

20 h
MF

44 h

3 (2 ± 2)

108 (72.41 ± 2.95)

4 (2.74 ± 1.95)

4 (2.61 ± 1.24) 122 (78.77 ± 3.29)

2 (1.27 ± 0.78)

1 (0.69 ± 0.69) 18 (12.06 ± 4.21) 6 (3.98 ± 1.64) 107 (70.64 ± 6.26)

4 (2.65 ± 1.94)

Result of 2-way ANOVA
Time of CCs removal

0.551

-

0.652

-

0.653

-

GDF9 supplementation

0.582

-

0.845

-

0.592

-

-

-

-

-

-

-

Interaction76Supplementary table 4. Effect of follicle size and GDF9 addition on oocyte nuclear maturation after IVM of SF and MF oocytes when denuded at 20 h ofculture.

Follicle
type

GDF9

GDF9

Nuclear stage of oocytes after 44 h of IVM (n (% ± SEM))

1st part

2nd part

IVM

IVM

-

-

151

28 (18.38 ± 3.34)A 4 (0.65 ± 0.65) 13 (10.48 ± 2.11) 3 (2.07 ± 1.41) 101 (59.24 ± 5.83)B

14 (9.18 ± 4)

+

+

153

21 (13.67 ± 2.07)A 3 (1.92 ± 1.29) 16 (10.38 ± 3.09) 5 (3.31 ± 1.06)

92 (60.16 ± 4.84)B

15 (9.87 ± 4.76)

-

-

149

12 (8.03 ± 3.04)B 1 (0.65 ± 0.65) 13 (8,78 ± 2.07) 7 (4.81 ± 2.10) 109 (73.06 ± 1.79)A

6 (4.00 ± 1.64)

+

+

155

14 (9.01 ± 3.88)B 1 (0.63 ± 0.63) 12 (7.72 ± 2.99) 4 (2.61 ± 1.24) 122 (78.77 ± 3.29)A

2 (1.27 ± 0.78)

Oocytes
examined (n)

SF

MF

GV

PMI

MI

A/T

MII

Deg

Result of 2-way ANOVA
Effect of follicle size

< 0.05

-

0.518

-

< 0.01

0.065

Effect of GDF9 addition

0.686

-

0.518

-

0.445

0.489

Interaction

0.551

-

0.874

-

0.579

0.473

A, B

: Different superscript letters indicate significant differences amongst oocytes from different size of follicles.

77Supplementary table 5. Nuclear maturation status of oocytes from SF when denuded after 20 h of IVM and supplemented with VEGF protein and denuded at
the end of IVM.
CCs
removaltime (h)20
20
44

Nuclear stage after IVM

VEGF

Oocyteaddition

number

GV

PMI

MI

-

170

30 (17.82 ±3.71)

5 (3.01 ± 1.02)

15 (8.48 ± 5.38) b

29 (17.80 ± 2.66) 7 (4.37 ± 1.24)b

3 (1.86 ± 0.76) 100 (61.16 ± 3.89)a11 (6.72 ± 1.47)

123 (75.48 ± 3.48)ab

6 (3.59 ± 0.58)

131 (78.43 ± 1.45)

+
-

163
167

A/T

13 (8.08 ± 3.02)

30 (17.99 ± 0.98) 8 (4.84 ± 1.99) 49 (29.34 ± 4.49)

MII

Deg

5 (2.86 ± 1.28) 104 (61.30 ± 2.56) a 11 (6.54 ± 1.22)

7 (4.13 ± 1.92)

67 (40.17 ± 1.89)

GVBD
129 (75.64 ± 3.69)

Results of 2-way ANOVA
Cumulus cell removal

0.849

-

< 0.005-< 0.001-

0.529

VEGF addition

0.946

-

0.640

-

0.975

-

0.971

-

-

-

-

-

-

-

Interaction78Supplementary table 6. Nuclear maturation status of oocytes from MF when denuded after 20 h of IVM and supplemented with VEGF protein and denuded
at the end of IVM
CCs

Nuclear stage after IVM

VEGF

Oocyteaddition

number

GV

PMI

MI

20

-

162

8 (4.85 ± 1.53)

3 (1.78 ± 0.73)

11 (6.83 ± 0.73)

20

+

161

17 (10.73 ± 4.14) 2 (1.27 ± 0.78)

8 (4.96 ± 2.13)

44

-

162

11 (6.85 ± 1.23)

removaltime (h)

A/T

MII

Deg

GVBD

7 (4.27 ± 0.66)

147 (90.87 ± 1.81)

132 (81.79 ± 4.98)

2 (1.25 ± 0.77)

142 (88.02 ± 4.34)

2 (1.20 ± 0.73) 20 (12.46 ± 2.88) 2 (1.27 ± 0.78) 124 (76.38 ± 5.29)

3 (1.84 ± 0.76)

149 (91.31 ± 1.86)

1 (0.67 ± 0.67) 132 (81.59 ± 1.64)
0Results of 2-way ANOVA
Cumulus cell removal

0.400

-

0.167

-

0.504

-

0.903

VEGF addition

0.150

-

0.268

-

0.813

-

0.694

-

-

-

-

-

-

-

Interaction79Supplementary table 7. Nuclear maturation status of oocytes from SF and MF when denuded after 20 h of IVM and supplemented with VEGF protein
FolliclVEGF

e typeaddition number

SF+

Nuclear stage after IVM

Oocyte
GV

PMI

MI

A/T

MII

Deg

GVBD

170

30 (17.82 ±3.71) 5 (3.01 ± 1.02) 15 (8.48 ± 5.38) 5 (2.86 ± 1.28) 104 (61.30 ± 2.56) B

11 (6.54 ± 1.22)

129 (75.64 ± 3.69)

163

B

11 (6.72 ± 1.47)

123 (75.48 ± 3.48)

A

7 (4.27 ± 0.66)

147 (90.87 ± 1.81)

0132 (81.79 ± 4.98) A

2 (1.25 ± 0.77)

142 (88.02 ± 4.34)

29 (17.80 ± 2.66) 7 (4.37 ± 1.24) 13 (8.08 ± 3.02) 3 (1.86 ± 0.76) 100 (61.16 ± 3.89)-

162

8 (4.85 ± 1.53)

3 (1.78 ± 0.73) 11 (6.83 ± 0.73) 1 (0.67 ± 0.67) 132 (81.59 ± 1.64)+

161

17 (10.73 ± 4.14) 2 (1.27 ± 0.78) 8 (4.96 ± 2.13)

MF

Results of 2-way ANOVA
Follicle size

< 0.005

-

0.966

-

< 0.001-< 0.005

VEGF addition

0.286

-

0.940

-

0.831

-

0.764

Interaction

0.316

-

0.302

-

0.829

-

0.797

80

81