Journal of Applied Animal Science Vol.11 (Supplement) 201813

In vitro growth of bovine oocytes
Takashi Miyano
Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan
Corresponding author: miyano@kobe-u.ac.jp

Keywords: bovine oocyte, in vitro culture, oocyte growth

Abstract

layer of flat-shaped granulosa cells (pre-granulosa cells).

A large number of small oocytes are contained inLarger-sized follicles are developing follicles which

the mammalian ovary. However, only a small number ofcontain a growing oocyte and multi-layered cuboidaloocytes grow to their final size, mature, and are ovulated.

granulosa cells. In cattle, approximately a hundred

In vitro growth (IVG) of small oocytes would provide athousand primordial follicles are contained in a female

potential source of mature eggs for livestock production

animal (Erickson, 1966; Gosden and Telfer, 1987). Aand contribute to assisted reproduction in humans. In 2016,

small population of oocytes in the primordial follicles

baby mice were successfully produced from oogonia ingrow and reach the final size, in turn, a species-specificfetal gonads, and even from ES cells and iPS cells. On thenumber of fully grown oocytes resume meiosis andother hand, there are significant hurdles to overcome

mature to the second metaphase (MII) after gonadotropic

before full establishment of IVG systems for domestic

stimulation. Thus, only a limited number of oocytes

species. IVG culture systems have been improved, and now

are ovulated from the ovaries throughout the female

bovine growing oocytes collected from early antral

reproductive life.

follicles are able to grow to their final size and acquire

We don't know whether all of the mammalianfull developmental capacity. IVG of small oocytes will

oocytes in the ovaries have the ability to grow up andprovide a new understanding of the mechanisms

develop to babies as in amphibians and fish. In vitro

regulating oogenesis and folliculogenesis in the complex

growth (IVG) culture systems of mammalian small

mammalian ovary.

oocytes are thought to provide new fundamental
knowledge that may help to answer the question.

Introduction

Furthermore, IVG of small oocytes would provide a newFemale amphibians and fish spawn thousands ofsource of mature eggs for livestock production by using

eggs which have grown in their ovaries as oocytes.

existing assisted reproductive technologies, such as

Female mammals also contain a huge number of oocytes

in vitro maturation (IVM) and in vitro fertilization (IVF)

in their ovaries. However, as the mammals are viviparousof oocytes (Fig. 1). In this review, I introduce the recent

and have a long reproductive life, only a selected number

progress in IVG systems, and discuss about the hurdlesof oocytes grow up to the final size, mature and arehave yet to overcome before establishment of IVG

ovulated.

systems for domestic species.

Oocytes grow in the ovarian follicles. In theovary, follicles vary in size and morphology, and themajority of the follicles have a small uniform size. Theseare primordial follicles composed of an oocyte and a14

Journal of Applied Animal Science Vol.11 (Supplement) 2018calf in 1999 (Yamamoto et al., 1999). Briefly, early antral
follicles (0.5-0.7 mm in diameter) were dissected frombovine ovaries, and oocyte-cumulus complexes whichcontained pieces of parietal granulosa cells (OCGs)

Figure 1. Schematic flow of in vitro growth (IVG), in vitro

were collected from the follicles. OCGs were embedded

maturation (IVM) and in vitro fertilization (IVF) of

in collagen gels and cultured for 2 weeks in TCM199

mammalian oocytes.

containing fetal calf serum and hypoxanthine. After
IVG culture, surviving oocytes were further cultured

Baby mice production by IVG

for maturation and subsequently inseminated withIn 1996, Eppig and O'Brien demonstrated thatspermatozoa. Resulting blastocysts were transferred tomouse oocytes from primordial follicles grew to the final

recipient cows and one cow delivered a live calf. The

size by IVG, undergo fertilization in vitro, and develop toresults first demonstrated that in vitro-grown bovine

a live born pup (Eppig and O'Brien, 1996). They used aoocytes acquired full developmental ability to produce

combination of two culture systems for the oocyte

live young.

growth. First, they cultured newborn mouse ovaries
containing only primordial follicles in organ culture for

Problems in the IVG system

8 days and recovered growing oocytes surrounded by

For the large species, no IVG systems supporting

granulosa cells (OGCs). The OGCs were further cultured

the entire developmental course from the primordial to thefor 14 days, and recovered fully-grown oocytes were

fully-grown stage have yet been established. Althoughmatured, and fertilized in vitro. The resulting 2-cell

production of calves by IVG using growing oocytes fromembryos derived from non-growing oocytes in primordial

early antral follicles has been reported, the yield wasfollicles were then transferred into pseudo pregnant

quite low. To establish the IVG systems which enablefemales, and one baby mouse was obtained. The efficiency

growing oocytes even at the mid-growth stage to grow toof the procedure was low, but the birth suggested thetheir final size and acquire full developmental capacity,

possibility that small oocytes in primordial follicles

we have to overcome some problems as follows.

provide a potential source of oocytes for IVF. The culture
methods have been improved. In 2016, baby mice were1) Detachment of granulosa/cumulus cells fromoocytes

produced from oogonia in fetal gonads (Morohaku et al.,In the ovary, oocytes grow accompanied with2016), and from ES cells and iPS cells (Hikabe et al., 2016)

follicular development. In the follicle, there is bidirectional("in vitro differentiation" in Fig. 1).

communication between oocytes and surrounding
granulosa cells (cumulus cells after antral stage) (Matzuk

Calf production by IVG

et al., 2002). These somatic cells contact with oocytes

In contrast to the mouse results, there arethrough cytoplasmic processes penetrating the zonasignificant hurdles to overcome before full establishment

pellucida and establish gap junctions with the oolemma.

of IVG systems for domestic species. Mouse oocytes

Gap junctions efficiently transfer small molecules such as

grow from 15-20 μm in diameter (excluding the zonaenergy substrates, nucleotides, and amino acids into thepellucida) to 75 μm, while bovine oocytes grow fromoocyte (Brower and Schultz, 1982). On the other hand,

30 μm to 120-125 μm (Hyttel et al., 1997). We developed

oocytes secrete oocyte specific growth factors, growth

an IVG system that supported growth of bovine growing

differentiation factor 9 (GDF9) and bone morphogenetic

oocytes (90-99 μm) for 2 weeks and produced a baby

protein 15 (BMP15) to regulate granulosa cells.

Journal of Applied Animal Science Vol.11 (Supplement) 201815

Therefore, it is essential for IVG of oocytes to maintainthe meiotic arrest. They require additional oocyte-specificthe communication between the oocyte and its

phosphodiesterase 3A inhibitor, such as cilostamide

companion granulosa/cumulus cells during the culture

and milrinone (Alam et al., 2018).
3) Low survival rate and maturation rate ofperiod.
Cultured granulosa cells prefer the plastic dishoocytesrather than the oocyte. When bovine OCGs are cultured

PVP medium maintains viability of bovine

in a dish, granulosa cells migrate away from the oocytes

growing oocytes to some extent. Steroid hormones, suchand spread onto the bottom of the dish, with the oocytes

as 17β-estradiol and androstenedione further increase

subsequently becoming denuded and degenerated. Whenoocyte viability (Taketsuru et al., 2012). Androstenedione

OCGs are embedded in collagen gels and cultured,also promotes the acquisition of oocyte meiotic

granulosa cells surround cumulus cells to continuecompetence efficiently (Makita and Miyano, 2015).

enclosing the oocytes, and the OCGs form antrum-like

Gaseous phase in the IVG culture (20% O2 or 5% O2)

structures in the gels. Hirao et al. (2004) improved IVG

also affects oocyte viability (Hirao et al., 2012). Now,

culture medium which contained a high concentration of

we culture bovine growing oocytes in the medium

polyvinylpyrrolidone (PVP). In the medium, granulosa

containing a mixture of estradiol and androstenedione

cells proliferated and the complexes made dome-like(Makita and Miyano, 2014), under the 5% O2 for thestructures, and a high percentage of oocytes grew to thefirst-half period of IVG then in 20% O2. In the system,

full size in the structures. Although both collagen gels and70-80% of oocytes grow to the full size after 2 weeks;

PVP prevent the detachment of granulosa/cumulus

about 80% of the in vitro grown oocytes mature to MII,

cells from the oocytes, PVP medium is superior to thefertilize normally, and develop to blastocysts at the similarcollagen-gel system. Collagen gels lose transparency andpercentage to in vivo grown oocytes (Makita et al., 2016).

shrink after a long-term culture. His group reported thesecond successful production of baby calf by this newculture system.
2) Oocytes resume meiosis before growing to thefull size

Conclusions
As described here, in vitro-grown bovine oocytes
from early antral follicles acquire full developmental
ability to produce live young. Although IVG technologies

In the IVG culture of bovine oocytes, supplemen-are still at the experimental stage, the efficiency istation with hypoxanthine is essential (Harada et al.,increasing. As IVM and IVF systems have provided new1997). Hypoxanthine is a component of follicular fluids

understandings of the mechanisms regulating maturationand is identified as a meiotic-arresting substance by means

and fertilization of mammalian oocytes, IVG systemsof an inhibitory action on cAMP-phosphodiesterase

will provide the answer to the basic questions in oocyte/(Downs et al., 1985; Eppig and Downs, 1987). Hypoxan-follicle physiology including a classical question ofthine promotes association between oocytes and granulosa"oocyte/ follicle selection" in the ovary.

cells, and increases the percentages of surviving oocytes.
Furthermore, it maintains oocytes are at the germinal

Acknowledgements

vesicle stage. If growing oocytes resume meiosis before

I am grateful to Dr. Yuji Hirao (NARO Institutegrowing to the full size during IVG, they degenerate.

of Livestock and Grassland Science, Tsukuba, Japan) for

Therefore, oocyte meiotic arrest must be maintained in

his helpful suggestions for oocyte culture. The authorIVG. For bovine growing oocytes having a larger size

thanks the staff of Animal Bio-Technology Center, Kobe

(105-115 μm), hypoxanthine is not enough to maintainfor supplying bovine ovaries. This work was supported

16

Journal of Applied Animal Science Vol.11 (Supplement) 2018

in part by a Japan Society for the Promotion of Science
KAKENHI grant (17K08137) to T.M.

Hirao, Y., Itoh, T., Shimizu, M., Iga, K., Aoyagi, K.,
Kobayashi, M., Kacchi, M., Hoshi, H. and Takenouchi,
N. 2004. In vitro growth and development of bovine

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