Impact of Insulin-Like Growth Factor-Ion bovine Follicular dinamics

March 2024

Ahmad Farid Rawan
Graduate School of Environmental and Life Science(Doctor’s Course)

OKAYAMA UNIVERSITY

3

OKAYAMA UNIVERSITY
Graduate School of Environmental and Life Science
SUMMARY OF DISSERTATION
Submitted in fulfillment of the requirement of the Ph.D. Course
By
Ahmad Farid Rawan
Laboratory of Reproductive Physiology
March 2024

Examining Committee
Dr.

Chairman of examining committee _______________

Dr. Koji KIMURA

Laboratory of Reproductive Physiology _______________(Academic Supervisor)

Dr. Tsuji TAKEHITO

Laboratory of Molecular Genetics of Mammals ___________(Academic Co-Supervisor)

Dr. Toshimitsu HATABU

Laboratory of Animal Physiology _______________(Academic Co-Supervisor)

4

CONTENTS
DEDICATION…………...………………………………….……………………….

1

PREFACE………………………………………………….………………….……..

2

ACKNOWLEDGEMENTS ………………………………….……………………...

3

PUBLICATIONS AND PRESENTATIONS ….……………………………………

4-5

TABLE LIST………………………………………………………………………...

6

FIGURE LIST………………………………………………………………………..

7

CHAPTER 1

GENERATION INTRODUCTION …..……………………………

8-13

CHAPTER 2

INTRODUCTION.……….……………………………………….

14-16

MATERIALS AND METHODS …………………………………... 17-24
RESULTS …………………………………………………………..

25-38

DISCUSION.…………...…………………..……………………….

39-43

SUMMARY………………………………………………………… 44
CHAPTER 3

INTRODUCTION…………………………………………………

45-46

MATERIALS AND METHODS…………………………………… 47-51
RESULTS…………………………………………………………...

52-59

DISCUSSION………………………………………………………. 60-63
SUMMARY………………………………………………………… 64
CHAPTER 4

CONCLUSION……………………………………………………... 65

ABBREVATIONS……………….…………………………………………………..

66-68

REFERENCES………………………………………………………………………

69-83

5

I dedicate this thesis to my mother, who passed away in June 2020.
May Allah grant my mother Jannatul-Firdaus
Ameen
I love you, Mom. I want to be with you in Jannah.

6

PREFACE
The experiments described in this dissertation were carried out at the Laboratory of
Reproductive Physiology, Graduate School of Environmental and Life Science (Ph.D. course),
Okayama University, Japan, from November 2021 to March 2024, under the supervision ofProfessor Koji KIMURA.
This dissertation has not been previously submitted in whole or in part to a council, auniversity, or any other professional institute for a degree, diploma, or other professional
qualification.
Ahmad Farid Rawan
March, 2024

7

ACKNOWLEDGMENTS
Thank you, Allah, for all the blessings I have received and for giving me the ability tofulfill the requirements to obtain the Ph.D. degree from Okayama University, Japan. I would
like to express my deepest respect and gratitude to my supervisor Dr. Koji KIMURA Professorof the Graduate School of Environmental and Life Science, Okayama University, Japan, for his
invaluable time and unrestricted support of the technical and scientific aspects of this study, andprovision of valuable comments on this manuscript.
I would like to owe my endless and heartfelt gratitude to my Lab mates for theirfriendly and warm guidance during the time of my stay, while I cannot list all the names here,
you are always on my mind. I would also like to extend special thanks to Mr. Yosuke SUGINO,
who consistently provided kind assistance with my daily life in Okayama, Japan.
I am pleased to express my deepest gratitude to the Japan International Cooperation
Agency (JICA), for their support. The research reported here was made possible through JICA’s
assistance, and I deeply appreciate their collaboration in the promotion of Afghanistan.
I would like to extend my sincere gratitude to Dr. Mohammad Hamid KADWAL,
Associate Professor, and ex-vice chancellor of Nangarhar University for his encouragement andsupport during my pursuit of a Ph.D. course in Japan. I also offer Heartfelt gratitude to allacademic and administrative staff of the Veterinary Science Faculty for their endless spiritualsupport and for encouraging me throughout my studies.
Finally, I must express my profound gratitude and appreciation to my beloved family
members for their love and support throughout my life. They have made numerous sacrifices
on my behalf, offering unconditional love and care. Words cannot adequately convey how much
I love them. I also want to extend my deepest thanks to them for taking care of my children
even better than I could. To my family, I love you all.
8

Publications and Presentations
Publication:
Ahmad Farid Rawan, Hikmatullah Langar, Maho Munetomo , Yuki Yamamoto, Kohei
Kawano, Koji Kimura. Effects of Insulin-like growth factor-1 on the mRNA expression of
estradiol receptors, steroidogenic enzymes, and steroid production in bovine follicles. J Reprod
Dev 2023; 69: 337–346.
In preparation for submission for publication
Ahmad Farid Rawan, Hikmatullah Langar, Koji Kimura (xxx). Effects of Insulin-like
Growth Factor-1 on Growth Regulatory Factors of Granulosa Cells in Bovine Small Follicles.

Oral presentations:Ahmad Farid Rawan, Hikmatullah Langar, Yosuke Sugino, Yuki Yamamoto, and KOJI
KIMURA (2022). Effects of Insulin-like growth factor-1 on the abundance of the aromatase
enzyme, expression of mRNA estradiol receptors, and steroid hormones production in bovine
follicles. The 9th JASID Western Japan Research Meeting, August 26. 2022, Hiroshima
University, Japan.
Ahmad Farid Rawan, Hikmatullah Langar, MUNETOMO Maho, YUKI Yamamoto, Koji
KIMURA (2022). Effects of Insulin-Like Growth Factor-1 on the Expression of Estradiol
Receptors and Androgens Production in Bovine Small Follicles. The 115th Society for
Reproduction Development (SRD) Meeting, 12-14.9.2022, Tokyo Agriculture University,
Tokyo, Japan.

9

Ahmad Farid Rawan, Hikmatullah Langar, Kojic KIMURA (2023). Effects of Insulin-Like
Growth Factor-1 on Growth Regulatory Factors in Bovine Small Follicles. The 116th Society
for Reproduction Development (SRD) Meeting, 24-27.9.2023, Kobe University, Kobe, Japan.
Ahmad Farid Rawan, Hikmatullah Langar, Kohei KAWANO, Koji KIMURA (2023).
Insulin-like Growth factor-1 Regulates the Gene Expression of Insulin-Like Growth Factor-1
Receptors, Steroidogenic Enzymes, and Steroid Production of Granulosa and Theca cells in
Bovine Small Follicles. International Symposium on Animal Bioscience, 3.11.2023, Okayama
University, Okayama, Japan.

Poster Presentations
Ahmad Farid Rawan, Hikmatullah Langar, Yuki Yamamoto, and Koji KIMURA (2022).
Insulin-like growth factor-1 regulates the aromatase enzyme abundance, estrogen receptors
expression, and androgen production in bovine follicles, The 4th International Conference on
Sustainable Agriculture and Environment in Vietnam, 17-19.11.2022 (online).
Ahmad Farid Rawan, Hikmatullah Langar, Koji KIMURA (2023). Effects of Insulin-likegrow factor-1 on the mRNA expression of oestradiol receptors, steroidogenic enzymes, andsteroid hormone production in bovine follicles, International Symposium on Environmental andLife Science, 24.1.2023, Okayama University, Japan.

10TABLE LIST
Table 1: Sequences of primers used for quantitative RT-PCR…………...……………

27

Table 2: Sequences of primers used for quantitative RT-PCR………………...………

54

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FIGURE LIST
CHAPTER 2

Page

Fig 1. Purity of granulosa and theca cells……………...…………………………….

28

Fig 2. ESR2 mRNA expression in uncultured granulosa cells…………………….… 29
Fig 3. Effects of IGF-1 on the expression of mRNA of ESR2 in cultured granulosa

30

cells…………………………………………………………………………………...
Fig 4. Effects of IGF-1 on the expression of mRNA of FSHR in cultured granulosa

31

cells………………………………………………………………………………….
Fig 5. Effects of IGF-1 on the expression of mRNA of CYP19A1 in cultured

32

granulosa cells……………………………………………………………………….
Fig 6. Effects of IGF-1 on mRNA expression of LHCGR and steroidogenic

33-36

enzymes in cultured TCs……………………………………………………………..
Fig 7. Effect of IGF-1 on steroid hormone production in cultured TCs……………..

37-38

Fig 8. Illustration of conclusion……………………………………………………...

43

CHAPTER 3
Fig 1. Expression of mRNA of IGF1R, PCNA and steroidogenic enzymes in

55-56

uncultured GCs from the three largest follicles……………………………………...
Fig 2. Effects of IGF-1 expression of mRNA of IGF1R, PCNA and steroidogenic

57-58

enzymes in cultured GCs……………………………………………………………..
Fig 3. Effects of IGF-1 on granulosa cell proliferation……………………………… 59

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CHAPTER 1
GENERAL INTRODUCTION
Studying and understanding reproductive physiology has allowed the development of
new biotechnologies that improved animal production and helped in the solution of classic
physiological problems that affect the reproduction of domestic animals (1).

THE OVARIES AND FOLLICLESThe term “ovary” is derived from the Latin word “ovum,” meaning egg (2). In cattle,
ovaries are located at the side and slightly below the uterine horns in the pelvic cavity, whereas
in multiparous cows they are situated in the abdominal cavity (3). The ovaries have two parts,
cortex and medulla. The surface epithelium, tunica albuginea, numerous ovarian follicles, andcorpus luteum (CL) constitute the cortex, while the medulla consists of numerous blood vessels,
lymphatics, and nerves (4).
Follicles are blister-like structures and fluid-filled, containing developing oocytes or
eggs (5). There are two different pools of follicles on the ovaries of cattle, the non-growing poolthat contains primordial follicles, and the growing pool that contains primary, secondary, andtertiary follicles (6). The primordial follicles enter the growth phase by leaving the arrested pooland grow into primary follicles, whereby the oocytes increase in size and the surrounding
squamous pre-granulosa cells become cuboidal and proliferate to form a layer of the cells
around the growing oocyte (7). A follicular wave is defined as the synchronous growth of agroup of small antral follicles, from which a single follicle is selected to become the dominantfollicle (8,9). Rajakoski in 1960 proposed for the first time, that follicle development in cattle
occurs in a follicular wave pattern (10). The number of follicular waves that occur during the13

bovine estrous cycle can vary between two or three waves (8). During folliculogenesis andrecruitment for follicle selection, a cohort of small follicles usually 4-6 mm in diameter begin
their final growth (11). The recruitment of a cohort of follicles is associated with the initiationof expression of messenger-ribonucleic acid (mRNA) for cholesterol side-chain cleavage
enzyme (P450scc) and aromatase (CYP19A1) in granulosa cells (12). This stage is initiated by
the high-level production of follicle-stimulating hormone; FSH (13). Three to six follicles witha diameter of 4 to 5 mm occur after the recruitment of follicles into a follicular wave (14). The
beginning of the difference in growth rates between the two largest follicles is termed follicle
deviation (15). In cattle, follicle deviation occurs when the largest follicle reaches a mean
diameter of 8.5 mm (16). For mono-ovulatory species, such as humans, horses, and cattle, only
one single follicle is selected as a dominant within each follicular wave and it is able to ovulate(17). Follicle selection in monovular species is the process wherein only one follicle develops
from a wave of growing follicles and continues to grow and ovulate. Several physiologicalchanges have been associated with follicular selection, including deviation in follicular growth
rates, decreased circulating FSH, increased circulating estradiol-17β (E2), expression ofluteinizing hormone receptors (LHCGR) on granulosa and theca cells, and increased free
insulin-like growth factor (IGF)-1 (16, 18, 19, 20) in follicular fluid. One of the recruitedfollicles is subsequently selected from the cohort for continued growth (11), whereas thesubordinate follicles become atretic (11). The selected follicle acquires LHCGR in the granulosa
cells and more free IGF-1 compared with the subordinate follicles (11). The selected follicle
becomes larger than other follicles and becomes capable of surviving in a low FSH environment(11). The dominant follicle grows approximately 1 to 2 mm per day and reaches its maximum
size of 10 to 20 mm (8).

14GONADOTROPHIN HORMONES
The gonadotropin-releasing hormone (GnRH) is secreted by the hypothalamus andstimulates the secretion of both FSH and luteinizing hormone (LH) by gonadotropic cells of the
adenohypophysis (21). The process of follicle development is tightly regulated by FSH and LH,
and intraovarian regulators (22). It has been shown that antral follicles (≥ 4 mm) are responsive
to the FSH and emerged by an increase in the circulating FSH concentration in blood (13, 23).
At the beginning of selection, all the growing follicles ≥ 5 mm contribute to the decline in FSH
concentrations, it is not known whether these growing follicles play a direct role in the initialdecline in FSH concentrations (24, 25). The suppression of FSH secretion prevents the
emergence of a new group of growing follicles and it enables only future dominant follicle tosurvive (25). Regardless of whether the dominant follicle continues to use the low
concentrations of FSH, the follicle apparently begins to use LH at the beginning of deviation(16, 23). Thus, the deviation process has been proposed as the morphological manifestation of
the dominant follicle selection (26, 27). LHCGR starts arising in GCs of the future dominantfollicle around the beginning of deviation, which induces the steroidogenic capacity in thedominant follicle (25). In a normal cycle, the elevation in pulsatile secretion of LH influences
the final maturation and development of antral follicles (28). The role of LH in the follicular
selection process has shown that the selected follicle opportunistically switches from being
predominantly FSH-dependent to being supported by LH (29). LH stimulates androgensynthesis within the ovarian theca cells (TCs). Androgens also indirectly maintain follicular
health as they are the indispensable substrate for E2 production, which is essential for follicle
growth and survival.

15

ESTRADIOL AND ESTRADIOL RECEPTORS
Estrogen is a primary female sex hormone and is primarily synthesized by the ovarianfollicle (30). Theca interna cells produce androstenedione (A4) and testosterone (T) which
diffuse into the granulosa cells. The granulosa cells synthesize E2 from androgenic precursors
produced by the theca interna cells (31). E2 is the primary signal to the brain that induces theexpression of estrus, but only in the absence of progesterone (P4) (32). E2 also plays importantroles in growth and development, and the regulation of reproductive cycles. E2 has beenreported to play the following roles within the ovary, including 1) stimulating the development
and maturation of rat ovarian follicles (33); 2) increasing the expression of FSH receptor(FSHR) and LHCGR by granulosa cells in rats (34) and 3) modulating steroid production by
granulosa and theca cells, as demonstrated in cattle (35). E2 has an effect on follicle
development along positive or negative feedback mechanisms to the hypothalamus and pituitary
gland. As the ovarian follicles grow and differentiate, increasing amounts of E2 are produced,
which up-regulate the synthesis and release of the pituitary gonadotrophins (FSH and LH),
thereby promoting ovarian follicular growth (36). E2 stimulates proliferation and induces theexpression of FSHR, LHCGR, aromatase, and IGF-1 production in granulosa cells (33).
Circulating E2 concentrations begin to increase at the time of deviation as a result of increased
E2 production by granulosa cells of the future dominant follicle (15, 37). In cattle, follicularfluid concentrations of E2 began to increase differentially in large follicle than in subordinatedfollicle near the expected beginning of deviation (38, 18).
E2 is known to elicit rapid effects on cellular function by binding to a membrane-boundestradiol receptor (ER) (39). The ER is a member of the superfamily of steroid nuclear receptors(40, 41, 42). ER is located within the nucleus (43, 44). Two major forms of ER are presently
known: ESR1 and ESR2 (45). ESR1 predominates in theca and is upregulated as follicles
become more reactive to E2 (46). ESR2 was observed in granulosa cells of primary, secondary,16

and mature follicles but not in theca cells (47). The bovine ESR2 mRNA expression in the
granulosa cells decreases with increasing follicular size (48). By contrast, the mRNA expressionof ESR1 in theca tissue increases continuously during the final growth of the bovine follicles(49). A previous study has shown that the expression of ESR2 mRNA is high in bovine smallfollicles (≤4 mm), and there may be a role for ER in accelerating the growth of small follicles(50). ER plays an important role in maintaining ovarian granulosa cell differentiation, follicle
and oocyte growth, development, and ovulation.

IGF SYSTEMThe IGF system in the ovary provides an integrated, cooperating, local network that isregulated by auto/paracrine and endocrine mechanisms to promote growth and differentiationin the ovary (51). The IGF system is composed of different elements which are two ligands(IGF-I and IGF-II) and two types of receptors (IGF1R and IGF2R). The IGF1R mediates mostof the somatomedin-like actions of both IGF-I and II (52). The TCs have greater levels of IGF2mRNA in dominant follicles. It appears that IGF2 acts through IGF1R, even though specific
IGF2R exists in TCs and GCs (53). The components of the IGF system in mammals include
IGF-1 and IGF-2, along with their receptors. This superfamily comprises six high-affinity IGFbinding proteins (IGFBP-1, -2, -3, -4, -5, and -6). Additionally, there are several low-affinity
IGFBP-related proteins (IGFBP-rP) (52). Four IGFBPs (BP-2, -3, -4 and -5) have been detectedin the follicular fluid of cattle (38).
The indispensable tetralogy for follicle selection and final maturation of antral follicles
in bovine follicles involves activating the IGF system (36). For the acquisition of follicle
dominance free IGF1 is required, which decreases in the subordinate follicle but remains
elevated in the dominant follicle (18) due to the breakdown of IGFBP by the IGFBP protease17(54). The intrafollicular factors that are candidates for activation of deviation include thoserelated to the IGF system (55).
Increasing free IGF-1 in follicular fluid is one of the physiological changes that areassociated with other factors in bovine follicle selection (18). The IGFs function as modulatorsof gonadotropin action on granulosa and theca cells (29). The intrafollicular effects of the
gonadotropins are mediated by the IGF-1 system and E2 (56). IGF-1 can stimulate granulosa
cell proliferation and synergize with gonadotrophins to promote the differentiation of follicle
cells (57) and induce the expression of FSHR and aromatase in granulosa cells during thepreantral-to-antral transition (58). In vitro studies with bovine granulosa cells indicate that IGF1 stimulates the production of E2 and A4 (59). The elevated levels of E2 and IGF-1 in thefollicular fluid of large follicles compared to subordinate follicles indicate their roles infollicular deviation in cattle (29, 18). Taken together, IGF-1 plays a crucial role in follicular
growth.
The aims of the present study are to investigate the effects of IGF-1 on various factors
involved in follicular growth and deviation such as ESR2, LHCGR, FSHR, IGF1R, proliferativecell nuclear antigen (PCNA), steroidogenic mRNA expression, steroid hormones production,and cell proliferation in bovine follicles.

18

CHAPTER 2
Effects of Insulin-like growth factor-1 on the mRNA expression of estradiol
receptors, steroidogenic enzymes, and steroid production in bovine follicles

INTRODUCTION
The selection of a single dominant follicle is associated with differential growth rates ina cohort of developing follicles that maintain the ovulatory capabilities of cattle. The growthrate change between the largest and the second largest follicles is defined as follicular deviation(16). During the common growth phase that precedes deviation, the follicle-stimulating
hormone (FSH) concentrations decrease (60), and the follicular fluid concentrations of estradiol
17β (E2) begin to increase in the future dominant follicle (18). E2 promotes follicular growth
and aromatase activity in granulosa cells (GCs) (35, 61). The action of E2 is mediated by
estradiol receptors (ER) (39). One of ERs, ESR2 was detected mainly in GCs, whereas ERα intheca cells (TCs) (62). Previous studies have shown that ESR2 knockout mice exhibited delayedfollicular growth (63) and fail to exhibit efficient ovulation even when treated with exogenous
gonadotropins (64). These findings suggest that ESR2 plays a crucial role in folliculardevelopment and ovulation.
Insulin-like growth factor-1 (IGF-1) plays a central role in bovine follicular growth. It
has been proposed that IGF-1 is required for follicular dominance in cattle (16) and may be
involved in the mechanism of deviation by increasing the E2 concentration in follicles in cattle(18) or responsiveness of GCs to their specific gonadotropin receptors (65). IGF-1 increases the
FSH responsiveness of GCs by increasing the FSH receptor (FSHR) expression in mice (66).
In an in vitro study, IGF-1 increased the expression of luteinizing hormone receptors (LHCGR)

19

in GCs of small follicles in cattle (60). However, no study has been conducted to determine theeffects of IGF-1 on mRNA expression of ESR2 in bovine GCs either in in vitro or in vivo.
Androgens are substrates for E2 synthesis, which is crucial for follicular growth andselection. During steroidogenesis, androstenedione (A4) is synthesized by the actions ofsteroidogenic acute regulatory (STAR) (67), cytochrome P450 side chain cleavage (CYP11A1),
cytochrome P450 17α-hydroxylase (CYP17A1) (68), 17𝛽-hydroxysteroid dehydrogenase(HSD17B) and 3β-hydroxysteroid dehydrogenase (HSD3B) in TCs under the LH stimulationand is used as the substrate for E2 synthesis by cytochrome P450 aromatase (CYP19A1) under
the FSH stimulus interacting with specific receptors in GCs (68, 69). The biosynthesis of P4 isregulated by HSD3B (70), and P4 is converted to testosterone (T) by the action of HSD17B in
TCs (71). In previous studies, IGF-1 was found to enhance the mRNA of CYP11A1, HSD3B,
and CYP19A1 in bovine GCs in vitro (72, 73). IGF-1 also significantly promoted the mRNA ofSTAR, CYP19A1, and HSD3B in mice cultured TCs (74). However, the effects of IGF-1 onsteroidogenic enzymes in bovine TCs are not well known. Several studies have shown that A4
and T stimulate the growth and development of follicles in cows (75). The in vitro culture ofmouse preantral follicles with T and A4 enhances follicular growth and development (76, 77).
IGF-1 in cattle enhances androgen synthesis from TCs (78). The in vivo effects of IGF-1 onfollicular fluid factors were studied in heifers where the second largest follicle was injected with
IGF-1 at the expected beginning of deviation and increased the follicular fluid concentrationsof A4 after 3 h (79). However, little is currently known about the effects of IGF-1 on steroid
hormone production in bovine TCs.
Taking together the findings described above, we hypothesized that IGF-1 increases theexpression of ESR2 mRNA in GCs and A4 production in the theca cells of small follicles, which
are the predominant factors for the development and selection of follicles. Therefore, in thepresent study, we aimed to determine the effects of IGF-1 on ESR2 expression in bovine GCs
20

of small and mid-sized follicles. In addition, the effects of IGF-1 on mRNA expression ofsteroidogenic proteins and steroid hormones (A4, P4, and T) production were examined inbovine GCs and TCs derived from small and medium follicles.

21

MATERIALS AND METHODS
Collection of bovine ovaries
The ovaries of healthy cows were collected from a local abattoir after the exsanguinationand transported to the laboratory on ice to obtain TCs and GCs from small (<6 mm) and medium(7-8 mm) diameter follicles, as previously described (80). The ovaries were washed with sterile
saline and 70% ethanol and kept in physiological saline at 4°C until the GCs aspiration andfollicles dissection for the collection of TCs. For unculture, GCs were collected from each pairof ovaries between days 1-4 post-ovulation, at which point the first follicular wave is normally
initiated (81); therefore, it is easy to select the healthy growing follicles in the ovaries.

Theca and granulosa cells isolation and culture
Bovine TCs were enzymatically harvested from dissected small and medium follicles
as described previously (82, 83). Briefly, follicles were dissected from ovaries, and the tissues
surrounding the follicles were removed using forceps. The dissected follicles were cut into
hemispheres and gently scraped with forceps to remove GCs and a part of the theca cell layer.
The thin theca layer thus obtained was rinsed with DMEM (Sigma-Aldrich, St. Louis, MO,
USA) containing 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.1% bovine serum
albumin (BSA, Nacalai Tesque, Kyoto, Japan) then the theca layer was minced in DMEM andtreated with 6600 units/ml collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan),
8000 units/ml DNase (DNP2, BBI Solutions, Newport, UK), 1 mg/ml dispase (Godo Shusei
Co., Ltd., Chiba, Japan) and 1mg/ml hyaluronidase (Nacalai Tesque). Cell dissociation wasallowed to continue for 60 min at 37°C for continuous stirring at 800 rpm. After incubation, thecell suspension was separated from undigested tissue by filtration through metal meshes (10022µm x 2, 80 µm x 2), and the dispersed theca cells were centrifuged (800 x g, 5 min at 4°C) andresuspended in 50 mM Tris-NH4CL for hemolysis after discarding the supernatant. Dispersed
cells were washed two times with DMEM (Sigma-Aldrich) containing 100 IU/ml penicillin,
100 µg/ml streptomycin, and 0.1% BSA and cell viability was estimated at 90–95% by trypan
blue exclusion test.
The GCs and follicular fluid were collected aseptically from small and medium follicles
using a 2.5 ml disposable syringe with a 24-gauge needle and transferred to a plastic Petri dishfilled with phenol red-free DMEM/Ham's F-12, 1:1 (v/v); (Invitrogen, Carlsbad, CA, USA)
containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 20 µg/ml amphotericin B(Sigma Aldrich, Inc.), and 50 IU heparin sodium salt (Nacalai Tesque). GCs with follicular
fluid were centrifuged (800 x g, 5 min at 4°C) and resuspended in Tris-NH4CL for hemolysis
after discarding the supernatant. The cell suspensions were centrifuged again and resuspended
in DMEM (Sigma-Aldrich) containing 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.1%
BSA and then were filtered through metal meshes (100 µm x 2, 80 µm x 2) to remove cumulusoocyte complexes (COCs). After centrifugation, the supernatant was discarded, and the washing
step was repeated twice. The cell suspensions were then resuspended in an appropriate volumeof phenol red-free DMEM/F-12 containing 10% FBS and 20 µg/ml gentamicin (SigmaAldrich). The viability of GCs was determined at 75-80% by the exclusion of trypan blue. The
dispersed GCs and TCs separately were cultured at 1.0 x 105 viable cells per 1 ml in phenol redfree DMEM/F-12 culture medium containing 10% FBS in 75-cm2 culture flasks (20 ml/flask;
Greiner Bio-One, Frickenhausen, Germany) and cultured under 95% air and 5% CO2 at 38.5°C
for 2–3 days. The medium was changed at 24 h intervals. When the cultured cells reached 80–
90% confluence, they were passaged with 0.1% bovine trypsin (Sigma-Aldrich) and sterile
phosphate-buffered saline (PBS). Dispersed cells in four replicate wells were preincubated with23

and without IGF-1 by culturing for 24 h in a serum-free medium in the absence (control) and in
the presence of various concentrations of IGF-1.

Total RNA extraction and quantitative RT-PCR
Total RNA was extracted from cultured and non-cultured GCs and TCs using RNAiso
plus (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The extracted
RNA from each sample was quantified and 2 µg of each total RNA was reverse transcribedusing ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo, Osaka, Japan).
The mRNA expressions of bovine ESR2, FSHR, CYP19A1, LHCGR, STAR, CYP11A1,
CYP17A1, HSD3B, HSD17B, β-actin (ACTB), and Glyceraldehyde 3-phosphate dehydrogenase(GAPDH) were determined according to a previously described method (84). The expressionof each mRNA was quantified using Brilliant III Ultra-Fast SYBER Green qPCR Master Mix
with Low ROX (Agilent Technologies, Santa Clara, CA, USA), starting from 2 ng of reversetranscribed total RNA. To standardize the relative level of ESR2, FSHR, and CYP19A1 mRNA
expression in GCs, ACTB was used as an internal control, and to standardize the relative levelsof LHCGR, STAR, CYP11A1, CYP17A1, HSD3B and HSD17B in TCs, GAPDH was used as aninternal control. Five housekeeping genes, MRPL4, H2A, GAPDH, ACTB, and 18S, were tested
to determine the optimal reference gene for quantitative RT-PCR data from isolated andcultured bovine GCs and TCs separately. ACTB for GCs and GAPDH for TCs were selected as
reference genes using NormFinder (85) because the expression of ACTB and GAPDH was themost stable among the samples compared to other genes. For the quantification of mRNA
expression levels, PCR was performed using the AriaMx Real-Time PCR System (Agilent
Technologies). The amplification was conducted with an initial hot start at 95ºC for 30 sec,
followed by 45 cycles of denaturation at 95ºC for 10 sec, annealing at the temperature indicated24

in Table 1 for 10 sec, and extension at 72ºC for 15 sec, followed by dissociation program (95°C
for 1 min, temperature indicated in Table 1 to 95ºC at 0.5°C intervals, 5 sec per interval). Serial
dilutions (20–20,000,000 copies) of each PCR product extracted from the agarose gel were usedas standards to analyze the respective mRNA expression levels.

Enzyme immunoassay (EIA)
The concentrations of A4, P4, and T in the culture medium were determined using
enzyme immunoassays, as described previously (86). The A4 standard curve ranged from3.9062 to 4000 ng/ml. The intra- and inter-assay coefficients of variation of the TCs of small
and medium follicles were, on average 5.81% and 11.52%, 5.14% and 15.01% respectively.
The P4 standard curve ranged from 0.0078125 to 2 ng/ml. The intra- and inter-assay coefficientsof variation of the small and medium follicles TCs averaged were 4.22% and 12.57%, 3.53%,
and 10.64% respectively. The T standard curve ranged from 0.625 to 12.8 ng/ml. The intra- andinter-assay coefficients of variation of TCs of small and medium follicles averaged were 4.16%
and 16.34%, 7.8%, and 14.32% respectively. To fit the range of standard concentrations, theculture media was diluted 10–40 times in assay buffer.

DNA assay
The DNA content of the cultured TCs was measured as previously described (87).
Briefly, after disrupting the cells with an ultrasonic homogenizer, cell lysates were incubatedwith Hoechst 33258 (Sigma-Aldrich). After 10 min of incubation, the fluorescence of eachsample and standard was measured using a microplate fluorometer (Fluoroskan Ascent,Labsystems, Waltham, USA). The standard curve ranged from 0.625 to 30 µg/ml.
25

Experiment.1: The ESR2 mRNA expression in uncultured granulosa cells
The three largest healthy follicles (i.e. well vascularization and having a transparent
follicular wall with fluid) were dissected from paired ovaries in a dish placed on ice andclassified into three size categories according to their diameter (small, <6 mm; medium 7–8 mm
and large, ≥9 mm) on days 1-4 post ovulation. GCs were obtained by hemisecting follicles atroom temperature in a dish placed on ice. GCs were collected from each follicle after
centrifugation, immediately mixed with 400 µl of RNAiso plus (Takara Bio Inc), and stored at-80°C until the extraction of total RNA for quantitative RT-PCR.

Experiment 2: Effects of IGF-1 on the mRNA expression of ESR2, FSHR andCYP19A1 in cultured granulosa cells
Cultured GCs of small and medium follicles were seeded at a density of 1.0 X 105 viable
cells in 1 ml phenol red-free DMEM/F-12 to each well of 24-well plates (Greiner Bio-One).
The cells were incubated at 38.5oC, and 2 days after seeding, they were incubated with medium(v/v) containing 0.1% BSA, 5 µg/ml holotransferrin (Sigma-Aldrich), and 2 µg/ml insulin(Sigma- Aldrich) and treated with various concentrations (10, 50, and 100 ng/ml) of IGF-1 (BioVision, Waltham, MA, USA) for 24 h. In our study, the concentrations of IGF-1 were selectedbased on previous studies (60, 72). In each group, the cells were mixed with 400 µl of RNAiso
plus in a 1.5 ml tube and stored at -80°C for subsequent RNA extraction.

26

Experiment 3: Effects of IGF-1 on the mRNA expression of LHCGR, STAR,
CYP11A1, HSD17B1, CYP17A1 and HSD3B in cultured theca cells
The culture condition for experiment 3 was the same as for experiment 2, but in thisexperiment, the cultured TCs were treated with various concentrations (10, 50, and 100 ng/ml)
of IGF-1 for 24 h. In each group, the cells were mixed with 400 µl of RNAiso plus in a 1.5 ml
tube and stored at -80°C for subsequent RNA extraction.

Experiment 4: Effects of IGF-1 on steroid hormones (A4, P4 and T) production by
cultured bovine theca cells
To determine the effects of IGF-1 on A4, P4, and T production, cultured TCs from small
and medium follicles were seeded at a density of 0.5 x105 viable cells in 250 µl phenol red-free
DMEM/F-12 in 48 well plates (250 µl/well; Greiner Bio-One). The cells were incubated at
38.5oC, and 2 days after seeding, they were incubated with medium (v/v) containing 0.1% BSA,
5 µg/ml holotransferrin (Sigma-Aldrich), and 2 µg/ml insulin (Sigma- Aldrich) and treated withdifferent concentrations (10, 50, and 100 ng/ml) of IGF-1 for 24 h. After incubation, the medium
was collected in a 1.5 ml tube, the concentrations of A4, P4, and T were measured by enzyme
immunoassay, and the DNA content was measured to standardize the androgen and P4
concentrations.

27

Purity of granulosa and theca cells
Isolated GCs and TCs were separately cultured at 1.0 x 105 viable cells per 1 ml in
phenol red-free DMEM/F-12 culture medium containing 10% FBS in 6 well plates and cultured
under 95% air and 5% CO2 at 38.5°C for 2 days. The GCs and TCs were checked under the
microscope (FSX100, Olympus, Tokyo, Japan) to determine the purity of isolated cells. TCs
were thin, long, and spindle-shaped while GCs were thick, flat-shaped, and smaller than TCs.
The purity of isolated GCs and TCs were 95% and 90%, respectively (Fig. 1).

28

STATISTICAL ANALYSISAll experimental data are shown as the mean ± SEM and normality andhomoscedasticity were confirmed using the Shapiro-Wilk test and Brown-Forsythe test using
R (88) respectively and analyzed by ANOVA followed by Tukey’s multiple comparison tests
using R. The statistical level of significance was considered at P <0.05.

29

RESULTS
Experiment 1. ESR2 mRNA expression in uncultured granulosa cells
The three largest healthy follicles were dissected from each pair of ovaries (Days 1–4
post-ovulation). The ESR2 mRNA expressions in GCs from small (<6 mm), medium (7–8 mm),and large (≥9 mm) follicles were shown in Fig. 2. The ESR2 mRNA expression in the GCs was
low in large follicles (≥9 mm), moderately higher in medium follicles (7–8 mm) and highest insmall follicles (<6 mm) (p<0.05). The ESR2 mRNA expressions were higher in small follicles
than in medium and large-sized follicles.

Experiment 2. Effects of IGF-1 on the expression of mRNA of ESR2, FSHR, andCYP19A1 in cultured granulosa cells
GCs were treated with different doses of IGF-1 for 24 h. Results showed that the mRNA
expression of ESR2 (Fig. 3), FSHR (Fig. 4), and CYP19A1(Fig. 5) significantly increased(p<0.05) at a dose of 100 ng/ml of IGF-1 in cultured GCs of small follicles. However, the otherdoses did not have any effect on the mRNA expression of ESR2, FSHR, and CYP19A1 in GCs
of small follicles. In medium follicles, IGF-1 (10–100 ng/ml) increased the ESR2 mRNA
expression, but only IGF-1 (100 ng/ml) significantly (p<0.05) increased the expression of
CYP19A1 mRNA. The FSHR mRNA expression in cultured GCs of medium follicles was notaffected by IGF-1 treatment for 24 h.

30Experiment 3: Effects of IGF-1 on the mRNA expression of LHCGR, STAR,
CYP11A1, CYP17A1, HSD17B1 and HSD3B in cultured theca cells
The cultured TCs were treated with selected doses of IGF-1 for 24 h. IGF-1 (100 and
50-100 ng/ml) increased significantly (p<0.05) the mRNA expression of LHCGR and HSD17B
in small and medium follicles, respectively. IGF-1 (100 ng/ml) increased the expression ofSTAR mRNA in TCs of small follicles while IGF-1 (10–100 ng/ml) enhanced STAR mRNA
expression in TCs of medium follicles. IGF-1 (50 and 100 ng/ml) significantly raised (p<0.05)
the CYP11A1 mRNA expression in the TCs of small and medium follicles, respectively. IGF-1(10-100 ng/ml) increased the CYP17A1 mRNA in TCs of small follicles, while IGF-1 (50-100
ng/ml) significantly increased the CYP17A1 mRNA in TCs of medium follicles. IGF-1 (100,50-100 ng/ml) significantly enhanced the HSD3B and HSD17B1 mRNA in TCs of small andmedium follicles, respectively (Fig. 6).

Experiment 4. Effects of IGF-1 on androstenedione, progesterone, and testosterone
production in cultured bovine theca cells.
The cultured TCs were treated with different doses of IGF-1 (10, 50, and 100 ng/ml) for
24 h. IGF-1 (50 ng/ml) increased A4 production in TCs of small follicles, while IGF-1 (10-50
ng/ml) significantly enhanced (p<0.05) the production of A4 in TCs of medium follicles (Fig.
7A). Furthermore, IGF-1 significantly enhanced P4 production in TCs (50-100 ng/ml in smallfollicles, 100 ng/ml in medium follicles, respectively) (Fig. 7B). However, there was no effectof IGF-1 on the production of T in TCs of either small or medium follicles (Fig. 7C).

31

Table 1. Sequences of primers used for quantitative RT-PCR

Forward and reverse primers
Accession no.
Product size (bp) Temperature (oC)5'-GCCAGCCGTCAGTTCTGTAT-3'
ESR2
NM_174051
121
58.5
5'-CAACTGCTCCCACTAGCCTT-3'5'-TGCAGTCGAACTGAGGTTTG-3'
FSHR
NM_174061.1
154
60
5’-ATGTAGTTTGGGCAGGTTGG-3'5’-CAACAGCAGAGAAGCTGGAAGACA-3’
CYP19A1
NM_174305.1
225
60
5’-CACCAACAACAGTCTGGATTTCCCT-3'
5'GAGAAGAGCTACGAGCTTCCTGACG-3'
ACTB
NM_173979.3
10660
5'-AGGATTCCATGCCCAGGAAGGAAGG-3'5'-GATAGAAGCTAATGCCTTTGACAAC-3’
LHCGR
NM_174381.1
197
60
5'-CCAGAATGAAATTAAATTCAGAGGAG-3'
5’-CAACAGCAGAGAAGCTGGAAGACA-3’
STAR
NM_174189.3
133
60
5’-CACCAACAACAGTCTGGATTTCCCT-3’5'-TAAACCTGGTCACCGACTGC-3’
HSD17B1
NM_001035395.2
126
57
5'-CTGCCAGGGATGTAGGCAAA-3'
5’-TAAGCAAGATGCTGCAAATG-3’
CYP11A1
NM_176644.2
137
59
5’-CTTGGCAGGAATCAGGTAAT-3'
5'-CACCCTCAAGATTGTCAGCA-3’
GAPDH
NM_001034034.2
103
60
5'-GGTCATAAGTCCCTCCACGA-3'
Gene

32

Fig. 1. Isolated bovine granulosa (GCs, A) and theca cells (TCs, B) were separately cultured at
1.0 x 105 viable cells per 1 ml in phenol red-free DMEM/F-12 culture medium containing 10%
FBS for 2 days and checked under the microscope. TCs were thin, long, and spindle-shaped
while GCs were thick, flat-shaped, and smaller than TCs.

33

Fig. 2 Gene expression of E2 receptor β (ESR2) mRNA relative to the amount of β-actin (ACTB)
in isolated GCs from the three largest follicles in each pair ovary between days 1 and 4 postovulation (follicles/group). The expression values are presented as means ± SEM. The follicles
were grouped by range as follows: small (<6mm), medium-sized (7-8 mm), and large (≥9 mm)
in diameter. Different superscript letters (a-c) indicate significant differences (p< 0.05;
ANOVA) among groups followed by Tukey’s multiple comparison test.

34

Fig. 3. Effects of IGF-1 on mRNA expression of ESR2 in GCs. The GCs from small- andmedium-sized follicles were exposed to various concentrations of IGF-1 (10, 50, and 100 ng/ml)
for 24 h. The genes were quantified relative to β-actin (ACTB) in cultured GCs from small- andmedium-sized follicles. The expression values are presented as means ± SEM of five replicates.
Different superscript letters indicate statistically significant differences (p < 0.05; ANOVA) as
determined by Tukey’s multiple comparison test.

35

Fig. 4. Effects of IGF-1 on mRNA expression of FSHR in GCs. The GCs from small- andmedium-sized follicles were exposed to various concentrations of IGF-1 (10, 50, and 100 ng/ml)
for 24 h. The genes were quantified relative to β-actin (ACTB) in cultured GCs from small- andmedium-sized follicles. The expression values are presented as means ± SEM of five replicates.
Different superscript letters indicate statistically significant differences (p < 0.05; ANOVA) as
determined by Tukey’s multiple comparison test.

36

Fig. 5. Effects of IGF-1 on mRNA expression of CYP19A1 in GCs. The GCs from small- andmedium-sized follicles were exposed to various concentrations of IGF-1 (10, 50, and 100 ng/ml)
for 24 h. The genes were quantified relative to β-actin (ACTB) in cultured GCs from small- andmedium-sized follicles. The expression values are presented as means ± SEM of five replicates.
Different superscript letters indicate statistically significant differences (p < 0.05; ANOVA) as
determined by Tukey’s multiple comparison test.

37

38

Fig. 6. Effects of IGF-1 on mRNA expression of LHCGR (A), STAR (B) and CYP11A1 (C) in
TCs. The TCs from small- and medium-sized follicles were exposed to various concentrationsof IGF-1 (10, 50, and 100 ng/ml) for 24 h. The genes were quantified relative to GAPDH in
cultured TCs from small- and medium-sized follicles. The expression values are presented as
the means ± SEM of five replicates. Different superscript letters indicate statistically significant
differences (p < 0.05; ANOVA) as determined by Tukey’s multiple comparison test.

39

40

Fig. 6. Effects of IGF-1 on mRNA expression of CYP17A1 (D), HSD17B1 (E), and HSD3B (F)in TCs. The TCs from small- and medium-sized follicles were exposed to various
concentrations of IGF-1 (10, 50, and 100 ng/ml) for 24 h. The genes were quantified relative to
GAPDH in cultured TCs from small- and medium-sized follicles. The expression values arepresented as the means ± SEM of five replicates. Different superscript letters indicate
statistically significant differences (p < 0.05; ANOVA) as determined by Tukey’s multiple
comparison test.

41

42

Fig. 7. Androstenedione (A), Progesterone (B), and Testosterone (C) production by theca cellsof small- and medium-sized follicles. Theca cells were cultured with various concentrations of
IGF-1 (10, 50, and 100 ng/mL) for 24 h. All values are presented as the means ± SEM of five
replicates. Different superscript letters indicate significant differences (p < 0.05; ANOVA),
followed by Tukey's multiple comparison test.

43

DISCUSSION
The intraovarian actions of ESR2 are vital to fully differentiated follicles and arenecessary for the enzymatic activity to convert androgen precursors to E2 (89), which is crucial
for follicular growth and deviation. The ESR2 mRNA receptor mediates E2 actions in GCs(63,69,70). In the present study, the expression of ESR2 mRNA in GCs decreased with follicularsize. This result agrees with previous studies that the mRNA ESR2 decreased in bovine GCs oflarge follicles (50, 90, 48). These results suggest that higher ESR2 mRNA expression in GCs ofsmall and medium follicles is crucial for their growth and deviation. In a previous study, IGF1 increased E2 production in cultured GCs of small follicles (60), which may require greater
ESR2 mRNA expression to maintain its bioactivity. The effect of IGF-1 on ESR2 mRNA
expression in GCs has not been thoroughly investigated in cattle previously. Therefore, we
examined the effect of IGF-1 on ESR2 mRNA expression in bovine GCs. Our results showedthat IGF-1(100 ng/ml) increased ESR2 mRNA expression in the GCs of small follicles, whereas
low levels of IGF-1 (10-100 ng/ml) increased ESR2 mRNA expression in GCs of medium
follicles. These results indicate that (i) the effect of IGF-1 on ESR2 mRNA in the GCs is dosedependent, (ii) the stimulatory effect of IGF-1 on ESR2 mRNA supports our hypothesis thatIGF-1 increases ESR2 mRNA expression in GCs of small to maintain E2 bioactivity forfollicular growth and deviation, and (iii) the effects of IGF-1 may depend on the presence of
IGF1R in GCs, requiring further investigation into IGF-1 effects on mRNA expression of
IGF1R in GCs of different stages of follicles.
The steroid hormone synthesis is controlled by ESR2 (89), FSHR (91), and CYP19A1(72) in the GCs of cattle. IGF-1 plays a central role in these interactions with respect tosteroidogenesis (92, 56). In the present study, we investigated the effects of IGF-1 on gene
expression that regulate E2 synthesis in GCs. Our previous study showed that IGF-1 (1-100

44

ng/ml) significantly increased E2 production in GCs of small follicles in vitro (60). In this study,
IGF-1 (100 ng/ml) increased CYP19A1 and FSHR mRNA expressions and even a low dose of
IGF-1 (10 ng/ml) stimulated CYP19A1 mRNA expression in GCs of small follicles, whichconverts A4 to E2 in GCs. IGF-1 (100 ng/ml) increased CYP19A1 mRNA expression, but IGF1 did not affect FSHR mRNA expression in GCs of medium follicles. In previous studies, IGF1(10 ng/ml) increased the mRNA FSHR in mice (74) and IGF-1 (50 ng/ml) enhanced FSHR
and CYP19A1 mRNA expressions in bovine GCs (2-8 mm) treated for 48 h (72, 73). The species
or culture protocol can influence the expression of mRNA of genes. These results clearly statedthat IGF-1 significantly enhanced the mRNA of CYP19A1 and FSHR in GCs of small follicles
which regulate E2 synthesis that plays a vital role in follicular development.
Androgen is synthesized via steroidogenesis-related enzymes in TCs which are the precursors
for E2 (93-94). The binding of LH to its receptor on TCs stimulates the activities of thesteroidogenic enzymes necessary for A4 synthesis (19). In our study, IGF-1 significantly
increased the expression of LHCGR mRNA in TCs in both sized follicles, consistent withprevious reports in cultured follicles of mice (74) and in bovine GCs (60). Our study also
demonstrated the effects of IGF-1 on three genes regulating A4 synthesis in TCs, namely STAR,
CYP11A1, and HSD17B (13, 95-97, 71). IGF-1 (100 ng/ml) increased the mRNA of STAR,
CYP11A1, and HSD17B in TCs of small follicles, whereas lower doses of IGF-1 enhanced these
enzymes in TCs of medium follicles, comparable with previous studies that IGF-1 enhanced
mRNA of STAR in cultured follicles of mice (74) and CYP11A1 mRNA in GCs of porcine 98).
IGF-1 had little effect on CYP11A1 mRNA in bovine-cultured TCs in the presence of LH (72).
Interestingly, in the present study, IGF-1 alone increased the expression of mRNA of CYP11A1
in TCs. The conversion of pregnenolone to dehydroepiandrosterone (DHEA) is regulated by
CYP17A1, and DHEA is subsequently converted to A4 by HSD17B and HSD3B (68). Previous
studies have reported a significant increase in the expression of CYP17A1 mRNA in bovine
45theca cells (TCs) cultured with IGF-1 supplemented medium for 24 hours (99). Additionally,
CYP17A1 mRNA in bovine small-sized follicle TCs was found to increase in response to IGF1 and LH in vitro (100). However, IGF-I alone has no effect on CYP17A1 mRNA in cultured
rat TCs (100). IGF-1 has been shown to increase HSD3B mRNA in bovine granulosa cells (GCs)(74) and in TCs of rats (91). In the present study, IGF-1 increased CYP17A1 and HSD3B mRNAin cultured TCs of both small and medium-sized follicles. The differences in results may
indicate that the species or experimental treatment protocol can influence the expression of
CYP17A1 mRNA in TCs. These results indicate that IGF-1 enhances gene expression involvedin A4 biosynthesis.
In fact, IGF-1 (50 ng/ml) and IGF-1 (10-100 ng/ml) increased A4 production in TCs ofsmall and medium follicles respectively. This study provides more information that IGF-1
enhances A4 production in TCs of small follicles, which play a key role in follicular growth,and indicates that enhancing A4 production and related gene expression in TCs of small andmedium follicles are IGF-1 dose-dependent. Pregnenolone is converted to P4 via HSD3B in
TCs (68). In vivo, no differential changes in P4 concentration were observed in the two largest
follicles before the beginning of deviation in heifers (18, 101). IGF-1 increased P4 production
in bovine TCs from large follicles (101, 58). In our study, IGF-1 enhanced the P4 production in
TCs of small and medium follicles. However, P4 is not crucial for follicular growth but may
serve as a great precursor for A4 synthesis in TCs. IGF-1 did not increase T production in
cultured TCs from either small or medium follicles. These results show that IGF-1 has thecapability to increase the expression of steroidogenic enzymes and A4 production in TCs ofsmall follicles which is a great source for E2 synthesis in GCs that play a crucial role in follicular
growth and deviation (Fig. 8).
In conclusion, IGF-1 usually affects pre-antral or early antral follicles. This study showsthat IGF-1 is involved in some gene expressions and hormone production which play vital roles46

in various aspects of follicular growth of small and medium antral follicles. This study showsthat the effects of IGF-1 in small and medium follicles were in a dose-dependent manner. The
high dose of IGF-1 (100 ng/ml) was more effective in enhancing the targeted variables in smallfollicles whereas the lower doses were in medium follicles. Indeed, these results supported ourhypothesis that IGF-1 increased the expression of ESR2 mRNA in GCs and A4 production in
TCs of small follicles which enhance follicular growth and is required for follicular deviation.

47

Fig. 8. Illustration describing our conclusion. IGF-1 increased A4 production via enhancing
gene expression of steroidogenic enzymes and LHCGR in TCs and promoted mRNA of ESR2
in GCs of small follicles, indicating that IGF-1 is the best choice for enhancing factors involvedin bovine follicular deviation and selection.

48

SUMMARY
Insulin-like growth factor-1 (IGF-1) plays a crucial role in follicular growth andstimulates steroid hormone production in bovine follicles. Steroid hormones are synthesized by
the actions of steroidogenic enzymes, namely STAR, CYP11A1, HSD3B, CYP17A1, HSD17B1
and CYP19A1 in both theca cells (TCs) and granulosa cells (GCs) under the stimulation of
gonadotropins. Especially, estradiol 17β (E2) plays a key role in follicular development andselection via estrogen receptors β (ESR2) in GCs. We examined the mRNA expression of ESR2
in GCs of developing follicles and the effect of IGF-1 on the mRNA expression of ESR2,
CYP19A1, FSHR and on LHCGR, STAR, CYP11A1, HSD17B, HSD3B, CYP17A1 in cultured
GCs and TCs, respectively. We also checked the effect of IGF-1 on androstenedione (A4),progesterone (P4) and testosterone (T) production in TCs. Small follicles (<6 mm) expressed
the highest levels of ESR2 mRNA and medium (7-8 mm) expressed higher than large follicles(≥9 mm) (p < 0.05). IGF-1 increased the mRNA expression of ESR2, CYP19A1, and FSHR in
GCs of both sizes of follicles, except for FSHR mRNA in medium follicles (p < 0.05). IGF-1
significantly increased mRNA expression of LHCGR, STAR, CYP11A1, CYP17A1, HSD3B and
HSD17B in TCs of small and medium follicles (p < 0.05). Moreover, IGF-1 increased A4 andP4 production but did not affect the T production in TCs of small and medium follicles. Taken
together, IGF-1 up-regulates the steroidogenic enzymes and steroid hormone production
suggesting that IGF-1 is crucial for follicle development and selection.

49

CHAPTER 3
Effects of Insulin-like Growth Factor-1 on Growth Regulatory Factors ofGranulosa Cells in Bovine Small Follicles
INTRODUCTION
In follicular development, the endocrine, paracrine, and autocrine factors are involved,
which control several processes including the proliferation and differentiation of the follicular
cells (102). It has been shown that IGF-1 is important for antral formation, where it is involvedin follicle growth, stimulation of somatic cell proliferation, and steroidogenesis (56, 103). In in
vitro culture of preantral follicles, IGF-1 stimulated the follicular growth in bovines (104). The
essential role of IGF-I was confirmed in experiments using IGF-I knockout mice, whose
follicles were arrested at the early antral stage and failed to respond to gonadotrophins (66).
In cattle, the mRNA of IGF-1 receptors (IGF1R) is expressed in granulosa cells (GCs)throughout follicle development (105, 106) playing roles as a mitosis inducer (107) andapoptosis suppressor (108), and for the formation of the preovulatory follicle (19). However,
the regulation of the bioactivity of IGF-1 is still under concern. It has been reported that thebioactivity of IGF-1 is controlled by its association with at least six IGF-binding proteins (110).
In contrast, another study revealed that IGF-1 mediates their biological actions via IGF1R (105).
The unbound or the free fraction of IGF-1 may be the dominant biologically active form (110).
As follicles undergo growth, there is a reduction in the level of IGF binding protein- 2 (IGFBP2), and more IGF-1 would be available for binding with the IGF1R (111). These findings showthat the bioactivities of IGF-1 may be mediated via IGF1R mRNA in the GCs of growing
follicles. However, the effects of IGF-1 on mRNA expression of IGF1R in GCs remain
unknown. To assess the effect of IGF-1 on the expression of IGF1R mRNA, we hypothesizedthat IGF-1 promotes the expression of IGF1R mRNA in GCs of small follicles.

50

The initiation of follicular growth depends on sensitive markers such as proliferatingcell nuclear antigen (PCNA), a protein expressed by actively growing and proliferating cells(112) which has been suggested to play a key role in ovarian follicle development (113). The
follicle growth was initiated in vitro, and its initiation was further confirmed throughimmunolocalization of PCNA in cultured and freshly isolated pieces of the ovarian cortex (114).
IGF-1 is one of the growth factors in bovine that stimulates both follicular growth (115), andproliferation (116). Despite their evident significance, the specific role of IGF-1 on theexpression of PCNA mRNA in GCs remains unknown. Therefore, we investigated the effectsof IGF-1 on PCNA mRNA expression and on GC number in small follicles.
The steroidogenic potential of GCs can be regulated with the interaction of locally
produced steroidogenic enzymes which are crucial for follicular growth and selection. IGF-1
plays a central role in these interactions with respect to steroidogenesis (92, 56). The previous
studies, using diverse culture systems with different types of follicular cells from various
follicular diameters of bovine ovaries, have demonstrated that IGF-1 enhances the mRNA
expression of steroid-related enzymes (117, 73, 92). In the present study, we also examined theeffect of IGF-1 on STAR, CYP11A1, CYP17A1, HSD3B, and HSD17B1 mRNA expression in
cultured GCs of small follicles.

51

MATERIALS AND METHODS
Collection of bovine ovaries
Freshly excised ovaries of healthy cows were collected from a local abattoir to obtainGCs from small (<6 mm) diameter follicles, as previously described (80). For unculture, GCs
were collected from each pair of ovaries between days 1-4 post-ovulation (81).

Granulosa cells isolation and culture
The GCs and follicular fluid were collected aseptically from small follicles using a 2.5
ml disposable syringe with a 24-gauge needle and transferred to a plastic Petri dish filled withphenol red-free DMEM/Ham's F-12, 1:1 (v/v); (Invitrogen) containing 10% calf serum, 20µg/ml amphotericin B (Sigma Aldrich), and 50 IU heparin sodium salt (Nacalai Tesque). The
further procedure for isolation GCs was identical to the one explained in Chapter Two’s
methods and materials section. Dispersed GCs in four replicate wells were preincubated withand without IGF-1 by culturing for 24 h in a serum-free medium in the absence (control) and in
the presence of various concentrations of IGF-1.

Total RNA extraction and quantitative RT-PCR
Total RNA was extracted from cultured and fresh GCs using RNAiso plus (Takara Bio
Inc) according to the manufacturer’s instructions. The extracted RNA from each sample wasquantified and 2 µg of each total RNA was reverse transcribed using ReverTra Ace qPCR RT
Master Mix with gDNA remover (Toyobo).

52

The mRNA expression of IGF1R, PCNA, STAR, CYP11A1, CYP17A1, HSD3B, andHSD17B1 mRNA, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-actin (ACTB)
were determined according to a previously described method (83). The expression of eachmRNA was quantified using Brilliant III Ultra-Fast SYBER Green qPCR Master Mix with Low
ROX (Agilent Technologies), starting from 2 ng of reverse-transcribed total RNA. To
standardize the relative level of genes in uncultured GCs, GAPDH was used as an internal
control, and to standardize the relative levels of genes in cultured GCs, ACTB was used as aninternal control. The internal control was selected using NormFinder (84). For the quantificationof mRNA expression levels, PCR was performed under the following conditions using the
AriaMx Real-Time PCR System (Agilent Technologies) The amplification was conducted withan initial hot start at 95ºC for 30 sec, followed by 35 cycles of denaturation at 95ºC for 10 sec,
annealing at the temperature indicated in Table 2 for 10 sec, and extension at 72ºC for 15 sec,
followed by dissociation program (95°C for 1 min, temperature as indicated in Table 1, to 95ºC
at 0.5°C intervals, 5 sec per interval). Serial dilutions (20–20,000,000 copies) of each PCR
product extracted from the agarose gel were used as standards to analyze the respective mRNA
expression levels. The sequences of primers were listed in Table 2.

Cell proliferation
The DNA content of the GCs was measured as previously described (87). Briefly, after
disrupting the cells with an ultrasonic homogenizer, cell lysates were incubated with Hoechst
H 33258 (Sigma-Aldrich). After 10 min of incubation, the fluorescence of each sample andstandard was measured using a microplate fluorometer (Fluoroskan Ascent). The standard curve
ranged from 0.625 to 30 µg/ml.

53

Experiment.1: The IGF1R, PCNA, STAR, CYP11A1, HSD3B, CYP17A1 andHSD17B1 mRNA expression in uncultured granulosa cells
The three largest healthy follicles were dissected from paired ovaries and classified into
three size categories according to their diameter (small, <6 mm; medium-sized, 7–8 mm; andlarge, ≥9 mm) on days 1-4 post ovulation as described previously (118). GCs were collectedfrom each follicle after centrifugation, immediately mixed with 400 µl of RNAiso plus (Takara
Bio Inc) and stored at -80°C until the extraction of total RNA for quantitative RT-PCR.

Experiment 2: Effects of IGF-1 on the mRNA expression of IGF1R, PCNA, andsteroidogenic-related enzymes in cultured granulosa cells
Cultured GCs of small were seeded at a density of 1.0 X 105 viable cells in 1 ml phenol
red-free DMEM/F-12 to each well of 24-well plates (Greiner Bio-One). The cells wereincubated at 38.5oC, and after the cells reached confluence, they were incubated with medium(v/v) containing 0.1% BSA, 5 µg/ml holotransferrin (Sigma-Aldrich), and 2 µg/ml insulin(Sigma- Aldrich) and treated with different concentrations (10, 50, and 100 ng/ml) of IGF-1(Bio Vision) for 24 h. In each group, the cells were mixed with 400 µl of RNAiso plus in a 1.5
ml tube and stored at -80°C for subsequent RNA extraction.

Experiment 3: Effects of IGF-1 on granulosa cell proliferation
The isolated GCs of small follicles were cultured at 1.0 x 105 viable cells per 1 ml in
phenol red-free DMEM/F-12 culture medium containing 10% calf serum in 75-cm2 culture
flasks (20 ml/flask) under 95% air and 5% CO2 at 38.5°C for 24 h. GCs were passaged with54

0.1% bovine trypsin (Sigma-Aldrich) and sterile phosphate-buffered saline (PBS). GCs wereseeded at a density of 5.0 X 103 viable cells in 1 ml phenol red-free DMEM/F-12 to each wellof 24-well plates. The cells were incubated at 38.5oC with medium (v/v) containing 0.1% BSA,
5 µg/ml holotransferrin (Sigma-Aldrich), and 2 µg/ml insulin (Sigma- Aldrich) and treated with
(10, 50 and 100 ng/ml) IGF-1 for 0, 12, 24 and 48 h. In each group, the cells were mixed with
100-150 µl of DNA assay buffer in 1.5 ml tube and stored at -30°C for subsequent DNA assay.

55

STATISTICAL ANALYSISAll experimental data are shown as the mean ± SEM and normality andhomoscedasticity were confirmed using the Shapiro-Wilk test and Brown-Forsythe test using
R (88), respectively and analyzed by ANOVA followed by Tukey’s multiple comparison tests
using R. In Fig. 3, the raw data were subjected to a logarithmic transformation (LOG 10) toensure parametric properties, the two-way ANOWA test was performed among the two factors
at the same time. The statistical level of significance was considered at P<0.05.

56

RESULTS
Experiment 1. The IGF1R, PCNA, STAR, CYP11A1, HSD3B, CYP17A1 andHSD17B1 mRNA expression in uncultured granulosa cells
The three largest healthy follicles (i.e. well vascularization and having a transparent
follicular wall with fluid) were dissected from each pair of ovaries (Days 1–4 post-ovulation).
The IGF1R, PCNA, and steroidogenic-related enzymes mRNA expression in GCs from small(<6 mm), medium (7–8 mm), and large (≥9 mm) follicles were investigated. The IGF1R andCYP17A1 mRNA expression in the GCs was low in small follicles, slightly higher in medium
follicles, and highest in large follicles (Fig. 1A, B; p<0.05). The mRNA expressions of STAR,
HSD3B and HSD17B1 were significantly highest in medium follicles among these three groups(Fig. 1C, D, E). The PCNA and CYP11A1 mRNA expressions were higher in large follicles
compared to the small and medium follicles (Fig. 1F, G). The IGF1R mRNA expression wasslightly higher in GCs of large follicles than in medium and small follicles (Fig. 1A).

Experiment 2: Effects of IGF-1 on the mRNA expression of IGF1R, PCNA, andsteroidogenic-related enzymes in cultured granulosa cells
GCs were treated for 24 h with various doses of IGF-1. IGF-1 (10-100 ng/mL)
significantly increased (p<0.05) the mRNA expression of IGF1R, whereas the mRNA
expression of PCNA significantly increased at the dose of 100 ng/ml of IGF-1 (Fig. 2A, B).
However, the other doses of IGF-1 did not have any effect on the mRNA expression of PCNA
in GCs. IGF-1 (50-100 ng/ml) enhanced the mRNA expression of STAR and CYP11A1 while
IGF-1 (100 and 10-100 ng/ml) significantly increased the mRNA expression of HSD3B and57

CYP17A1, respectively (Fig. 2C, D, E, F; p<0.05). IGF-1 did not affect the mRNA expressionof HSD17B1 in cultured GCs of small follicles for 24 h (Fig. 2G).

Experiment 3: Effects of IGF-1 on granulosa cell proliferation
The 24 h cultured GCs were treated with IGF-1(10, 50 and 100 ng/ml) for 0, 12, 24 and48 h. IGF-1 (50-100 ng/ml) increased significantly (p<0.05) the proliferation of GCs at 48hcompared to 0 and 12 h of the incubation time base. In contrast, IGF-1 (100 ng/ml) significantly
increased the GC proliferation in comparison to the control group with a dose base. The otherdoses of IGF-1 did not affect the proliferation of GCs in small follicles (Fig. 3).

58

Table 2. Sequences of primers used for quantitative RT-PCR

Forward and reverse primers
Accession no.
Product size (bp) Temperature (oC)5'-GATCCCGTGTTCTTCTACGTTC-3'
IGF1R
X54980101595'-AAGCCTCCCACTATCAACAGAA-3'
5’AGGGCTTCGACACTTACCG-3'
PCNA
NM_176644.2
137
59
5’-CTTGGCAGGAATCAGGTAAT-3'
5'GAGAAGAGCTACGAGCTTCCTGACG-3'
ACTB
NM_173979.3
10660
5'-AGGATTCCATGCCCAGGAAGGAAGG-3'
5’-TCCCGGATGAGCCTTCCTAT-3’
HSD3B
NM_174343.3
116
58
5’-ACTAGGTGGCGGTTGAAGCA-3’5’-GACGTTGTGGTCAACCTGTG-3’
CYP17A1
NM_174304
142
59
5’-CTGCTCCAAAGGGCAAGTAG-3’
5’-CAACAGCAGAGAAGCTGGAAGACA-3’
STAR
NM_174189.3
133
60
5’-CACCAACAACAGTCTGGATTTCCCT-3’5'-TAAACCTGGTCACCGACTGC-3’
HSD17B1
NM_001035395.2
126
57
5'-CTGCCAGGGATGTAGGCAAA-3'
5’-TAAGCAAGATGCTGCAAATG-3’
CYP11A1
NM_176644.2
137
59
5’-CTTGGCAGGAATCAGGTAAT-3'
5'-CACCCTCAAGATTGTCAGCA-3’
GAPDH
NM_001034034.2
103
60
5'-GGTCATAAGTCCCTCCACGA-3'
Gene

59

60

Fig. 1. Gene expression of IGF1R (A), CYP17A1 (B), STAR (C), HSD3B (D), HSD17B1 (E),
PCNA (F), CYP11A1 (G) mRNA relative to the amount of GAPDH in isolated granulosa cells
from the three largest follicles in each pair ovary between Day 1 and Day 4 post-ovulation.
Expression values are presented as means ± SEM, n=3-5. Follicles were grouped by range asfollows: <6mm, 7-8 mm, and ≥9 mm in diameter. Different superscripts (a-b) indicate
significant differences (p< 0.05; ANOVA) followed by Tukey’s multiple comparison test.

61

62

Fig. 2. Effects of IGF-1 on mRNA expression in granulosa cells. The granulosa cells from smallfollicles were treated with various concentrations of IGF-1 (10, 50, and 100 ng/ml) for 24 hours.
The gene expression of IGF1R (A), PCNA (B), STAR (C), CYP11A1 (D), HSD3B (E), CYP17A1(F), HSD17B1 (G) mRNA, relative to the amount of ACTB in cultured granulosa cells fromsmall follicles (Cont, control; IGF-1, insulin-like growth factor-1). Expression values arepresented as means ± SEM, n=3-5. Different superscript letters indicate significant differences(p< 0.05; ANOVA) followed by Tukey’s multiple comparison test.

63* P< 0.01

Fig. 3. The cells proliferation of granulosa cells of small follicles. Granulosa cells were treatedwith various concentrations of IGF (50, and 100 ng/ml) for 0, 12, 24, and 48 hours. All values
are presented as means ± SEM, n=5. Asterisk indicate significant differences (p<0.01; two-way
ANOVA) followed by Tukey’s multiple comparison test.

64

DISCUSSION

The effects of IGF-1 on mRNA expressions of IGF1R, PCNA, steroidogenic enzymes(STAR, CYP11A1, CYP17A1, HSD17B1, and HSD3B), and cell proliferation were studied in
GCs isolated of small follicles. IGF1R is essential for the formation of preovulatory follicles,
the lack of IGF1R signaling in GCs of follicles that develop to the secondary stage fail to
respond to FSH (109) and the low FSH receptor expression is the reason for reduced follicle
growth in IGF-1 knockout mice (59). These findings show that IGF1R is crucial for follicular
growth and suggest that the effects of IGF-1 are controlled by IGF1R in GCs as previously
reported (39). Our results demonstrated that the expression of mRNA IGF1R increased withfollicle diameter and significantly increased the expression in fresh GCs of large follicles,
indicating that IGF1R is involved in the growth of all stages of dominant follicles. The
expression of mRNA IGF1R was lowest in GCs of small follicles. While the GCs of smallfollicles were treated with IGF-1, the mRNA expression of IGF1R was significantly increasedin GCs treated with IGF-1 for 24 h. Interestingly, the low dose of IGF-1 (10 ng/ml) increased
the mRNA IGF1R in GCs. These results align with the previous study, where IGF-1 (50 ng/ml)
increased IGF1R mRNA in bovine GCs isolated from follicles (2-8 mm) (72). Although theculture system and incubation period differed, taking together the present results support the
hypothesis that IGF-1 boosts IGF1R mRNA in GCs of small follicles which can mediate theeffect of exogenous IGF-1 in bovine follicles.
The success of studies on the initiation of follicular development depends on theavailability of sensitive markers to detect the onset of follicular growth. One such marker is
PCNA, a protein expressed by growing and proliferating cells (112). During follicular growth,
IGF-1 works in synergy with FSH and LH to regulate the proliferation and differentiation of
granulosa cells (119, 120). PCNA is an essential protein found in proliferating eukaryotic cells
65and plays crucial roles in DNA replication, repair, and control of cell proliferation (121). The
present study showed that the mRNA expressions of PCNA were lower in GCs of small follicles
compared to large follicles. Interestingly, IGF-1 (100 ng/ml) significantly increased PCNA
mRNA expression in cultured GCs of small follicles. These results are similar to a previous
study conducted on human primary follicles, where IGF-1 induced the expression of PCNA
mRNA in GCs of primary follicles (122). This result indicates that IGF-1 enhances the levelsof PCNA mRNA in GCs of small follicles, which play a key role in the proliferation of GCs
during follicular growth. This implies that the effects of IGF-1 on proliferation may be due tothe enhanced mRNA expression of PCNA.
IGF-1 is potent and regulates A4 production, the main substrate for E2 essential forfollicular growth (59). In the present study, we did not measure the A4 production. Our previous
studies showed that IGF-1 increased A4 production in GCs (59) and TCs (118) of smallfollicles. In the present study, we observed the expression of mRNA for steroidogenic enzymes
in fresh granulosa cells isolated from small, medium, and large-sized follicles. We found thatthe expressions of STAR, CYP11A1, HSD3B, CYP17A1, and HSD17B1 mRNA were lower in
the GCs of small follicles than in medium and large-sized follicles. Next, we examined theeffect of IGF-1 on the mRNA expression of mentioned steroidogenic enzymes. IGF-1 enhanced
the expressions of mRNA for STAR in GCs of small follicles and is responsible for cholesteroltransport to the inner layer of mitochondria. This result supports the previous study that IGF-1
significantly increased the expression of STAR mRNA in cultured follicles of mice (74). IGF-1(50-100 ng/ml) increased CYP11A1 mRNA expression in cultured GCs of small follicles, whichconvert the cholesterol of pregnenolone. This result is also consistent with previous reports thatIGF-1 increased the CYP11A1 mRNA in the GCs of porcine (98). The next step for A4 synthesisis the conversion of pregnenolone to DHEA which is regulated by CYP17A1(71). In the present
study, IGF-1 increased the expression of mRNA of CYP17A1 in cultured GCs of small follicles,66but IGF-1 did not affect the expression of HSD17B1 mRNA. IGF-1 significantly elevated
HSD3B in cultured GCs of small follicles which regulate the conversion of DHEA to A4. These
results agree with past studies that IGF-1 increased HSD3B in TCs of rats (123), in bovine GCs(30), and in bovine TCs (118). The A4 is mainly synthesized in TCs under the stimulation of
LH while the GCs also play a role in the synthesis of A4 (35). The GCs produce only a small
amount of A4 (124). Previous reports indicate that the bovine follicle wall, containing both TCs
and GCs, produced significantly higher quantities of A4 than did isolated TCs (125). The
minimal secretion of A4 by GCs suggests that GCs somehow enhance the ability of theca cells
to produce A4, the substrate for E2 synthesis (126). Taken together, these findings show thatIGF-1 enhances the mRNA expression of steroidogenic enzymes that regulate E2 and A4
production, leading to follicular growth and selection (18, 27).
IGFs comprise a family of peptides that promote cell proliferation, and differentiationand produce insulin-like metabolic effects. In a previous study, the CGs of large follicles weretreated with IGF-1 for 2 days to check whether IGF-1 enhances the proliferation of GCs in vitro,and the results showed that IGF-1 (100 ng/ml) significantly increased the number of GCs
compared to the control group (73). In the present study, the GCs of small follicles were treatedwith IGF-1 for 12, 24, and 48 hours. IGF-1 (100 ng/ml) significantly increased the proliferationof GCs at 48-hour incubation. These results are consistent with the above reports and indicate
the importance of IGF-1 in follicular growth.
In conclusion, IGF-1 increased the expression of IGF1R mRNA which can mediate thebioactivity of IGF-1 in GCs. IGF-1 up-regulated key steroidogenic enzymes such as STAR,
CYP11A1, HSD3B, and CYP17A1 mRNA which are essential for A4 synthesis, a crucial
substrate for E2 biosynthesis. IGF-1 Enhanced the mRNA expression of PCNA and increased
the proliferation percentage of GCs of small follicles. These results indicate that IGF-1 has an
excellent capacity to enhance factors that are involved in follicular growth. Further investigation67

is required to confirm whether IGF-1 directly increases the mRNA expression of PCNA orindirectly through the enhanced IGF1R mRNA in GCs of bovine follicles.

68

SUMMARY

Insulin-like growth factor-1 (IGF-1) hormone plays a crucial role in follicular growth,
antral formation, and steroidogenesis. IGF-1 is known to play a key role in the acquisition andmaintenance of functional dominance. However, the biological effect of IGF-1 is a topic ofinterest, whether it is managed by IGF-1 receptors (IGF1R) or IGF-binding proteins. We
examined the expression of IGF1R, PCNA, STAR, CYP11A1, CYP17A1, HSD17B1 and HSD3B
mRNA in uncultured granulosa cells (GCs) of the three largest follicles from paired ovaries andthe effects of various concentrations (10, 50, and 100 ng/ml) of IGF-1 on the expression of
IGF1R, STAR, CYP11A1, CYP17A1, HSD17B1 and HSD3B mRNA in cultured GCs. We also
investigated the effects of IGF-1 on the proliferation of GCs. Small follicles (<6 mm) expressedsignificantly lower IGF1R, PCNA, and steroidogenic enzymes than large follicles (≥9 mm)(p<0.05). IGF-1 (10-100 ng/ml) significantly increased IGF1R mRNA in cultured GCs. IGF-1(50-100 ng/ml) increased STAR, CYP11A1 mRNA, and IGF-1 (10-100 ng/ml) enhanced
CYP17A1 mRNA in GCs. IGF-1 had no effect on HSD17B1 mRNA expression in GCs. IGF-1(100 ng/ml) increased the HSD3B mRNA in GCs of small follicles (p < 0.05). Interestingly,
IGF-1 (100 ng/ml) significantly increased the PCNA mRNA expression in cultured GCs and
GC number of small follicles. These results showed that IGF-1 increased IGF1R mRNA in GCs
which may mediate the action of IGF-1 required for the follicular growth. IGF-1 increased thesteroidogenic enzyme mRNA which are essential for E2 synthesis and IGF-1 increased the GC
number. Overall, these findings indicate that IGF-1 is critical for follicular growth and selection.

69

CHAPTER 4
CONCLUSION
The present study investigated the effects of IGF-1 on the factors involved in follicular
growth and selection in cattle. Both granulosa cells (GCs) and theca cells (TCs) from small andmedium-sized follicles were used in the present study. Based on the findings, the first series ofexperiments demonstrated that IGF-1 plays a significant role in enhancing the expression ofmRNA steroidogenic-related enzymes and promoting the steroid hormone (A4 and P4)
production in TCs substrate for E2 production. Furthermore, IGF-1 enhanced the FSHR,
CYP19A1 and ESR2 mRNA, suggesting its involvement in mediating the synthesis andbioactivity of E2 in GCs of small follicles. These findings emphasize that IGF-1 plays a crucialrole in regulating the complex molecular processes related to steroids and the development offollicles. The second series of experiments using GCs of small follicles indicated that IGF-1
enhanced other factors involved in follicular growth including mRNA expression of IGF1R,
PCNA, STAR, SYP11A1, CYP17A1, HSD3B, and promoting cell proliferation. These findings
underscore the involvement of IGF-1 in fostering the factors crucial for the growth of follicles
and indicate how IGF-1 controls the complicated processes involved in follicle development.
Overall results suggested that IGF-1 plays a critical role in promoting various factors
essential for follicular growth. In TCs, it enhances the expression of steroidogenic enzymes andhormones, while in GCs, it boosts factors such as IGF1R, PCNA, steroidogenesis enzymes
mRNA, and cell proliferation. These findings highlight that IGF-1 actively regulates important
processes in both cell types, contributing to follicular growth and selection.

70Abbreviation

Complete word (s)A4Androstenedione

ANOVA

Analysis of variance

ACTB

β-actin

BSA

Bovine serum albumin

CYP17A1

Cytochrome P450 17A1

CYP19A1

Cytochrome P450 aromatase

CYP11A1

P450 side-chain cleavage

COC

Cumulus-oocyte complexes

CDH1

E-cadherin

CDH2

N-cadherin

CL

Corpus luteum

CO2

Carbon dioxideoC

Degree Celsius

DHEA

dehydroepiandrosterone

DMEM

Dulbecco’s modified Eagle’s medium

DNA

Deoxyribonucleic acid

EIA

Enzyme immune assay

EMT

Epithelial-to-mesenchymal transition

E2

Estradiol 17β

ER

Estrogen receptor

ESR1

Estrogen receptor α

ESR2

Estrogen receptor β

FBS

Fetal bovine serum

FSHR

Follicle-stimulating hormone receptor

71

FSH

Follicle simulating hormone

Fig

FigureGAPDH

Glyceraldehyde 3-phosphate dehydrogenase

GCs

Granulosa cell

GnRH

gonadotropin-releasing hormone

HSD17B17β hydroxysteroid dehydrogenase

HSD3B

3β hydroxysteroid dehydrogenase

H2SO4

Sulphuric acid

HRP

Horseradish peroxidase

IGF-1

Insulin-like growth factor-1

IGFBP

IGF binding protein

IGF1R

Insulin-like growth factor-1 receptor

LH

Luteinizing hormone

LHCGR

Luteinizing hormone receptorml

Mili literµl

Microliterµg

Micro grammm

Mili meter diametermRNA

messenger RNA (ribonucleic acid)ng

Nanogram

P4

Progesterone

PBS

Phosphate-buffered saline

P450SCC (CYP11A1)

P450 side chain cleavage

PGE2

Prostaglandin E2

72

PCNA

Proliferative cell nuclear antigen agent

RT-PCR

Reverse Transcription Polymerase Chain
Reactionrpm

Revolution (s) per minute

StAR

Steroidogenic acute regulatory

SEM

Standard error of the mean

SDStandard deviation

T

Testosterone

TCs

Theca cell73

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