Abstract

This review aims to discuss the main factors involved in the development of early antral follicles until gonadotropin dependence. This follicular phase is characterized by intense proliferation of granulosa cells, formation of a fluid-filled cavity, morphological differentiation of cumulus cells, mural granulosa cells and recruitment of theca cells. The interaction between oocyte, granulosa and theca cells is crucial for follicular growth and hormone production. Growth factors produced by the oocyte, such as growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), regulate granulosa cell proliferation and differentiation and antral cavity development, as well as stimulate the production of follicle-stimulating hormone (FSH) receptors in granulosa cells. In response to FSH, granulosa cells secrete C-type natriuretic peptide (CNP), which acts through its receptor to increase cyclic guanosine monophosphate (cGMP) production and consequently follicular development. Granulosa cells also produce insulin-like growth factor-1 (IGF-1) and increase aromatase enzyme activity, which results in greater sensitivity to gonadotropins and follicular steroidogenesis. The absence of IGF-1 signaling causes cessation of follicular growth at the early antral stage. Many other local factors are involved in the regulation of follicular development. Therefore, this review brings relevant data for a better understanding of the mechanisms involved in the control of early antral follicle growth, emphasizing the role of endocrine and paracrine factors, the oocyte-granulosa cell interaction and the processes of follicular atresia. The challenges for the establishment of efficient culture systems for in vitro growth of early antral follicles are also discussed.

Keywords:
Oocyte; granulosa cells; theca cells; gonadotropins; cumulus cells; folliculogenesis

Resumo

Esta revisão tem como objetivo discutir os principais fatores envolvidos no desenvolvimento de folículos antrais iniciais até a dependência de gonadotrofinas. Essa fase folicular é caracterizada por intensa proliferação de células da granulosa, formação de uma cavidade preenchida por líquido, diferenciação morfológica das células do cumulus, células da granulosa murais e recrutamento de células da teca. A interação entre oócito, células da granulosa e da teca é determinante para o crescimento folicular e produção hormonal. Fatores de crescimento produzidos pelo oócito, fator de crescimento e diferenciação-9 (GDF-9) e proteína morfogenética óssea-15 (BMP-15), regulam a proliferação e diferenciação de células da granulosa, e o desenvolvimento da cavidade antral, bem como estimulam a produção de receptores do hormônio folículo estimulante (FSH) nas células da granulosa. Em resposta ao FSH, as células da granulosa secretam o peptídeo natriurético tipo C (CNP), que atua através de seu receptor para aumentar a produção de monofosfato de guanosina cíclico (GMPc) e consequentemente o desenvolvimento folicular. As células da granulosa também produzem o fator de crescimento semelhante à insulina 1 (IGF-1) e aumentam a atividade da enzima aromatase, o que resulta em maior sensibilidade às gonadotrofinas e esteroidogênese folicular. A ausência de sinalização do IGF-1 causa cessação do crescimento folicular no início do estágio antral. Muitos outros fatores locais estão envolvidos na regulação do desenvolvimento folicular. Por tanto essa revisão traz dados relevantes para uma melhor compreensão dos mecanismos envolvidos no controle do crescimento de folículos antrais iniciais, enfatizando o papel dos fatores endócrinos e parácrinos, a interação oócito-células da granulosa e os processos de atresia folicular. Os desafios para o estabelecimento de sistemas de cultivo eficientes para o crescimento in vitro de folículos antrais iniciais também são discutidos.

Palavras-chave:
Oócito; células da granulosa; células da teca; gonadotrofinas; células do cumulus; foliculogênese

1. Introduction

Oocyte developmental competence refers to the ability of a female gamete to mature, to be fertilized, and to support embryonic development until the blastocyst stage(¹1 Sirait B, Wiweko B, Jusuf AA, Iftitah D, Muharam R. Oocyte competence biomarkers associated with oocyte maturation: Frontiers in Cell and Developmental Biology. 2021. 30(9): 710292. DOI: https://doi.org/10.3389/fcell.2021.710292
https://doi.org/10.3389/fcell.2021.71029... ). According to Dode et al.(²2 Dode MAN, Rodovalho NC, Ueno VG, Alves RGO. Effect of follicle size in nuclear and cytoplasmic maturation of oocytes from zebu cows, Pesquisa Agropecuaria Brasileira. 2000; 35: 207-214. DOI: https://doi.org/10.1590/S0100-204X2000000100023
https://doi.org/10.1590/S0100-204X200000... ), this competence is acquired gradually during preantral and early antral follicular growth. To reinforce this information, oocytes from 3.0 mm antral follicles are able to complete nuclear maturation in vitro, while those from smaller follicles (1.0 and 2.0 mm) have reduced competence(³3 Pavlok A, Lucas-Hahn A, Niemann H. Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles. Molecular Reproduction and Development. 1992; 31: 63-67. DOI: https://doi.org/10.1002/mrd.1080310111
https://doi.org/10.1002/mrd.1080310111... ,⁴4 Nemcová L, Hulínska P, Ješeta M, Kempisty B, Kanka J, Machatková M. Expression of selected mitochondrial genes during in vitro maturation of bovine oocytes related to their meiotic competence. Theriogenology. 2019; 133: 104-112. DOI: https://doi.org/10.1016/j.theriogenology.2019.05.001
https://doi.org/10.1016/j.theriogenology... ). This non-competence of oocytes from small antral follicles is due to the reduced expression of genes that activate signaling pathways to increase the oocyte’s ability to respond to the increase in gonadotropins(⁵5 Varmosfaderani SR, Hajian M, Jafarpour F, Zadegan FG, Nasr-Esfahani MH. Granulosa secreted factors improve the developmental competence of cumulus oocyte complexes from small antral follicles in sheep. PloS one. 2020; 15: e0229043. DOI: https://doi.org/10.1371/journal.pone.0229043
https://doi.org/10.1371/journal.pone.022... ). Responsiveness to gonadotropins enables the follicles to grow until selection and dominance(⁶6 Oliveira MEF, Ferreira RM, Mingoti GZ. Local and systemic control of bovine follicular growth and selection. Revista Brasileira de Reprodução Animal. 2011; 35: 418-432. Disponível em http://www.cbra.org.br
http://www.cbra.org.br... ,⁷7 Silva JR, Figueiredo JR, Van Den Hurk R. Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis. Theriogenology. 2009; 71(8):1193-208. DOI: https://doi.org/10.1016/j.theriogenology.2008.12.01
https://doi.org/10.1016/j.theriogenology... ).

In the course of follicular development, proliferation and morphological differentiation of granulosa cells are of great importance to prepare the follicle to respond to gonadotropins and to create a favorable environment for oocyte development(⁸8 Baumgartem SC, Stocco C. Granulosa Cells. Encyclopedia of Reproduction. 2018; 2: 8-13. DOI: https://doi.org/10.1016/B978-0-12-801238-3.64623-8
https://doi.org/10.1016/B978-0-12-801238... ). Granulosa cells produce several autocrine and paracrine factors that may be involved in oocyte growth and antrum formation(⁹9 Vasconcelos GL, Saraiva MVA, Costa JJN, Passos MJ, Silva AWB, Rossi RODS, et al. Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles. Reproduction, Fertility and Development. 2012; 25: 1194-1203. DOI: http://dx.doi.org/10.1071/RD12173
http://dx.doi.org/10.1071/RD12173... ). Additionally, oocyte-derived factors stimulate the expression of follicle-stimulating hormone receptors (FSHR) in granulosa cells, enabling them to become responsive to gonadotropins(¹⁰10 Kim JW, Kang KM, Yoon TK, Shim SH, Lee WS. Study of circulating hepcidin in association with iron excess, metabolic syndrome, and BMP-6 expression in granulosa cells in women with polycystic ovary syndrome. Fertility and Sterility. 2014;102(2):548-554.e2. DOI: https://doi.org/10.1016/j.fertnstert.2014.04.031
https://doi.org/10.1016/j.fertnstert.201... ). The follicle-stimulating hormone (FSH) induces proliferation and viability of the oocyte-cumulus-granulosa complex and may also induce granulosa cell differentiation(¹¹11 Sakaguchi K, Huang W, Yang Y, Yanagawa Y, Nagano M. Relationship between in vitro growth of bovine oocytes and steroidogenesis of granulosa cells cultured in medium supplemented with bone morphogenetic protein-4 and follicle stimulating hormone. Theriogenology. 2017; 97: 113-123. DOI: http://dx.doi.org/10.1016/j.theriogenology.2017.04.030.
http://dx.doi.org/10.1016/j.theriogenolo... ). Furthermore, oocyte-derived factors also stimulate antral cavity formation by increasing the expression proteoglycans, as a result of interaction with FSH(⁹9 Vasconcelos GL, Saraiva MVA, Costa JJN, Passos MJ, Silva AWB, Rossi RODS, et al. Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles. Reproduction, Fertility and Development. 2012; 25: 1194-1203. DOI: http://dx.doi.org/10.1071/RD12173
http://dx.doi.org/10.1071/RD12173... ). Thus, understanding the endocrine, paracrine and autocrine mechanisms that control the interaction between follicular cells and the oocyte during early antral follicles favors the development of strategies to promote their development in vitro(¹²12 Paulino LRFM, de Assis EIT, Azevedo VAN, Silva BR, da Cunha EV, Silva JRV. Why Is It So Difficult To Have Competent Oocytes from In vitro Cultured Preantral Follicles? Reproductive Sciences. 2022; 29(12):3321-3334. DOI: https://doi.org/10.1007/s43032-021-00840-8
https://doi.org/10.1007/s43032-021-00840... ).

The present review provides an overview of the main factors that control the development of early antral follicles up to gonadotropin dependence, i.e., regulation of granulosa cell proliferation, steroidogenesis, atresia, interaction between oocyte and granulosa cells, as well as strategies to promote the development of early antral follicles in vitro.

2. Endocrine control of early antral follicle development

Follicle development from the preantral to the early antral stage is mainly controlled by intraovarian regulators, but it can be stimulated by FSH. The specific receptors for FSH are expressed in granulosa cells of secondary and early antral follicles(¹³13 Pangas SA, Rajkovic A. Matzuk MM. CHAPTER 10 - Follicular development: mouse, sheep, and human models. In: Jimmy D. Neill, Knobil and Neill’s Physiology of Reproduction (Third Edition), Academic Press, 2006, pages 383-423, ISBN 9780125154000.). When secondary follicles are formed, granulosa cells express FSHR, and theca cells express the luteinizing hormone receptor (LHR)(¹⁴14 Orisaka M, Jiang JY, Orisaka S, Kotsuji F, Tsang BK. Growth differentiation factor 9 promotes rat preantral follicle growth by up-regulating follicular androgen biosynthesis. Endocrinology. 2009; 150: 2740-2748. DOI: https://doi.org/10.1210/en.2008-1536
https://doi.org/10.1210/en.2008-1536... ). In domestic and human species, antrum formation is observed when the follicles have around 0.2 mm(¹⁵15 Gougeon A. Dynamics of follicular growth in the human: A model from preliminary results. Human Reproduction. 1986; 1: 81-87. DOI: https://doi.org/10.1093/oxfordjournals.humrep.a136365
https://doi.org/10.1093/oxfordjournals.h... ) and become dependent on gonadotropin when they reach 3.0 mm in cow(¹⁶16 Campbell BK, Scaramuzzi RJ and Webb R. Control of antral follicle development and selection in sheep and cattle. Journal Reproduction Fertility, Supplement, 1995; 49: 335–350. DOI: https://hdl.handle.net/102.100.100/231568?index=1
https://hdl.handle.net/102.100.100/23156... ), 4.0 mm in sheep(¹⁷17 Webb RB, Nicholas JG, Gong BK, Campbell CG, Gutierrez HA, Garverick DG. Mechanism regulating follicular development and selection of the dominant follicle. Reproduction, Supplement. 2003; 23: 123- 234. DOI: https://cir.nii.ac.jp/crid/1573387450361202816
https://cir.nii.ac.jp/crid/1573387450361... ), 3.0 mm in goat(¹⁸18 Berlinguer F, Leoni GG, Succu S, Spezzigu A, Madeddu M and Satta V. Exogenous melatonin positively influences follicular dynamics, oocyte developmental competence and blastocyst output in a goat model. Journal of Pineal Research. 2009; 46: 383-391. doi https://doi.org/10.1111/j.1600-079X.2009.00674.x
https://doi.org/10.1111/j.1600-079X.2009... ) and 5.0 mm in human(¹⁹19 Zeleznik AJ, Plant TM. Control of the Menstrual Cycle. Knobil and Neill’s Physiology of Reproduction 2014: 1307 1362. DOI: https://doi.org/10.1016/B978-0-12-571136-4.50008-5
https://doi.org/10.1016/B978-0-12-571136... ). Follicular growth and maturation beyond this stage, which includes follicle recruitment, selection, dominance, and ovulation, is gonadotropin-dependent(²⁰20 Kumar TR, Wang Y, Lu N, Matzuk MM. Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nature Genetics.1997; 15: 201- 204. DOI: https://doi.org/10.1038/ng0297-201
https://doi.org/10.1038/ng0297-201... ,²¹21 McGee EA, Hsueh AJ. Initial and cyclic recruitment of ovarian follicles. Endocrinology Review. 2000; 21: 200-214. DOI: https://doi.org/10.1210/edrv.21.2.0394
https://doi.org/10.1210/edrv.21.2.0394... ). Acquisition of FSH dependence during this interval of growth is crucial to determining follicular fate, i.e., growth or atresia. The C-type natriuretic peptide (CNP) is secreted by granulosa cells of secondary and antral follicles in response to FSH stimulation. CNP acts through its receptor (NPRB) expressed in granulosa cells of secondary follicles and increases Guanosine 3’,5’-cyclic monophosphate (cGMP) production to stimulate follicle development(³³33 Sato Y, Cheng Y, Kawamura K, Takae S, Hsueh AJ.. C-type natriuretic peptide stimulates ovarian follicle development. Molecular endocrinology.2012, 26(7), 1158-1166. DOI: https://doi.org/10.1210/me.2012-1027
https://doi.org/10.1210/me.2012-1027... ). Gene expression analyses indicated increases in transcripts for CNP receptors (NPP and NPRB) during early folliculogenesis in mice, in association with increases in ovarian CNP peptides(³³33 Sato Y, Cheng Y, Kawamura K, Takae S, Hsueh AJ.. C-type natriuretic peptide stimulates ovarian follicle development. Molecular endocrinology.2012, 26(7), 1158-1166. DOI: https://doi.org/10.1210/me.2012-1027
https://doi.org/10.1210/me.2012-1027... ) (Figure 1).

Figure 1.
Factors that regulate the development of preantral follicles up to gonadotropin-dependent stages.

Growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein 15 (BMP-15), both secreted by the oocyte, promote proliferation of granulosa cells and the recruitment of theca cells, events that are required for the transition of follicles from the primary to the secondary stage(³⁴34 Alam Md, Hasanur JL, Takashi M. GDF9 and BMP15 induce development of antrum-like structures by bovine granulosa cells without oocytes. The Journal of Reproduction and development. 2018; 64(5): 423-431. DOI: http://doi.org/10.1262/jrd.2018-078
http://doi.org/10.1262/jrd.2018-078... ). Factors produced by secondary follicles, including vascular endothelial growth factor (VEGF), transforming growth factor (TGF), insulin-like growth factor (IGF), fibroblast growth factor-2 and -7 (FGF-2 and FGF-7), BMPs and activin, are necessary for survival and further development. At the antral stage, locally synthesized peptides play a key role in the regulation of follicular development through endocrine and paracrine mechanisms(¹⁷17 Webb RB, Nicholas JG, Gong BK, Campbell CG, Gutierrez HA, Garverick DG. Mechanism regulating follicular development and selection of the dominant follicle. Reproduction, Supplement. 2003; 23: 123- 234. DOI: https://cir.nii.ac.jp/crid/1573387450361202816
https://cir.nii.ac.jp/crid/1573387450361... , ³⁵35 Reinen PS, Coria MS, Barrionuevo MG, Hernández O, Callejas S, Palma GA. Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells/Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells. MVZ Córdoba. 2018; 3(23): 6778-6787. DOI: http://doi.org/10.21897/rmvz.1367.
http://doi.org/10.21897/rmvz.1367... ). Among these peptides, those of the IGF system, including IGF-1, IGF-2 and the IGF binding proteins (IGFBPs) and some members of the FGF family, such as FGF-2, FGF-7 (or KGF), FGF-8 and FGF-10(³⁵35 Reinen PS, Coria MS, Barrionuevo MG, Hernández O, Callejas S, Palma GA. Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells/Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells. MVZ Córdoba. 2018; 3(23): 6778-6787. DOI: http://doi.org/10.21897/rmvz.1367.
http://doi.org/10.21897/rmvz.1367... ,³⁶36 Morikawa R, Jibak L,Takashi M. Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte–cumulus cell complexes in vitro. The Journal of Reproduction and Develoment. 2021; 67(4): 273-281. DOI: http://doi.org/10.1262/jrd.2021-026
http://doi.org/10.1262/jrd.2021-026... ), appear to be critical for late-stage follicle development (Figure 1).

Fushii et al.(²²22 Fushimi Y, Takagi M, Monniaux D, Uno S, Kokushi E, Shinya U. Effects of dietary contamination by zearalenone and its metabolites on serum anti-Müllerian hormone: Impact on the reproductive performance of breeding cows. Reproduction. 2021; 50: 834-839. DOI: https://doi.org/10.1111/rda.12599
https://doi.org/10.1111/rda.12599... ) recently showed that cumulus-oocyte complexes (COCs) cultured with FSH have the antral cavity formed one day earlier than those that do not receive this hormone, showing the importance of FSH in follicular development. Intraovarian regulators, IGF, activin, oocyte-derived factors, and gap junction membrane channel protein play a central role in the acquisition of FSH dependence at the early antral stage of follicle development(¹³13 Pangas SA, Rajkovic A. Matzuk MM. CHAPTER 10 - Follicular development: mouse, sheep, and human models. In: Jimmy D. Neill, Knobil and Neill’s Physiology of Reproduction (Third Edition), Academic Press, 2006, pages 383-423, ISBN 9780125154000.). Thecaderived androgens bind to androgen receptors (ARs) in granulosa cells(²³23 Tetsuka M, Whitelaw PF, Bremner WJ, Millar MR, Smyth CD, Hillier SG. Developmental regulation of androgen receptor in rat ovary. Journal of Endocrinology. 1995; 145: 535-543. DOI: https://doi.org/10.1677/joe.0.1450535
https://doi.org/10.1677/joe.0.1450535... ), thereby inducing FSHR expression and follicle growth during the preantral-to-antral transition(¹⁴14 Orisaka M, Jiang JY, Orisaka S, Kotsuji F, Tsang BK. Growth differentiation factor 9 promotes rat preantral follicle growth by up-regulating follicular androgen biosynthesis. Endocrinology. 2009; 150: 2740-2748. DOI: https://doi.org/10.1210/en.2008-1536
https://doi.org/10.1210/en.2008-1536... , ²⁴24 Vendola KA, Zhou J, Adesanya OO, Weil SJ, Bondy CA. Androgens stimulate early stages of follicular growth in the primate ovary. Journal of Clinical Investigation. 1998; 101: 2622-2629. DOI: https://doi.org/10.1172/JCI2081
https://doi.org/10.1172/JCI2081... , ²⁵25 Wang H, Andoh K, Hagiwara H. Effect of adrenal and ovarian androgens on type 4 follicles unresponsive to FSH in immature mice. Endocrinology. 2001; 142: 4930-4936. DOI: https://doi.org/10.1210/endo.142.11.8482
https://doi.org/10.1210/endo.142.11.8482... ). The AR deficiency in the mice ovary induces granulosa cell apoptosis, arrests antral follicle growth and results in premature ovarian failure(²⁶26 Shiina H, Matsumoto T, Sato T. Premature ovarian failure in androgen receptor-deficient mice. Proceedings of the National Academy of Sciences. 2006; 103: 224-229. DOI: https://doi.org/10.1073/pnas.0506736102
https://doi.org/10.1073/pnas.0506736102... ,²⁷27 Sen A, Hammes SR. Granulosa cell-specific androgen receptors are critical regulators of ovarian development and function. Molecular Endocrinology.2010; 24: 1393-1403. DOI: https://doi.org/10.1210/me.2010-0006
https://doi.org/10.1210/me.2010-0006... ,²⁸28 Walters KA, Middleton LJ, Joseph SR. Targeted loss of androgen receptor signaling in murine granulosa cells of preantral and antral follicles causes female subfertility. Molecular Biology Reports. 2012; 87: 151. DOI: https://doi.org/10.1095/biolreprod.112.102012
https://doi.org/10.1095/biolreprod.112.1... ). Thus, androgens play an important role in the growth, survival and acquisition of FSH dependence in early antral follicles(¹³13 Pangas SA, Rajkovic A. Matzuk MM. CHAPTER 10 - Follicular development: mouse, sheep, and human models. In: Jimmy D. Neill, Knobil and Neill’s Physiology of Reproduction (Third Edition), Academic Press, 2006, pages 383-423, ISBN 9780125154000.) (Figure 1).

Anti-Müllerian hormone (AMH) is a product of granulosa cells from small antral follicles onwards that has an inhibitory or retarding role in the development of antral follicles. The AMH reduces follicle sensitivity to FSH, decreasing the expression of FSHR. It inhibits cyclic FSH-dependent recruitment and appears to play a role in all gonadotropin-independent follicular growth. Despite having a regulatory relationship between androgens and AMH, it is not possible to guarantee that their effects are mediated by estradiol, via testosterone aromatization(²⁹29 Dewailly D, Robin G, Peigne M, Decanter C, Pigny P, Catteau-Jonard S. Interactions between androgens, FSH, anti-Müllerian hormone and estradiol during folliculogenesis in the human normal and polycystic ovary. Human Reproduction Update.2016; 22: 709-724. DOI: https://doi.org/10.1093/humupd/dmw027
https://doi.org/10.1093/humupd/dmw027... ) (Figure 1).

Melatonin is found in the follicular fluid of human antral follicles and has important roles in the control of follicle development(³⁰30 Khan H L, Bhatti S, KhanYL, Abbas S, Munir Z, Sherwani IAR et al. Cell-free nucleic acids and melatonin levels in human follicular fluid predict embryo quality in patients undergoing in-vitro fertilization treatment. J. Gynecol. Obstet. Human Reproduction. 2020; 49: (1), 101624. DOI: https://doi.org/10.1016/j.jogoh.2019.08.007
https://doi.org/10.1016/j.jogoh.2019.08.... ). Its receptors have previously been detected in granulosa cells of preantral and antral follicles(³¹31 Barros VRP, Cavalcante AYP, Macedo TJS, Barberino RS, Lins TLB, Gouveia BB et al. Immunolocalization of melatonin and follicle-stimulating hormone receptors in caprine ovaries and their effects during in vitro development of isolated pre-antral follicles. Reproduction. 2013; 48: 1025–1033. DOI: https://doi.org/10.1111/rda.12209
https://doi.org/10.1111/rda.12209... ). Regarding the effects of melatonin, Barros et al.(³²32 Barros VRP, Monte APO, Santos JMS, Lins TLBG, Cavalcante, AYP, Gouveia, BB. Melatonin improves development, mitochondrial function and promotes the meiotic resumption of sheep oocytes from in vitro grown secondary follicles. Theriogenology. 2020; 144: 67-73. DOI: https://doi.org/10.1016/j.theriogenology.2019.12.006
https://doi.org/10.1016/j.theriogenology... ) demonstrated that this hormone is associated with meiotic competence of oocytes from early antral follicles. Melatonin maintains follicular survival, stimulates antral cavity formation and subsequent follicular and oocyte growth, as well as increases glutathione and metabolically active mitochondria levels after in vitro culture of sheep secondary follicles(³²32 Barros VRP, Monte APO, Santos JMS, Lins TLBG, Cavalcante, AYP, Gouveia, BB. Melatonin improves development, mitochondrial function and promotes the meiotic resumption of sheep oocytes from in vitro grown secondary follicles. Theriogenology. 2020; 144: 67-73. DOI: https://doi.org/10.1016/j.theriogenology.2019.12.006
https://doi.org/10.1016/j.theriogenology... ).

3. Oocyte-granulosa cell interaction during early antral follicle development

It is well known that during follicular development; members of the transforming growth factor beta (TGFβ) family and their receptors are involved in the control of oocyte growth and granulosa cell proliferation. Oocyte-derived TGFβ family members, such as GDF-9 and BMP-15, regulate granulosa cell proliferation and differentiation, as well as the development of the antral cavity(³³33 Sato Y, Cheng Y, Kawamura K, Takae S, Hsueh AJ.. C-type natriuretic peptide stimulates ovarian follicle development. Molecular endocrinology.2012, 26(7), 1158-1166. DOI: https://doi.org/10.1210/me.2012-1027
https://doi.org/10.1210/me.2012-1027... ,³⁴34 Alam Md, Hasanur JL, Takashi M. GDF9 and BMP15 induce development of antrum-like structures by bovine granulosa cells without oocytes. The Journal of Reproduction and development. 2018; 64(5): 423-431. DOI: http://doi.org/10.1262/jrd.2018-078
http://doi.org/10.1262/jrd.2018-078... ). Additionally, recent studies indicate that these factors regulate the expression of mRNA for LHR in cumulus cells(³⁵35 Reinen PS, Coria MS, Barrionuevo MG, Hernández O, Callejas S, Palma GA. Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells/Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells. MVZ Córdoba. 2018; 3(23): 6778-6787. DOI: http://doi.org/10.21897/rmvz.1367.
http://doi.org/10.21897/rmvz.1367... ). Oocyte-derived GDF-9 promotes the growth of cumulus-oocyte (COCs), while BMP-15 induces the expression of choriogonadotropin receptor mRNA (LHCGR) in cumulus cells, and FSH receptor expression in follicles. Such factors contribute to follicular development and oocyte maturation(³⁵35 Reinen PS, Coria MS, Barrionuevo MG, Hernández O, Callejas S, Palma GA. Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells/Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells. MVZ Córdoba. 2018; 3(23): 6778-6787. DOI: http://doi.org/10.21897/rmvz.1367.
http://doi.org/10.21897/rmvz.1367... ,³⁶36 Morikawa R, Jibak L,Takashi M. Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte–cumulus cell complexes in vitro. The Journal of Reproduction and Develoment. 2021; 67(4): 273-281. DOI: http://doi.org/10.1262/jrd.2021-026
http://doi.org/10.1262/jrd.2021-026... ). GDF-9 and BMP-15 bind to type II BMP receptor(³⁷37 Ma X, Huashan Y. BMP15 regulates FSHR via TGF-ß II receptor and SMAD4 signaling in the prepubertal ovary of Rongchang pigs. Research in Veterinary Science. 2022; 143: 66-73. DOI: http://doi.org/10.1016/j.rvsc.2021.12.013
http://doi.org/10.1016/j.rvsc.2021.12.01... ) and recruit type I activin-like kinase (ALK)5(³⁸38 Vitt UA, Mazerbourg S, Klein C, Hsueh AJ. Bone morphogenetic protein receptor type II is a receptor for growth differentiation factor-9. Biology of reproduction. 2002; 67: 473-480. DOI: http://doi.org/10.1095/biolreprod67.2.473
http://doi.org/10.1095/biolreprod67.2.47... ) and ALK6(³⁹39 Mazerbourg S, Klein C, Roh J, Kaivo-Oja N, Mottershead DG, Korchynskyi O, et al. Growth differentiation factor-9 signaling is mediated by the type I receptor, activin receptor-like kinase 5. M Endocrinology 2004;18.3: 653-665. DOI: http://doi.org/10.1210/me.2003-0393
http://doi.org/10.1210/me.2003-0393... ) to regulate downstream SMAD proteins in granulosa cells. Studies indicate that GDF-9 enhances growth and differentiation of preantral follicles in culture(⁴⁰40 Juengel JL, Bodensteiner KJ, Heath DA, Hudson NL, Moeller CL, Smith P, et al. Physiology of GDF9 and BMP15 signalling molecules. A reproduction science 2004; 82: 447-460. DOI: http://doi.org/10.1016/j. anireprosci.2004.04.021
http://doi.org/10.1016/j. anireprosci.20... ) and promotes theca cell androgen biosynthesis and proliferation(⁴¹41 Vitt UA, McGee EA, Hayashi M, Hsueh AJ. In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats. Endocrinology 2000; 141.10: 3814-3820. DOI: http://doi.org/10.1210/endo.141.10.7732
http://doi.org/10.1210/endo.141.10.7732... ). In addition to these factors, R-spondin2 protein is also an important paracrine factor that can promote granulosa cell proliferation(⁴²42 Spicer LJ, Aad PY, Allen DT, Mazerbourg S, Payne AH, Hsueh AJ. Growth differentiation factor 9 (GDF9) stimulates proliferation and inhibits steroidogenesis by bovine theca cells: influence of follicle size on responses to GDF9. Biology of Reproduction 2008;78.2: 243-253. DOI: http://doi.org/10.1095/biolreprod.107.063446
http://doi.org/10.1095/biolreprod.107.06... ). The FGF-2 and their respective receptors are also involved in early antral follicle development since(³⁶36 Morikawa R, Jibak L,Takashi M. Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte–cumulus cell complexes in vitro. The Journal of Reproduction and Develoment. 2021; 67(4): 273-281. DOI: http://doi.org/10.1262/jrd.2021-026
http://doi.org/10.1262/jrd.2021-026... , ⁴³43 Cheng Y, Kawamura K, Taka S, Deguchi M, Yang Q, Kuo C, et. Oocyte-derived R-spondin2 promotes ovarian follicle development. Jornal FASEB 2013;27: 2175-2184. DOI: http://doi.org/10.1096/fj.12-223412
http://doi.org/10.1096/fj.12-223412... , ⁴⁴44 Chang HM, Fang L, Cheng JC, Taylor EL, Sun YP, Leung PC. Effects of growth differentiation factor 8 on steroidogenesis in human granulosa-lutein cells. Fertility and sterility 2016;105: 520-528. DOI: http://doi.org/10.1016/j.fertnstert.2015.10.034
http://doi.org/10.1016/j.fertnstert.2015... ) FGF-2 alone or in association with VEGF-A influence steroidogenesis and proliferation of buffalo granulosa cells, by regulating mRNA expression of cytochrome P450 19A1 (CYP19A1), proliferating cell nuclear antigen (PCNA), and Bcl-2 Associated X-protein (BAX)(⁴⁴44 Chang HM, Fang L, Cheng JC, Taylor EL, Sun YP, Leung PC. Effects of growth differentiation factor 8 on steroidogenesis in human granulosa-lutein cells. Fertility and sterility 2016;105: 520-528. DOI: http://doi.org/10.1016/j.fertnstert.2015.10.034
http://doi.org/10.1016/j.fertnstert.2015... ,⁴⁵45 Mishra SR, Thakur N, Somal A, Parmar MS, Reshma R, Rajesh G, et al. “Expression and localization of fibroblast growth factor (FGF) family in buffalo ovarian follicle during different stages of development and modulatory role of FGF2 on steroidogenesis and survival of cultured buffalo granulosa cells. Research in Veterinary Science 2016; 108: 98-111. DOI: http://doi.org/10.1016/j.rvsc.2016.08.012
http://doi.org/10.1016/j.rvsc.2016.08.01... ) (Figure 2). Furthermore, Mattar et al.(⁴⁶46 Mattar D., Samir M., Laird M., Knight PG. Modulatory effects of TGF-ß1 and BMP6 on thecal angiogenesis and steroidogenesis in the bovine ovary. Reproduction. 2020;159(4), 397-408. DOI: https://doi.org/10.1530/REP-19-0311
https://doi.org/10.1530/REP-19-0311... ) reported that VEGF-A and FGF-2 promote the formation of endothelial cell networks during in vitro culture of theca cells. These structures support successive follicular development up to the preovulatory stage.

Figure 2.
Oocyte-granulosa cell interactions during early antral follicle development.

Granulosa cells play a role in the development of antral follicles by promoting the development of the oocyte-granulosa cell complex and providing adenosine triphosphate (ATP) to the oocytes(⁴⁸48 Yang Y, Kanno C, Huang W, Kang SS, Yanagawa Y, Nagano M. Effect of bone morphogenetic protein-4 on in vitro growth, steroidogenesis and subsequent developmental competence of the oocyte-granulosa cell complex derived from bovine early antral follicles. Reproductive Biology and Endocrinology 2016; 14:1: 1-8. doi; http://doi.org/10.1186/s12958-016-0137-1
http://doi.org/10.1186/s12958-016-0137-1... ). Moreover, Yang et al.(⁴⁸48 Yang Y, Kanno C, Huang W, Kang SS, Yanagawa Y, Nagano M. Effect of bone morphogenetic protein-4 on in vitro growth, steroidogenesis and subsequent developmental competence of the oocyte-granulosa cell complex derived from bovine early antral follicles. Reproductive Biology and Endocrinology 2016; 14:1: 1-8. doi; http://doi.org/10.1186/s12958-016-0137-1
http://doi.org/10.1186/s12958-016-0137-1... ) demonstrated the influence of BPM-4, derived from theca cells, on steroidogenesis in early antral follicles. The CNP is also a stimulating factor for early antral follicles(³⁰30 Khan H L, Bhatti S, KhanYL, Abbas S, Munir Z, Sherwani IAR et al. Cell-free nucleic acids and melatonin levels in human follicular fluid predict embryo quality in patients undergoing in-vitro fertilization treatment. J. Gynecol. Obstet. Human Reproduction. 2020; 49: (1), 101624. DOI: https://doi.org/10.1016/j.jogoh.2019.08.007
https://doi.org/10.1016/j.jogoh.2019.08.... ) (Figure 2). In association with the granulosa cells, the theca cells contribute to the synthesis of inhibin α; which is a hormone that inhibits the production of FSH(⁴⁹49 Laird, M., Glister, C., Cheewasopit, W., Satchell, L. S., Bicknell, A. B., Knight, P. G. Free inhibin a subunit is expressed by bovine ovarian theca cells and its knockdown suppresses androgen production. Scientific reports, 2019. 9(1), 19793. DOI: https://doi.org/10.1038/s41598-019-55829-w
https://doi.org/10.1038/s41598-019-55829... ). Yang et al.(⁴⁸48 Yang Y, Kanno C, Huang W, Kang SS, Yanagawa Y, Nagano M. Effect of bone morphogenetic protein-4 on in vitro growth, steroidogenesis and subsequent developmental competence of the oocyte-granulosa cell complex derived from bovine early antral follicles. Reproductive Biology and Endocrinology 2016; 14:1: 1-8. doi; http://doi.org/10.1186/s12958-016-0137-1
http://doi.org/10.1186/s12958-016-0137-1... ) demonstrated the influence of BPM-4, which comes from the theca cells, on steroidogenesis and initial antral follicles.

4. Control of granulosa cell proliferation and estradiol production during early antral follicle development

In early antral follicles, granulosa cells are highly proliferative but susceptible to apoptosis. The factors secreted by the oocyte, like GDF-9 and BMP-15, regulate granulosa cell proliferation and survival(⁵⁰50 Sun T, Diaz FJ. Ovulatory signals alter granulosa cell behavior through YAP1 signaling. Reproductive Biology Endocrinology. 2019; 17(1):113. DOI: https://doi.org/10.1186/s12958-019-0552-1
https://doi.org/10.1186/s12958-019-0552-... ,⁵¹51 McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Lawrence SB, et al. Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants. Reproduction. 2005; 129(4): 481-7. DOI: https://doi.org/10.1530/rep.1.0511
https://doi.org/10.1530/rep.1.0511... ). Furthermore, granulosa cell proliferation is dependent on cyclin D2 to activate cyclin-dependent kinase (CDK) family members CDK2, CDK4 and CDK6(⁵²52 Hoque SAM, Kawai T, Zhu Z, Shimada M. Mitochondrial protein turnover is critical for granulosa cell proliferation and differentiation in antral follicles. Journal of the Endocrine Society. 2018; 3(2): 324-339. DOI: https://doi.org/10.1210/js.2018-00329
https://doi.org/10.1210/js.2018-00329... ). In developing follicles, FSH stimulates granulosa cell proliferation and aromatization of androgens into estrogens. Estrogens also stimulate granulosa cell proliferation(⁵³53 Alam MH, Miyano T. Interaction between growing oocytes and granulosa cells in vitro. Reproductive Medicine and Biology. 2019; 19(1): 13-23. DOI: https://doi.org/10.1002/rmb2.12292
https://doi.org/10.1002/rmb2.12292... ). An increase in estradiol is associated with an increase in the expression of genes for aromatase, 3β-HSD and receptors for FSH and LH in granulosa cells (Figure 2)(⁵⁴54 Orisaka M, Hattori K, Fukuda S, Mizutani T, Miyamoto K, Sato T, et al. Dysregulation of ovarian follicular development in female rat: LH decreases FSH sensitivity during preantral-early antral transition. Endocrinology. 2013; 154(8): 2870-80. DOI: https://doi.org/10.1210/en.2012-2173
https://doi.org/10.1210/en.2012-2173... ).

Neuronal neuropeptide Y (NPY) is strongly present in granulosa cells, and the abundance of mRNA for NPY is higher in early antral follicles than in late antral follicles. In addition, NPY increases the proliferation of granulosa cells via NPY receptor Y5 (NPY5R) and mitogenactivated protein kinase (MEK)(⁵⁵55 Urata Y, Salehi R, Lima PDA, Osuga Y, Tsang BK. Neuropeptide Y regulates proliferation and apoptosis in granulosa cells in a follicular stage-dependent manner. Journal of Ovarian Research. 2020; 13(1): 5. DOI: https://doi.org/10.1186/s13048-019-0608-z
https://doi.org/10.1186/s13048-019-0608-... ). Baddela et al. (⁵⁶56 Baddela VS, Sharma A, Michaelis M, Vanselow J. HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells. Scientific Reports. 2020; 10(1): 3906. DOI: https://doi.org/10.1038/s41598-020-60935-1
https://doi.org/10.1038/s41598-020-60935... ) reported that, in granulosa cells, hypoxiainducible factor 1 (HIF1) transcriptionally regulates genes associated with steroidogenesis, such as steroidogenesis acute regulatory protein (StAR), 3B-hydroxysteroid dehydrogenase (HSD3B) and CYP19A1, and proliferation (CCND2 and PCNA). The onset of StAR mRNA expression occurs in early antral follicles of 1.0 mm in diameter(⁵⁷57 Braw-Tal R, Roth Z. Gene expression for LH receptor, 17 alpha-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary. Reproduction. 2005; 129(4): 453-61. DOI: https://doi.org/10.1530/rep.1.00464
https://doi.org/10.1530/rep.1.00464... ). Furthermore, FSH and LH, together with intraovarian cytokines, induce the expression of steroidogenic enzymes in granulosa cells, including StAR, CYP11a1, 3βHSD and CYP19a1, as shown in Figure 3(⁵⁸58 Samie KA, Tabandeh MR, Afrough M. Betaine ameliorates impaired steroidogenesis and apoptosis in mice granulosa cells induced by high glucose concentration. Systems Biology in Reproductive Medicine. 2020; 66(6): 400-409. DOI: https://doi.org/10.1080/19396368.2020.1811423
https://doi.org/10.1080/19396368.2020.18... ). The expression of mRNA for LHR is found in granulosa cells from follicles smaller than 5 mm(⁵⁹59 Douville G, Sirard MA. Changes in granulosa cells gene expression associated with growth, plateau and atretic phases in medium bovine follicles. Journal of Ovarian Research. 2014; 7: 50. DOI: https://doi.org/10.1186/1757-2215-7-50
https://doi.org/10.1186/1757-2215-7-50... ).

Figure 3.
Influence of FSH, LH estradiol and IGF-I on granulosa cells to induce proliferation and production of enzymes involved in steroidogenesis.

Follicular steroidogenic potential involves an extensive and highly coordinated series of developmental stages. During this process, after intense granulosa and theca cell proliferation (up to 100-fold), they differentiate into specialized endocrine cells. Ovarian steroids are synthesized by the cooperation of these cells. Theca cells synthesize androgens through the enzymatic activity of cytochrome P450 17A1 (CYP17A1)(⁵³53 Alam MH, Miyano T. Interaction between growing oocytes and granulosa cells in vitro. Reproductive Medicine and Biology. 2019; 19(1): 13-23. DOI: https://doi.org/10.1002/rmb2.12292
https://doi.org/10.1002/rmb2.12292... ). Follicles over 2 mm in diameter strongly expressed LH-R and CYP17A1 mRNAs in most thecal cells(⁵⁵55 Urata Y, Salehi R, Lima PDA, Osuga Y, Tsang BK. Neuropeptide Y regulates proliferation and apoptosis in granulosa cells in a follicular stage-dependent manner. Journal of Ovarian Research. 2020; 13(1): 5. DOI: https://doi.org/10.1186/s13048-019-0608-z
https://doi.org/10.1186/s13048-019-0608-... ). Androgens are then converted to estrogens by aromatase (CYP19) produced by granulosa cells. Moreover, progesterone is produced by granulosa cells and used by theca cells to synthesize androgens(⁶⁰60 Jeon MJ, Choi YS, Yoo JJ, Atala A, Jackson JD. Optimized culture system to maximize ovarian cell growth and functionality in vitro. Cell Tissue Research. 2021; 385(1): 161-171. DOI: https://doi.org/10.1007/s00441-021-03415-w
https://doi.org/10.1007/s00441-021-03415... ). StAR, CYP11a1 and CYP19a1 are the key enzymes in the hormone synthesis process(⁶¹61 Wang N, Zhao F, Lin P, Zhang G, Tang K, Wang A, et al. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis. International Journal of Molecular Sciences. 2017; 18(6): 1152. DOI: https://doi.org/10.3390/ijms18061152
https://doi.org/10.3390/ijms18061152... ) (Figure 3).

Granulosa cells express estradiol receptors, contributing to follicular development(⁶²62 Kishi H, Kitahara Y, Imai F, Nakao K, Suwa H. Expression of the gonadotropin receptors during follicular development. Reproductive Medicine and Biology. 2017; 17(1): 11-19. DOI: https://doi.org/10.1002/rmb2.12075
https://doi.org/10.1002/rmb2.12075... ). Autocrine and paracrine activities of estradiol in granulosa cells stimulate aromatase enzyme activity, increasing gonadotropin sensitivity and expression of IGF-1(⁵⁹59 Douville G, Sirard MA. Changes in granulosa cells gene expression associated with growth, plateau and atretic phases in medium bovine follicles. Journal of Ovarian Research. 2014; 7: 50. DOI: https://doi.org/10.1186/1757-2215-7-50
https://doi.org/10.1186/1757-2215-7-50... ). In the ovary, IGF-I stimulates follicular steroidogenesis and increases estradiol production. The absence of IGF-I results in interruption of follicular growth in the preantral/early antral stage since these follicles do not respond to gonadotropin(⁶⁰60 Jeon MJ, Choi YS, Yoo JJ, Atala A, Jackson JD. Optimized culture system to maximize ovarian cell growth and functionality in vitro. Cell Tissue Research. 2021; 385(1): 161-171. DOI: https://doi.org/10.1007/s00441-021-03415-w
https://doi.org/10.1007/s00441-021-03415... ,⁶¹61 Wang N, Zhao F, Lin P, Zhang G, Tang K, Wang A, et al. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis. International Journal of Molecular Sciences. 2017; 18(6): 1152. DOI: https://doi.org/10.3390/ijms18061152
https://doi.org/10.3390/ijms18061152... ). In granulosa cells, the stimulatory effect of FSH on CYP19 and protein kinase B (AKT) depends on IGF-I and on the expression and activation of IGF-IR(⁶¹61 Wang N, Zhao F, Lin P, Zhang G, Tang K, Wang A, et al. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis. International Journal of Molecular Sciences. 2017; 18(6): 1152. DOI: https://doi.org/10.3390/ijms18061152
https://doi.org/10.3390/ijms18061152... ). Furthermore, FSH induces estradiol production via FSHR-cAMP-dependent signaling to induce transcription of the CYP19A1 gene(⁷⁷77 Yamamoto K, Otoi T, Koyama N, Horikita N, Tachikawa S, Miyano T. Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro. Theriogenology. 1999; 52: 81-89. DOI: https://doi.org/10.1016/S0093-691X(99)00111-9
https://doi.org/10.1016/S0093-691X(99)00... ). After follicle recruitment, gonadotropins gradually reduce granulosa cell proliferation and induce their differentiation to produce estradiol(⁶⁸68 Meng L, Jan SZ, Hamer G, van Pelt AM, van der Stelt I, Keijer J, Teerds KJ. Preantral follicular atresia occurs mainly through autophagy, while antral follicles degenerate mostly through apoptosis. Biology of Reproduction. 2018; 1;99(4):853-863. DOI: https://doi.org/10.1093/biolre/ioy116
https://doi.org/10.1093/biolre/ioy116... ) (Figure 3).

5. Follicle atresia during development from early antral follicles

At birth, the ovaries contain thousands of follicles, but only a small portion develops up to ovulation, while the great majority (~99.9) are lost by atresia. Follicular atresia does not occur equally during follicular development, differing between preantral follicles and antral follicles(⁶⁸68 Meng L, Jan SZ, Hamer G, van Pelt AM, van der Stelt I, Keijer J, Teerds KJ. Preantral follicular atresia occurs mainly through autophagy, while antral follicles degenerate mostly through apoptosis. Biology of Reproduction. 2018; 1;99(4):853-863. DOI: https://doi.org/10.1093/biolre/ioy116
https://doi.org/10.1093/biolre/ioy116... ). Spanel-Borowski et al.(⁶⁹69 Spanel-Borowski K. Follicle stages and follicular atresia. In Atlas of the Mammalian Ovary Morphological Dynamics and Potential Role of Innate Immunity. Spanel-Borowski K, Germany, 2012, pp. 9-22. DOI: https://link.springer.com/book/10.1007/978-3-642-30535-1
https://link.springer.com/book/10.1007/9... ) reported two types of atretic patterns in ovarian follicles, namely type A, in which the oocyte degenerates while granulosa cells remain intact, and type B, in which the granulosa cells show signs of extensive degeneration while the oocyte remains initially unaffected. Type A is the predominant form of atresia in preantral follicles(⁷⁰70 Silva JR, Báo SN, Lucci CM, Carvalho FC, Andrade ER, Ferreira MA, Figueiredo JR. Morphological and ultrastructural changes occurring during degeneration of goat preantral follicles preserved in vitro. Animal Reproduction Science. 2001; 66(3-4):209-223. DOI: https://doi.org/10.1016/s0378-4320(01)00102-6
https://doi.org/10.1016/s0378-4320(01)00... ), while in late antral follicles only type B is observed, with apoptosis of granulosa cells in the presence of a more or less intact oocyte being characteristic of atresia in large antral follicles(⁶⁹69 Spanel-Borowski K. Follicle stages and follicular atresia. In Atlas of the Mammalian Ovary Morphological Dynamics and Potential Role of Innate Immunity. Spanel-Borowski K, Germany, 2012, pp. 9-22. DOI: https://link.springer.com/book/10.1007/978-3-642-30535-1
https://link.springer.com/book/10.1007/9... ). In early antral follicles, the first changes that indicate atresia occur in the oocyte, such as nuclear chromatin retraction and oocyte fragmentation, while changes are rarely found in the granulosa cells present in these follicles(⁷⁰70 Silva JR, Báo SN, Lucci CM, Carvalho FC, Andrade ER, Ferreira MA, Figueiredo JR. Morphological and ultrastructural changes occurring during degeneration of goat preantral follicles preserved in vitro. Animal Reproduction Science. 2001; 66(3-4):209-223. DOI: https://doi.org/10.1016/s0378-4320(01)00102-6
https://doi.org/10.1016/s0378-4320(01)00... ).

When the paracrine or endocrine environment is not suitable to support oocyte growth and/or proliferation and differentiation of follicular cells, atresia can occur through necrosis, necroptosis, autophagy and apoptosis pathways(⁷¹71 Pajokh M, Mesbah F, Bordbar H, Talaei-Khozani T. Different cell death types determination in juvenile mice ovarian follicles. Iranian Journal of Veterinary Research. 2018;19(4): 298-303. DOI: PMC6361600.
https://doi.org/PMC6361600... ) (Figure 4). The necrosis and necroptosis pathways have similar morphological features and are characterized by an increase in cell volume, permeabilization and rupture of the plasma membrane, which lead to cell death(⁷¹71 Pajokh M, Mesbah F, Bordbar H, Talaei-Khozani T. Different cell death types determination in juvenile mice ovarian follicles. Iranian Journal of Veterinary Research. 2018;19(4): 298-303. DOI: PMC6361600.
https://doi.org/PMC6361600... ). Generally, necrosis is initiated by non-cellular mechanisms such as ischemia, deficiency in ATP levels and trauma, leading to irreversible cell damage(⁷¹71 Pajokh M, Mesbah F, Bordbar H, Talaei-Khozani T. Different cell death types determination in juvenile mice ovarian follicles. Iranian Journal of Veterinary Research. 2018;19(4): 298-303. DOI: PMC6361600.
https://doi.org/PMC6361600... ). Necroptosis is initiated by tumor necrosis factor-α (TNFα) and operated through protein kinase-1 and 3, which interact with its receptors-interacting serine/threonine-protein kinase 1 and kinase 3, respectively, as well as by the domain-like protein of mixed lineage kinase (MLKL)(⁷¹71 Pajokh M, Mesbah F, Bordbar H, Talaei-Khozani T. Different cell death types determination in juvenile mice ovarian follicles. Iranian Journal of Veterinary Research. 2018;19(4): 298-303. DOI: PMC6361600.
https://doi.org/PMC6361600... ). Zhou et al.(⁷²72 Zhou J, Peng X, Mei S. Autophagy in ovarian follicular development and atresia. International Journal of Biological Sciences. 2019; 15: 726-737. DOI: https://doi.org/10.7150/ijbs.30369
https://doi.org/10.7150/ijbs.30369... ) showed that the process of autophagy is involved with atresia in secondary and early antral follicles. Autophagy is an evolutionarily conserved form of intracellular process that involves damaged proteins and organelles for degradation and recycling (Figure 4).

Figure 4.
Mechanisms involved in early antral follicle atresia.

It is believed that granulosa cell apoptosis in late antral follicles is triggered by insufficient FSH levels or reduced numbers of FSH receptors(⁷³73 Soares PHA, Junqueira FS. Reproductive features of the bovine female: Review. Pubvet. 2019; 13: 1-6. DOI: https://doi.org/10.31533/pubvet.v13n02a257.1-6
https://doi.org/10.31533/pubvet.v13n02a2... ). The absence of LH and the decline of FSH circulation cause a decrease in the growth of subordinate follicles, ultimately resulting in atresia(⁷⁴74 Landry DA, Sirard M. Follicle capacitation: A meta-analysis to investigate the transcriptome dynamics following FSH decline in bovine granulosa cells. Biology of Reproduction. 2018; 99: 877-887. DOI: https://doi.org/10.1093/biolre/ioy090
https://doi.org/10.1093/biolre/ioy090... ). FSH protects granulosa cells from oxidative damage and rescues granulosa cells from apoptosis. FSH is thought to rescue granulosa cells of antral follicles from apoptosis via activation of the phosphatidylinositol 3 kinase (PI3K)–AKT signal transduction pathway. The activation of the phosphoinositide 3-kinase (PI3K)/Akt, via binding of FSH to its receptor, leads to phosphorylation of the box O subfamily of forkhead transcription factors (FOXO), which influences, among other processes, the survival of granulosa cells(⁷⁵75 Meng L, Jan SZ, Hamer G, Van Pelt AM, Van der Stelt I, Keijer J. et al. Preantral follicular atresia occurs mainly through autophagy, while antral follicles degenerate mostlythrough apoptosis. Biology of Reproduction. 2018; 99: 853-863. DOI: https://doi.org/10.1093/biolre/ioy116
https://doi.org/10.1093/biolre/ioy116... ).

6. Strategies for in vitro development of early antral follicles

Several studies have investigated the relationship between follicular and oocyte size with the acquisition of oocyte developmental competence in vitro, and many studies have focused on the development of culture protocols that can support the development of oocytes from early antral follicles (Figure 5). Harada et al.(⁷⁶76 Harada M, Miyano T, Matsumura K, Osaki S, Miyake M, Kato S. Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine. Theriogenology. 1997; 48: 743-755. DOI: https://doi.org/10.1016/S0093-691X(97)00298-7
https://doi.org/10.1016/S0093-691X(97)00... ) demonstrated, for the first time, that 90.0 to 99.0 µm oocytes from early bovine antral follicles (0.5 to 0.7 mm) can grow and acquire developmental competence in vitro in the presence of hypoxanthine and FSH. Likewise, Yamamoto et al.(⁷⁷77 Yamamoto K, Otoi T, Koyama N, Horikita N, Tachikawa S, Miyano T. Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro. Theriogenology. 1999; 52: 81-89. DOI: https://doi.org/10.1016/S0093-691X(99)00111-9
https://doi.org/10.1016/S0093-691X(99)00... ) demonstrated that, in addition to being able to grow and acquire developmental competence in vitro, oocytes (90.0 to 99.0 µm) from small bovine follicles were capable of producing offspring after undergoing maturation, fertilization and subsequent in vitro cultivation.

Figure 5.
Schematic representation of the major advances with in vitro culture of early antral follicles.

When culturing isolated antral follicles with a diameter between 0.2 and 0.5 mm, it was observed that, like the COCs (0.4 and 0.7), the follicles can also grow in in vitro culture, and that the oocytes can achieve meiotic competence(⁷⁸78 Alm H, Katska-Ksiazkiewicz L, Rynska B, Tuchscherer A. Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles. Theriogenology. 2006; 65: 1422-1434. DOI: https://doi.org/10.1016/j.theriogenology.2005.08.014
https://doi.org/10.1016/j.theriogenology... ). In goat species, in addition to obtaining improved in vitro oocyte maturation, embryo production from oocytes from small antral follicles cultured in vitro was reported(⁷⁹79 de Sá NA, Ferreira AC, Sousa FG, Duarte AB, Paes VM, Cadenas J, et al. First pregnancy after in vitro culture of early antral follicles in goats: positive effects of anethole on follicle development and steroidogenesis. Molecular Reproduction and Development. 2020; 87: 966-977. DOI: https://doi.org/10.1002/mrd.23410
https://doi.org/10.1002/mrd.23410... ). Cadenas et al.(⁸⁰80 Cadenas J, Leiva-Revilla J. Vieira LA, Apolloni LB, Aguiar FLN, Alves BG, et al. Caprine ovarian follicle requirements differ between preantral and early antral stages after IVC in medium supplemented with GH and VEGF alone or in combination. Theriogenology. 2017; 87: 321-332. DOI: https://doi.org/10.1016/j.theriogenology.2016.09.008
https://doi.org/10.1016/j.theriogenology... ) showed that early antral follicles from goats cultured in a medium containing insulin (10 ng/mL) associated with growth hormone (50 ng/mL) are capable of maintaining the growth and maturation of oocytes in vitro at levels similar to in life. Similarly, when observing the effect of stimulation of recombinant human FSH (hrFSH) on early goat antral follicles, hrFSH improved antral follicle development in a concentration-dependent manner(⁸¹81 Ferreira, ACA, Cadenas J, Sá NA, Correia HH, Guerreiro DD, Lobo CH, et al. In vitro culture of isolated preantral and antral follicles of goats using human recombinant FSH: Concentration-dependent and stage-specific effect. Animal Reproduction Science. 2018; 196: 120-129. DOI: https://doi.org/10.1016/j.anireprosci.2018.07.004
https://doi.org/10.1016/j.anireprosci.20... ). Lopes et al.(⁸²82 Lopes EPF, Rodrigues GQ, de Brito DCC, Rocha RMP, Ferreira ACA, de Sá NA, et al. Vitrification of caprine secondary and early antral follicles as a perspective to preserve fertility function. Reproductive Biology. 2020; 20: 371-378. DOI: https://doi.org/10.1016/j.repbio.2020.05.001
https://doi.org/10.1016/j.repbio.2020.05... ) also demonstrated that early antral follicles isolated from goat ovarian stroma are able to grow and survive in vitro for a short period of time, after going through a vitrification process. Cordeiro et al.(⁸³83 Cordeiro EB, Silva BR, Paulino LR, Barroso PA, Barrozo LG, de Lima Neto MF, and Silva JR. Effects of N-acetylcysteine on growth, viability and reactive oxygen species levels in small antral follicles cultured in vitro. Asian Pacific Journal of Reproduction, 2023; 12(1), 42. DOI: https://doi.org/10.1016/j.anireprosci.2021.106801
https://doi.org/10.1016/j.anireprosci.20... ) have recently reported that the presence of N-acetyl-cysteine (NAC) in the medium culture of early antral follicles reduces the levels of reactive oxygen species (ROS) and maintains the integrity of oocytes during culture in cattle.

In vitro culture of COC and isolated follicles ensures bidirectional communication between oocytes and granulosa cells through transzonal projections (TZPs), which is crucial for the occurrence of molecular events necessary for follicle and oocyte development until ovulation. These events involve, in addition to chromosomal separation, characterizing nuclear maturation. Moreover, they involve the distribution of cytoplasmic organelles, the stock of mRNA, proteins and other factors that are essential for the oocyte to be able to resume meiosis and support fertilization and embryonic development(⁸⁴84 Jiang Y, He Y, Pan X, Wang P, Yuan X, Ma B. Advances in oocyte maturation in vivo and in vitro in mammals. International Journal of Molecular Sciences. 2023; 21;24(10):9059. DOI: https://doi.org/10.3390/ijms24109059
https://doi.org/10.3390/ijms24109059... ).

Some studies have already reported the birth of live calves from oocytes obtained from early antral follicles, but the viability and developmental competence of these oocytes in vitro can be improved(⁷⁷77 Yamamoto K, Otoi T, Koyama N, Horikita N, Tachikawa S, Miyano T. Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro. Theriogenology. 1999; 52: 81-89. DOI: https://doi.org/10.1016/S0093-691X(99)00111-9
https://doi.org/10.1016/S0093-691X(99)00... , ⁸⁵85 Huang W, Nagano M, Kang SS, Yanagawa Y, Takahashi Y. Effects of in vitro growth culture duration and prematuration culture on maturational and developmental competences of bovine oocytes derived from early antral follicles. Theriogenology 2013; 80:793–9. DOI: https://doi.org/10.1016/j.theriogenology.2013.07.004
https://doi.org/10.1016/j.theriogenology... , ⁸⁶86 Senbon S, Miyano T. Bovine oocytes in early antral follicles grow in serum- free media: effect of hypoxanthine on follicular morphology & oocyte growth. Zygote 2002;10:301–9. DOI: https://doi.org/10.1017/S0967199402004033
https://doi.org/10.1017/S096719940200403... ). Therefore, our research group has focused on the development of culture protocols that favor the acquisition of oocyte competence in vitro. Bezerra et al.(⁸⁷87 Bezerra FTG, Silva AWB, Rissi VB, Rosa PA, Cesaro MP, Costa JJN, Gonçalves PBD and Silva JRV. Cilostamide and follicular hemisections inhibit oocyte meiosis resumption and regulate gene expression and cAMP levels in bovine cumulus–oocyte complexes. Livestock Science, 2016; 184, 112–118. DOI: https://doi.org/10.1016/j.livsci.2015.12.014
https://doi.org/10.1016/j.livsci.2015.12... ) showed that follicular hemisections combined with cilostamide have a synergistic effect on the maintenance of oocytes in the germinal vesicle during in vitro culture. Barrozo et al.(⁸⁸88 Barrozo LG, Silva BR, Paulino LR, Barbalho EC, Nascimento DR, Costa FC, and Silva JR. N-acetyl cysteine reduces levels of reactive oxygen species and improves in vitro maturation of oocytes from medium-sized bovine antral follicles. Zygote, 2022; 30(6), 882-890. DOI: https://doi.org/10.1017/S0967199422000429
https://doi.org/10.1017/S096719942200042... ) showed that the presence of this NAC in the culture medium increased the percentage of meiotic resumption and the distribution of TZPs, as well as reduced ROS levels, indicating that the inclusion of antioxidants is important to optimize the IVM systems. He et al.(⁸⁹89 He Y, Meng K, Wang X, Dong, Z, Zhang, Y, Quan F. Comparison of bovine small antral follicle development in two-and three-dimensional culture systems. Annals of the Brazilian Academy of Sciences. 2020; 92, e20180935. DOI: https://doi.org/10.1590/0001-3765202020180935
https://doi.org/10.1590/0001-37652020201... ) suggested that the two-dimensional (2D) culture system is more suitable for the culture of oocytes from early antral follicles lasting up to four days, while in culture periods longer than four days, the three-dimensional (3D) system is more adequate.

Culture of isolated early antral follicles may also be a promising alternative for providing competent oocytes for use in in vitro maturation protocols(⁹⁰90 Bezerra FTG, Lima FEO, Paulino LRFM, Silva BR, Silva AWB, Souza ALP, Van Den Hurk R and Silva JRV. In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation-related transcripts in bovine oocytes. Molecular Reproduction and Development. 2019; 86 (12), 1874–1886. DOI: https://doi.org/10.1002/mrd.23284
https://doi.org/10.1002/mrd.23284... ), as this communication between oocytes and granulosa cells is maintained. However, the choice of follicle size is fundamental to the acquisition of competence in oocyte development. It has already been shown that oocytes from small antral follicles (1 and 2 mm) have significantly reduced competence compared to oocytes from larger antral follicles (>3 mm)(³3 Pavlok A, Lucas-Hahn A, Niemann H. Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles. Molecular Reproduction and Development. 1992; 31: 63-67. DOI: https://doi.org/10.1002/mrd.1080310111
https://doi.org/10.1002/mrd.1080310111... ,⁴4 Nemcová L, Hulínska P, Ješeta M, Kempisty B, Kanka J, Machatková M. Expression of selected mitochondrial genes during in vitro maturation of bovine oocytes related to their meiotic competence. Theriogenology. 2019; 133: 104-112. DOI: https://doi.org/10.1016/j.theriogenology.2019.05.001
https://doi.org/10.1016/j.theriogenology... ). Bezerra et al.(⁹⁰90 Bezerra FTG, Lima FEO, Paulino LRFM, Silva BR, Silva AWB, Souza ALP, Van Den Hurk R and Silva JRV. In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation-related transcripts in bovine oocytes. Molecular Reproduction and Development. 2019; 86 (12), 1874–1886. DOI: https://doi.org/10.1002/mrd.23284
https://doi.org/10.1002/mrd.23284... ) showed that the levels of mRNAs for transcripts involved in the oocyte development process, such as histone with oocyte-specific ligand (H1FOO), GDF-9 and poly (a) specific ribonuclease (PARN), increase in oocytes when follicles grow from secondary to small, medium and large antral follicles.

7. Conclusions

The development of early antral follicles up to gonadotropin dependence involves a wide range of processes, which can be decisive for follicular growth, steroidogenesis and acquisition of oocyte competence. The interplay between oocyte and follicle cells directly influences follicle and oocyte fate. In addition, the in vitro culture of early antral follicles opens new prospects for the use of their oocytes for in vitro fertilization and for a better understanding of the mechanisms involved in the control of early antral follicles.

Acknowledgments

The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for fellowships. J.R.V. Silva is a researcher at Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (grant number 308737/2018-0).

References

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