The mechanisms that control the preantral to early antral follicle transition and the strategies to have efficient culture systems to promote their growth in vitro

Summary

Preantral to early antral follicles transition is a complex process regulated by endocrine and paracrine factors, as well as by a precise interaction among oocyte, granulosa cells and theca cells. Understanding the mechanisms that regulate this step of folliculogenesis is important to improve in vitro culture systems, and opens new perspectives to use oocytes from preantral follicles for assisted reproductive technologies. Therefore, this review aims to discuss the endocrine and paracrine mechanisms that control granulosa cell proliferation and differentiation, formation of the antral cavity, estradiol production, atresia, and follicular fluid production during the transition from preantral to early antral follicles. The strategies that promote in vitro growth of preantral follicles are also discussed.

Introduction

The transition from preantral (multilaminar follicles) to early antral follicles requires the action of local factors, hormones, and efficient bidirectional communication between granulosa cells and oocytes. Several attempts to promote in vitro growth of preantral follicles up to maturation have been reported in various domestic species (bovine: Paulino et al., 2018; Antonino et al., 2019; Bezerra et al., 2019a; Paulino et al., 2020; Vasconcelos et al., 2021; caprine: Ferreira et al., 2018; Soares-Costa et al., 2018; Pontes et al., 2019; ovine: Macedo et al., 2019; Mbemya et al., 2019; Barros et al., 2020; Silva et al., 2021; Gomes et al., 2022; and swine: de Lima et al., 2017; Kere et al., 2020). Different from mice (Eppig and O’Brien, 1996; O’Brien et al., 2003), embryo production from in vitro cultured preantral follicles from human and domestic animals has not yet, however, been reported in the literature (Wu and Tian, 2007; Arunakumari et al., 2010; de Figueiredo et al., 2018, 2020; Paulino et al., 2022). The maintenance of bidirectional communication between the oocyte and the granulosa cells in cultured follicles, as well as the large quantity of messenger RNA (mRNA) and proteins that the oocyte needs to synthesize during its growth make it difficult the complete their development in vitro (Alam and Miyano, 2020; de Figueiredo et al., 2020; Paulino et al., 2022).

The endocrine and paracrine control that regulates the transition from preantral to antral follicles is complex and involves a precise interaction of several factors (Araújo et al., 2014; de Figueiredo et al., 2018, 2020). Any interference with this control can lead to ovarian disorders such as polycystic ovary syndrome (Abdel Aziz et al., 2021; Asghari et al., 2021). The present review highlights the mechanisms involved in endocrine and paracrine control during the transition from preantral to early antral follicles, as well as the importance of granulosa cell proliferation, antral cavity formation, and estradiol production. The strategies to promote in vitro growth of preantral follicles are also discussed.

Endocrine and paracrine control during the transition from preantral to early antral follicles

The transition from preantral to early antral follicles is a step in which follicle development is regulated by intraovarian factors, but the follicles are responsive to gonadotropins. The slow growth of preantral and early antral follicles is gonadotropin independent, but progression to a late antral follicular state requires follicle-stimulating hormone (FSH; Iber and Geyter, 2013). FSH is the main endocrine regulator of follicle development (Paulino et al., 2018, 2020; Vasconcelos et al., 2021) and its receptors are detectable in granulosa cells of preantral follicles in various species (murine: Camp et al., 1991; human: Oktay et al., 1997; Méduri et al., 2002; porcine: Durlej et al., 2011; bubaline: Sharma et al., 2011; caprine: Saraiva et al., 2011; Barros et al., 2013; Ferreira et al., 2018). This hormone stimulates granulosa cell proliferation and promotes follicular growth and antrum formation (Ferreira et al., 2018; de Figueiredo et al., 2020; Fushii et al., 2021). Other endocrine factors, such as melatonin have a functional role in preantral follicles by influencing their development, increasing the production of active mitochondria in oocytes and steroidogenesis in granulosa cells (Barros et al., 2013; Riaz et al., 2019; Barros et al., 2020). It is well established that FSH stimulates the production of aromatase (Cyp19a1), which synthesizes 17β-estradiol, an important hormone for granulosa cell proliferation (Fitzpatrick and Richards, 1994; Bishonga et al., 2001). Anti-Müllerian hormone (AMH) is also produced by granulosa cells of preantral and antral follicles (Umer et al., 2019; Gautam et al., 2021). It is already well established that this hormone prevents early depletion of follicles, but there is still much to elucidate in the role of this hormone during folliculogenesis. Rocha et al. (2021) have shown that there is an interaction between AMH and FSH, in which AMH reduces FSH-induced estradiol and progesterone production. Tanimoto et al. (2021) showed that, for the development of a viable follicle, blockage of AMH production by oestrogen is needed.

Oocyte-derived factors, such as growth differentiation factor-9 (GDF-9), bone morphogenetic protein 15 (BMP-15), and fibroblast growth factor-2 (FGF-2) are important regulators of preantral follicle growth by inducing granulosa cell proliferation and differentiation, and antral cavity formation (Reineri et al., 2018; Monte et al., 2019). Other factors, such as epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1), influence the development of preantral follicles by expanding oocyte diameter and inducing granulosa cell proliferation (Bezerra et al., 2019b; Paulino et al., 2020). Vascular endothelial growth factor (VEGF) is an important angiogenic factor that induces granulosa cell proliferation, an essential characteristic for the transition from preantral to antral follicles, and improves oocyte maturation (Araújo et al., 2011; da Silva et al., 2015; Cadenas et al., 2017). Activin is another intraovarian factor that accelerates the growth of preantral follicles, estradiol synthesis, and mRNA expression for FSH receptor in rat granulosa cells (Tanaka et al., 2019). Conversely, in bovine species, activin decreases the FSH stimulating action in the bovine preantral follicle cultured in vitro, which was associated with decreased levels of transcripts for hyaluronan synthases (HAS-1, HAS-2) and proliferating cell nuclear antigen (PCNA; Silva et al., 2014).

Granulosa cell proliferation and oocyte–granulosa cell interaction during the transition from preantral to early antral follicles

Granulosa cells form a favourable metabolic and hormonal environment for oocyte growth and maturation (Baumgarten and Stocco, 2018). Similarly, the oocyte can influence the proliferation of granulosa cells, providing follicular development through the production of growth factors such as BMP-15 and GDF-9 (de Figueiredo and de Lima, 2017; Baumgarten and Stocco, 2018). Orisaka et al. (2006) showed that GDF-9 controls follicular fate by promoting its survival and growth during the preantral to early antral transition, suppressing granulosa cell apoptosis and follicular atresia through PI3K/Akt activation.

During the development of the preantral follicle, GDF-9 and BMP-15 continue to stimulate the proliferation of granulosa cells, however, they decrease the production of progesterone and increase the expansion and reorganization of granulosa cells to form the antrum cavity. In addition, GDF-9 stimulates the expression of FSH receptors in granulosa cells, which become responsive to gonadotropins and influence the differentiation and recruitment of theca cells, which form a scaffold structure, supporting the vascular system (De Conto et al., 2021). Theca cells begin to produce luteinizing hormone (LH) receptors, steroidogenic enzymes and androgens (Young and McNeilly, 2010), while LH stimulates the production of androgens by theca cells, which are used by granulosa cells to produce 17β-estradiol (Xavier and Freitas, 2021). 17β-Estradiol increases the sensitivity of granulosa cells to gonadotropins and IGF-1, which stimulates follicular steroidogenesis (Ginther et al., 2001). BMP-4 and FSH also play an important regulatory role in the growth and steroidogenesis of preantral follicles. According to Sakaguchi et al. (2017), BMP-4 inhibits the luteinization of granulosa cells, while FSH increases their proliferation and the viability of the oocyte–cumulus–granulosa complex.

Granulosa cells produce several autocrine and paracrine factors that may be involved in the initiation of antrum formation such as kit ligand, activins, inhibins, hyaluronan, Versican, AMH and transforming growth factor α (TGFα). These factors also synchronize oocyte growth, granulosa cell proliferation and theca cell differentiation (Vasconcelos et al., 2013; Dumesic et al., 2015). In this sense, the interaction between the oocyte and granulosa cells, as well as the differentiation and proliferation of theca cells, can be determinants for the progress of preantral follicle growth, steroidogenesis and oocyte maturation (Chu et al., 2018; Alam and Miyano, 2020). AMH plays an inhibitory role in modulating the responsiveness of granulosa and theca cells to gonadotrophic stimuli, thereby regulating follicular progression from the gonadotropin-responsive to the gonadotropin-dependent stage (Campbell et al., 2012).

After antrum formation, granulosa cells are physically separated into mural granulosa cells, which organize along the follicle wall, and cumulus granulosa cells, which surround the oocyte (Baumgarten and Stocco, 2018; Zhang, 2018). Cumulus granulosa cells nourish the oocyte by providing specific amino acids, glycolysis products and cholesterol biosynthesis substrates through gap junctions (Baumgarten and Stocco, 2018). In addition, they prevent premature oocyte maturation and resumption of meiosis in the oocyte by maintaining high levels of cAMP in the oocyte (Russell and Robker, 2018; Zhang, 2018). The mechanisms that control the transition from preantral to early antral follicles are shown in Figure 1 and Table 1.

Figure 1. Factors that control the transition from preantral to early antral follicle.

Table 1. Role of growth factors in oocyte, granulosa and theca cells during the transition from preantral to early antral follicle

Mechanisms of antrum formation in preantral follicles

When the distance between the mural granulosa and the cumulus cells increases, the formation of the antral cavity occurs, marking the preantral to early antral follicles transition (Chu et al., 2018). The antral cavity is formed between the granulosa cells and requires a fluid ingress from the vascularization of theca cells via membrane proteins such as aquaporins (AQPs; Kawashima and Kawamura, 2018; Paz et al., 2018). Several AQPs, such as AQP5, AQP7, AQP8, and AQP9, are related to the influx of water through the follicle wall to form follicular fluid. These membrane proteins are expressed in the granulosa cells of different species (swine: Skowronski et al., 2009; ovine: Sales et al., 2015; bovine: Ishibashi et al., 2009). Paz et al. (2018) also demonstrated the presence of AQP3 in granulosa cells, and is also involved in the expansion of the antral cavity during the transition from preantral to the antral follicle. Antrum formation depends on the stimulation of local factors for the production and secretion of polysaccharides and proteins by the granulosa cells, which accumulate and generate an osmotic gradient that attracts fluid from the thecal vasculature (Baumgarten and Stocco, 2018). It is recognized that granulosa cells express the enzymes that synthesize hyaluronic acid, Versican and perlecan, which are responsible for the formation of follicular fluid (Schoenfelder and Einspanier, 2003; Clarke et al., 2006; Vasconcelos et al., 2013; Nagyova et al., 2020).

The development of the antral cavity is intensified by granulosa cell activity. Alam et al. (2018) showed that, even without the presence of an oocyte, GDF-9 and BMP-15 influence the production of antrum-like structures. GDF-9 is known to stimulate Versican and perlecan expression and interacts favourably with FSH to increase hyaluronan synthetase 2 expression (Vasconcelos et al., 2013; Silva et al., 2014). Hyaluronan, proteoglycans and their glycosaminoglycan side chains are osmotic solutes that act to increase the osmotic pressure inside the follicle, resulting in fluid accumulation (Rodgers and Irving-Rodgers, 2010). Some studies have also demonstrated that EGF increases mRNA levels for GDF-9 in in vitro cultured preantral follicles (Alam et al., 2018; Paulino et al., 2020), showing that EGF acts via GDF-9 to promote antral cavity formation. The mechanisms involved in the formation of antral cavity formation are shown in Figure 2.

Figure 2. Hormones and growth factors that control the formation of the antral cavity during the transition from preantral to early antral follicles.

Production of estradiol during the transition from preantral to early antral follicles

When preantral follicles reach six or seven layers of granulosa cells, the inner layer of theca becomes active and the formation of the antral cavity begins. Increased 17β-estradiol in follicular fluid is associated with increased mRNA expression for CYP19A1 in granulosa cells (García-Guerra et al., 2018).

As a growing follicle acquires sufficient aromatase activity as a result of FSH stimulation, estradiol production suppresses FSH secretion below that necessary to support the development of less mature follicles, which consequently suffer atresia (Zeleznik, 2004). Therefore, estradiol biosynthesis is a tightly regulated molecular process, dependent on the expression of key steroidogenic enzymes by FSH and intraovarian signalling molecules, including beta-catenin, an essential co-transcription factor for maximal induction of the aromatase mRNA transcript and subsequent estradiol production (Forrest et al., 2022). β-Catenin regulates the transformation of androgen to estradiol by increasing the transcription of CYP19A1 through functional interactions with steroidogenic factor-1 (SF1; Forrest et al., 2022). Furthermore, LH promotes preantral to antral follicular transition by upregulating follicular androgen biosynthesis (Orisaka et al., 2013).

Expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and cytochrome P450 family 17, subfamily A, member 1 (CYP17A1) mRNAs appear in large preantral follicles, concomitantly with theca differentiation. Followed by the expression of steroidogenic acute regulatory protein (StAR) in 1.0 mm antral follicles, granulosa cells from preantral and early antral follicles do not express StAR. Therefore, steroidogenesis in bovine follicles potentially begins in follicles ≥ 1.0 mm. Furthermore, the mRNA for CYP17A1 was located exclusively in theca internal cells, which indicates that the conversion of progestogens to androgens occurs only in the theca interna (Braw-Tal and Roth, 2005). Furthermore, the neonatal rat ovary is completely devoid of antral follicles at birth. By day 12 of age, small-sized to medium-sized antral follicles are present, in addition to follicles at all preceding stages of development. During the intervening period the ovary becomes steroidogenically active, and responsive to gonadotrophins on days 7–9 of age, suggesting that granulosa and theca cells become active at that time (Carson and Smith, 1986).

The expression of key enzymes involved in steroidogenesis is crucial for the proper development of the follicles. It has been observed that the mRNA encoding P450arom was not detectable until early antral cavity formation, in addition to being expressed only in granulosa cells (Yuan et al., 2008). The mechanisms involved in the production of estradiol during the transition from preantral to early antral follicles are shown in Figure 2.

Follicle atresia during the transition from preantral to early antral follicles

Follicular atresia is characterized by morphological changes in the oocyte, granulosa, and theca cells that culminate with follicular death and, consequently, regulate the capacity of females to generate mature gametes during the fertile period (Makarevich et al., 2018). This process promotes a drastic reduction in the ovarian follicle population during the reproductive lifespan. According to Zhang et al. (2018), crosstalk among cell apoptosis, autophagy and ferroptosis is involved in the control of atresia in ovarian follicles. In preantral follicles, degeneration initially occurs in the oocyte and, subsequently, in granulosa cells (Meng et al., 2018). The first signs of atresia in the oocyte are the retraction of nuclear chromatin and oocyte fragmentation, which triggers the process of irreversible elimination of ovarian follicles at this stage of development. In the granulosa cells of preantral follicles, changes are rarely observed.

Zoheir et al. (2021) showed that cellular DNA fragmentation is an important biochemical marker of apoptosis during follicular atresia. Other evidence confirmed that apoptosis is not the only death pathway active in the ovary. Gannon et al. (2012) reported a decrease in preantral follicle numbers without a concomitant increase in apoptosis, and no change in apoptosis markers caspase 3 and TUNEL. Recent studies have demonstrated that, in granulosa cells from cultured preantral follicles of buffaloes, transmembrane protein AQP8 is involved in the regulation of cycle progression and apoptosis (Cao et al., 2021). Furthermore, miRNAs have been shown to control several fundamental biological processes, including follicular atresia through their target genes and signalling pathways, and play a central role in the regulation of autophagy (Ma et al., 2020). Gannon et al. (2012) showed that degeneration of preantral follicles is associated with the activation of the autophagy cascade. Meng et al. (2018) reported that the standard pathway of degeneration in preantral follicles is through autophagy, and that the activation of this pathway occurs under normal physiological conditions. Considering that, in vivo, follicular atresia during preantral to early antral follicles transition is controlled by various intrafollicular regulators such as growth factors, cytokines, and steroids (Orisaka et al., 2009a), the development of strategies to interrupt atresia is a difficult task.

Despite early antral follicles (∼2.0 mm in diameter – human species) are still not dependent on gonadotropins, Orisaka et al. (2009b) showed that they are susceptible to apoptotic signals. In these follicles, with the progression of atresia, there is a reduction in the number of layers of granulosa cells, and invasion of fibroblasts and macrophages (Seneda et al., 2021). Meng et al. (2018) reported that atresia in these follicles was associated with the activation of the autophagy cascade. Increasing the levels of microtubule-associated light chain protein 3 (LC3) and of autophagy homeostasis-associated protein BECLIN1 is linked with the death of preantral and early antral follicles by the autophagy cascade (Gordy and He, 2012). Activation of this pathway occurs under normal physiological conditions and the presence of LC3, sequestosome 1 (SQSTM1/P62) and autophagy-related protein 7 (ATG7) are markers for autophagy (Gannon et al., 2012). During atresia of those follicles, Meng et al. (2018) also reported that a loss in mitochondrial antioxidant capacity in granulosa cells led to the activation of AMP-activated protein kinase alpha 1 (AMPK-α1) and AMPK-α2, while at the same time the expression of protein kinase B (AKT) and mammalian target survival of the rapamycin complex 1 (mTORC1) was decreased. AMPK is an important regulator of metabolism, an inhibitor of the mTORC1 complex, and a direct activator of autophagy (Emerling et al., 2009; Egan et al., 2011; Kim et al., 2011). Oxidative stress is able to reduce superoxide dismutase (SOD) expression in granulosa cells of preantral follicles, and to activate AMPK, which leads to the activation of autophagy. The mechanisms that regulate the preantral and early antral follicles atresia are shown in Figure 3.

Figure 3. Mechanisms that control production of estradiol (E2) during the transition from preantral to early antral follicles.

Strategies to promote in vitro growth of preantral follicles

Preantral follicles represent 90% of the ovarian follicle population and, therefore, the development of effective culture systems to promote their growth in vitro is an interesting field of study in regard to avoiding the process of atresia that naturally occurs in vivo during the late antral stages of development. Follicular growth, antrum formation and acquisition of oocyte competence have been obtained in some species through the addition of different substances such as hormones, growth factors and cytokines to the culture medium in species that require short culture periods, such as murine (O’Brien et al., 2003). However, in human and large animal species, the development of primordial to preovulatory follicles is a very long process (∼6 months; Paulino et al., 2022) and various researchers have been working to develop efficient culture systems.

The limitations of in vitro culture of preantral follicles come from the difficulty of maintaining the three-dimensional structure of follicles, and the many signals necessary to coordinate the stimulation of follicular growth (Paulino et al., 2022). Although bi-dimensional (2D) follicle culture has been successfully performed in many studies (da Cunha et al., 2018; Paulino et al., 2018, 2020; Vasconcelos et al., 2021), a major limitation of these systems is their inability to maintain follicle architecture, with the oocyte surrounded by granulosa cells. This is particularly problematic with follicles from large mammalian species that require longer term cultures (Simon et al., 2020). Given the importance of maintaining the follicle complex architecture, three-dimensional (3D) culture systems can help to maintain follicle complex architecture (Simon et al., 2020). For this system, various types of matrices are used to encapsulate follicles to maintain follicle architecture and cell–cell interactions, thereby creating a microenvironment similar to that of the in vivo ovary, improving somatic cell proliferation and oocyte growth (Belli et al., 2012). In general, matrices have been engineered from natural components such as collagen and alginate (Healy et al., 2021), or Matrigel (Hao et al., 2020), or from synthetic components such as polyethylene glycol (Green and Shikanov, 2016; Tomaszewski et al., 2021). Maintaining the follicular shape along with regulating the physical and chemical features of the microenvironment can be used as a tool to understand the underlying biology of follicle growth and maturation (Ghorbani et al., 2022; Khunmanee and Park, 2022).

Preantral follicles cultured in the 3D system had greater homogeneity of daily growth, higher rates of viability and antrum formation, as well as low rates of degeneration (Antonino et al., 2019; Panta et al., 2019). Preantral follicles from other species have been successfully cultured in a three-dimensional culture system using alginate and fibrin (monkey: Xu et al., 2013; Bulgarelli et al., 2018; cat: Chansaenroj et al., 2019; human: Chiti et al., 2017).

Multistep culture systems have also been developed to further mimic the physiologic environment of developing follicles (Green and Shikanov, 2016; Simon et al., 2020). These systems have been used for culturing early preantral follicles. The multistep method starts with the culture of ovarian cortical tissue for 3 weeks to initiate primordial follicle activation and to support follicle growth to the preantral stage. At the end of ovarian tissue culture, preantral follicles are isolated mechanically and cultured individually or in a group for 6 weeks (Xu et al., 2021). In this system, Xu et al. (2021) demonstrated that ∼50% of human follicles survived for 6 weeks. Most surviving follicles grew to the antral stage and produced the ovarian steroid hormones estradiol and progesterone, in addition to paracrine factors such as activin A, IGF-2 and VEGF. In addition, the cultured preantral follicles exhibited morphology similar to that of human follicles developed in vivo.

Many efforts have been made to elucidate the mechanisms involved in the growth of preantral follicles, as reviewed recently by Paulino et al. (2022). Although comprehension of the molecular regulation and composition of the microenvironment coordinating the events in preantral follicles remains incomplete, over time many studies have been conducted to optimize culture systems to support follicular growth (Healy et al., 2021). Preantral follicles from human and animal species require the development of a tightly regulated culture system with adequate nutrients, cytokines, growth factors, and developmental stage-dependent hormones to support cell survival and proliferation, as well as cellular function, which changes as follicles grow and oocytes mature (Simon et al., 2020; Paulino et al., 2022). Therefore, understanding the influence of several compounds for supplementation of culture medium for preantral follicles is fundamental. In this context, melatonin increases follicular and oocyte diameters, forms the antral cavity and preserves high rates of morphologically intact sheep preantral follicles for up to 18 days of culture (Barros et al., 2020).

Another alternative to improve follicular development in vitro is to supplement the culture medium with EGF, whose signalling regulates many cellular processes associated with survival (Sabbah et al., 2020). EGF has an important role in folliculogenesis, by promoting several processes, such as granulosa and theca cell proliferation (Jachter et al., 2022). The effects of adding EGF to the culture medium are directly associated with improved survival of bovine preantral follicles. In general, EGF-treated follicles reach a larger diameter at the end of the culture period (Paulino et al., 2020; Jachter et al., 2022). Hormones, such as progesterone, and cytokines, such as TNF-α, are able to maintain healthy follicle morphology and positively influence follicular growth and antrum formation in cattle (Paulino et al., 2018, 2020). Also in cattle, preantral follicles cultured in the presence of BMP-2 or BMP-4 showed a significant increase in follicular diameter and greater average daily growth (da Cunha et al., 2018).

Understanding the mRNA transcription of preantral follicles can provide important insights to detect follicular gene expression at several critical stages of its development under different culture conditions. Therefore, the identification of genes differentially expressed at each of growth and follicular stage development can be used to elucidate the processes involved in follicular growth (Paulini et al., 2022). In bovine species, an overexpression of mRNA for oocyte maturation factor Mos (c-MOS) and GDF-9 was observed when oocytes from preantral follicles were cultured in the presence of EGF. Furthermore, higher levels of cyclin B1 and GDF9 mRNA were observed in oocytes from follicles cultured with progesterone (Paulino et al., 2020). In addition, bovine preantral follicles cultured in the presence of alpha-lipoic acid expressed higher levels of transcripts for the FSH receptor, LHCGR, IGF-1, BMPR1a, TGFβ1, TGFβR1, ActRIIB, GDF9, and activin. The expression of pro-apoptotic genes BCL2 associated X (BAX) and c-Myc were also downregulated (Zoheir et al., 2017).

To mitigate the effects of oxidative stress induced by culture conditions, such as lower quality oocytes, follicular cell death, inactivation of antioxidant enzymes, and mitochondrial damage, there has been an increasing interest in the antioxidant potential of natural compounds (Lins et al., 2017). Preantral follicles from sheep cultured in medium supplemented with kaempferol showed high percentages of follicular survival, antrum formation, and greater follicular diameter. In addition, kaempferol preserves higher levels of metabolically active mitochondria (Santos et al., 2019). In cattle, the presence of eugenol in a culture medium promoted higher daily growth rates of bovine preantral follicles, in addition to stimulating the expression of mRNA for glutathione peroxidase 1 (GPX1; Vasconcelos et al., 2021). The strategies to promote in vitro growth of preantral follicles are represented in Figure 4.

Figure 4. Strategies to promote in vitro growth of preantral follicles.

Final considerations

The transition from preantral to antral requires intense and precise granulosa–oocyte interaction, any dysregulation can interfere with the acquisition of oocyte competence. Endocrine, paracrine and autocrine factors are essential for follicular growth, antral cavity formation, granulosa cell proliferation, differentiation, and future gonadotropin dependence. In vitro studies have contributed to a better understanding of the roles of hormones and growth factors during follicular development, addressing molecular events and bidirectional communications between the oocyte and surrounding granulosa cells.