The Role of Androgens in Mammals Folliculogenesis

Introduction: Steroid hormones production is a physiological process termed steroidogenesis. An important stage of this process is the conversion of androgens into estrogens through aromatase enzyme. Furthermore, androgens are important in the process of folliculogenesis, promoting follicular growth in different species. Thus, the aim of this review was to present the process of synthesis, mechanism of action, and importance of androgens in folliculogenesis. Additionally, the main results of in vitro culture of ovarian cells in the presence of these hormones were emphasized.Review: Folliculogenesis begins in prenatal life in most of species and can be defined as the process of formation, follicular growth, and oocyte maturation. Preantral follicles represent 95% of the follicular population and assisted reproductive technologies have been developed (e.g., Manipulation of Oocytes Enclosed in Preantral Follicles - MOEPF) in order to avoid the great follicle loss that occurs naturally in vivo by atresia. The MOEPF aim to obtain a large number of competent oocytes from preantral follicles and then subject to in vitro maturation, fertilization, and culture for embryo production. However, the development of an efficient medium to ensure the follicular survival and oocyte maturation is the major challenge of this biotechnology. To achieve the success on in vitro culture, the effects of substances as androgens on follicular development have been evaluated. Androgens are steroid hormones produced in theca cells (TC) that are fundamental for follicular growth. These cells provide all the androgens required by the developing follicles for conversion into estrogens by the granulosa cells (GC). Androgens receptors (AR) are localized in cell cytoplasm of all follicular categories, being more expressed in preantral follicles. The androgen pathway initiates through its connection to its receptor, making a complex androgen-AR, that in the nucleus helps on the process of gene transcription related with follicular survival. This mechanism is androgen receptor genomic activity. In addition to genomic action, there is an androgen receptor non-genomic activity. This occurs through activation of AR and its interaction with different signaling molecules located on the cell membrane, triggering events that aid in the follicular development. Regardless of the androgens actions, ovarian cells of several species subjected to in vitro culture have shown the importance of these hormones on the follicle development. Recent studies demonstrated that androgens addition on the culture medium stimulated the activation of preantral follicles (bovine and caprine), antrum formation (swine), survival (non-primate), and oocyte maturation (antral follicles; bovine). Also, some studies suggest that the addition of these hormones on in vitro culture is dose-dependent and species-specific.Conclusion: This review shows the role of androgens in different stages of follicular development and its action as a substrate for steroidogenesis and transcription of genes related to follicular survival and oocyte maturation. However, when these hormones should be added during in vitro follicular culture and which concentration is required remains unclear, being necessary more studies to elucidate these aspects.

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Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles

Pesquisa Veterinária Brasileira ◽

10.1590/1678-5150-pvb-5723 ◽

2018 ◽

Vol 38 (12) ◽

pp. 2284-2288

Author(s):

Camila Bizarro-Silva ◽

Suellen M. González ◽

Isabela Búfalo ◽

Andressa G. Lindquist ◽

Fabiana D. Sarapião ◽

...

Keyword(s):

In Vitro Culture ◽

Culture System ◽

Culture Medium ◽

Follicular Development ◽

Significance Level ◽

Primordial Follicles ◽

Preantral Follicles ◽

Medium Exchange ◽

Significant Difference

ABSTRACT: The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher’s test with a significance level of p<0.05. Out of a total of 399 follicles evaluated, 174 (43.6%) were primordial follicles, 225 (56.4%) were in development, and 63.76% were morphologically intact. In the in vitro culture performed over two days, there was no significant difference in relation to follicular integrity after medium replacement (p>0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements.

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Significance of pro-angiogenic estrogen metabolites in normal follicular development and follicular growth arrest in polycystic ovary syndrome

Human Reproduction ◽

10.1093/humrep/deaa098 ◽

2020 ◽

Vol 35 (7) ◽

pp. 1655-1665

Author(s):

Soledad Henríquez ◽

Paulina Kohen ◽

Xia Xu ◽

Claudio Villarroel ◽

Alex Muñoz ◽

...

Keyword(s):

Polycystic Ovary ◽

Follicular Development ◽

Male Factor Infertility ◽

Follicular Growth ◽

Estrogen Metabolites ◽

Male Factor ◽

Fertile Women

Abstract STUDY QUESTION Do alterations in pro- and anti-angiogenic estrogen metabolites in follicular fluid (FF) contribute to the follicular growth arrest and anovulation associated with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER FF of PCOS women with anovulation have reduced levels of pro-angiogenic estrogen metabolites (EMs) and vascular endothelial growth factor (VEGF) compared to that of fertile women with regular menstrual cycles, but exogenous gonadotropins increase the pro-angiogenic EMs and VEGF levels in PCOS women. WHAT IS KNOWN ALREADY PCOS is characterized by the arrest of follicular development that leads to chronic anovulation. Follicular arrest is generally associated with elevated plasma levels of luteinizing hormone (LH), androgens and anti-Mullerian hormone (AMH). There is also reduced angiogenesis in the follicles of PCOS women compared to those of normal cycling women. It is known that angiogenesis is a critical factor during follicular development. We and other investigators have explored the role of EMs in ovarian angiogenesis, particularly in human corpus luteum function, showing that 4-hydroxyestrone (4-OHE1) and 16-ketoestradiol (16-kE2) have pro-angiogenic effects while 2-methoxyestradiol (2-ME2) and 2-methoxyestrone (2-ME1) have anti-angiogenic effects. Additionally, 2-hydroxyestradiol (2-OHE2), which is produced in the ovary, has proliferative and pro-angiogenic properties. We hypothesized that EMs could be involved in angiogenesis necessary for ovarian follicular development in fertile women, and that dysregulation of these factors may contribute to follicular arrest in PCOS. The relationship between EMs, VEGF and AMH in the pathophysiology of follicular arrest in PCOS has not been previously studied at a follicular level in anovulatory women without ovulation induction. STUDY DESIGN, SIZE, DURATION This is a comparative experimental study of serum and FF collected from different sized follicles (antral ˂10 mm and dominant ˃16 mm) of women with and without ovarian stimulation. The study included women with regular menstrual cycles who were proven to be fertile (n = 20) and PCOS women with follicular arrest who were candidates for ovarian drilling (n = 17), as well as other patients requiring ovarian stimulation, i.e. control women undergoing IVF for male factor infertility (n = 12) and PCOS women undergoing IVF (n = 17). In vitro studies were carried out on granulosa-lutein cells (GCs) obtained from subsets of women undergoing IVF for male factor infertility (n = 6) and PCOS women undergoing IVF (n = 6). GCs were maintained in culture for up to 6 days. PARTICIPANTS/MATERIALS, SETTING, METHODS Intrafollicular estradiol, estrone and EMs concentrations were determined by high performance liquid chromatography–mass spectrometry. Testosterone in serum was measured by RIA, and LH, FSH and sex hormone-binding globulin in serum were measured with IRMA kits. AMH was determined in serum and FF by enzyme linked immunosorbant assay (ELISA). VEGF levels were measured in FF and conditioned medium by ELISA. Conditioned medium were obtained from cultured GCs. The angiogenic potential was assessed by in vitro angiogenic assays. MAIN RESULTS AND THE ROLE OF CHANCE Pro-angiogenic EMs (4-OHE1, 16-kE2 and 2-OHE2) and VEGF were lower in FF of antral follicles of PCOS women with follicular arrest compared those of fertile women with ovulatory cycles (P < 0.05). In contrast, higher concentrations of AMH were found in FF of antral follicles from PCOS women with follicular arrest compared to those of fertile women with ovulatory cycles (P < 0.05). Exogenous gonadotropins used in IVF increased pro-angiogenic EMs and VEGF production in PCOS women, reaching similar profiles compared to control women receiving gonadotropins in their IVF treatment for male factor infertility. The pro-angiogenic EM 2-OHE2 increased the angiogenic potential and VEGF levels of GCs from PCOS women compared to the basal condition (P < 0.05). These findings suggest that there is a role for pro-angiogenic EMs in the control of follicular VEGF production. LIMITATIONS, REASONS FOR CAUTION The limitations include the possibility that in vitro analysis of GCs might not reflect the in vivo mechanisms involved in the pro-angiogenic action of 2-OHE2 since GCs obtained at the time of oocyte retrieval belong to a very early stage of the luteal phase and might not be representative of GCs during follicular growth. Therefore, our findings do not conclusively rule out the possibility that other in vivo mechanisms also account for defective angiogenesis observed in PCOS. WIDER IMPLICATIONS OF THE FINDINGS The present study highlights the significance of EMs, angiogenic factors and AMH and their interaction in the pathophysiology of follicular development in PCOS. This study provides new insights into the role of pro-angiogenic factors in follicular arrest in PCOS. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by CONICYT/FONDECYT 1140693 and NIH grant R01HD083323. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A

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Influence of follicle-stimulating hormone concentrations on the integrity and development of bovine follicles cultured in vitro

Zygote ◽

10.1017/s0967199418000497 ◽

2018 ◽

Vol 26 (5) ◽

pp. 417-423 ◽

Cited By ~ 1

Author(s):

C. Bizarro-Silva ◽

M.M. Santos ◽

J.R. Gerez ◽

S.M. González ◽

L.A. Lisboa ◽

...

Keyword(s):

Ovarian Tissue ◽

Follicle Stimulating Hormone ◽

Follicular Development ◽

Follicular Growth ◽

Minimal Essential Medium ◽

Preantral Follicles ◽

Mean Diameter ◽

Stimulating Hormone

SummaryThis study investigated the in vitro culture of bovine follicles included in ovarian tissue for 2 or 6 days (D2 or D6), with the addition of different concentrations of follicle-stimulating hormone (FSH) (0, 10, 50, 100 or 200 ng/ml). Data were compared for follicular development, morphological integrity and diameter of follicles and oocytes. Ovaries (n = 10) from Nelore cows (n = 5) were divided into fragments (n = 11 per ovary) and were immediately fixed in Bouin’s solution (D0) or were individually cultured for 2 or 6 days in one of the described concentrations of FSH and then processed for histology. Compared with the rates of follicular development at D2 for minimal essential medium (MEM) (75.0%) and 50 ng/ml of FSH (71.1%), the best rates of follicular development at D2 were obtained with 10 (84.7%), 100 (87.5%) and 200 ng/ml of FSH (85.0%; P<0.05). After 6 days of cultivation, there were no differences among treatments regarding follicular growth. The morphological integrity of preantral follicles was better maintained by 100 ng/ml FSH for 2 and 6 days of cultivation (51.2 and 40.4%, respectively; P<0.05) than that for MEM (D2: 30.9%, D6: 20.8%), 10 (D2: 39.2%, D6: 22.8%), 50 (D2: 30.4%, D6: 28.8%) and 200 ng/ml FSH (D2: 45.2%, D6: 36.8%). FSH at 100 ng/ml provided the highest mean diameter averages: 34.5±10.8 µm at D2 and 33.2±12.5 µm at D6 (P<0.05). We concluded that the medium supplemented with 100 ng/ml FSH during in vitro culture provided appropriate conditions for the development and morphological integrity of preantral follicles in cattle.

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Activin-A promotes the development of goat isolated secondary follicles in vitro

Zygote ◽

10.1017/s0967199413000294 ◽

2013 ◽

Vol 23 (1) ◽

pp. 41-52 ◽

Cited By ~ 6

Author(s):

Cleidson Manoel Gomes da Silva ◽

Simone Vieira Castro ◽

Luciana Rocha Faustino ◽

Giovanna Quintino Rodrigues ◽

Ivina Rocha Brito ◽

...

Keyword(s):

Growth Rate ◽

Formation Rate ◽

Follicular Development ◽

Activin A ◽

Mrna Levels ◽

Follicular Growth ◽

P450 Aromatase ◽

Estradiol Levels

SummaryThe role of activin-A in follicular development and on the mRNA expression levels of different genes in goat secondary follicles was evaluated. Goat secondary follicles (≥150 μm) were cultured for 18 days under control conditions or with the addition of either 50 or 100 ng/ml activin-A (Experiment 1). The mRNA levels for the genes that code for activin-A, ActR-IA, ActR-IB, ActR-IIA, ActR-IIB, follicle stimulating hormone receptor (FSH-R) and P450 aromatase were measured in each condition (Experiment 2). We observed that after 6 days of culture, the antrum formation rate was higher in cultures with added activin-A than in the cultured control (P < 0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate in the final third of the culture (days 12–18), resulting in a higher percentage of meiosis resumption (P < 0.05). On day 6, the addition of activin-A (50 ng/ml) increased the levels of ActR-IA mRNA compared with the cultured control (P < 0.05). After 18 days, the addition of 50 ng/ml activin-A significantly increased the levels of its own mRNA compared with the non-cultured control. Moreover, this treatment reduced the mRNA levels of P450 aromatase in comparison with the cultured control (P < 0.05). Higher levels of P450 aromatase mRNA were found for both activin-A treatments compared with the non-cultured control (P < 0.05). No difference in estradiol levels was detected among any of the tested treatments. In conclusion, the addition of activin-A to culture medium stimulated early antrum formation as well as an increase in the daily follicular growth rate and the percentage of meiosis resumption.

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Comparison of three in vitro culture systems for maturation of early preantral mouse ovarian follicles

Zygote ◽

10.1017/s0967199405003151 ◽

2005 ◽

Vol 13 (2) ◽

pp. 167-175 ◽

Cited By ~ 21

Author(s):

Nathalie Mousset-Siméon ◽

Pierre Jouannet ◽

Laëtitia Le Cointre ◽

Christiane Coussieu ◽

Catherine Poirot

Keyword(s):

Oocyte Maturation ◽

Follicular Development ◽

Optimal Growth ◽

Ovarian Follicles ◽

Meiotic Maturation ◽

Follicular Growth ◽

Hydrophobic Membrane ◽

Culture Systems ◽

Nuclear Maturation

The aim of this study was to compare three different culture systems for in vitro follicular growth and oocyte maturation in ovarian follicles of mice in order to assess the technique with the optimal growth and improved rate of meiotic maturation. The three systems tested were culture under oil, on a hydrophobic membrane and on agar respectively. Early preantral follicles were cultured for 12 days in α-MEM GlutaMAX medium. Follicular growth, oocyte meiotic maturation, oocyte extrusion, atresia and estradiol production were analysed. Follicular development showed two phases in the three systems, with slow growth before day 5 and subsequent acceleration. The percentage of follicles transferred into oocyte maturation medium was significantly higher after culture under oil. The proportion of oocytes that achieved nuclear maturation (metaphase II) was higher when follicles were cultured under oil or on a hydrophobic membrane than on agar. Our results support the use of culture under oil for in vitro follicular growth from the early preantral stage in order to obtain metaphase II oocytes. Fertilization ability of these oocytes and the capacity to obtain healthy mice in a reproducible manner warrants further investigation.

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SET Improved Oocyte Maturation by PP2A and Inhibited Oocyte Apoptosis in Mouse Oocytes

10.21203/rs.3.rs-591644/v1 ◽

2021 ◽

Author(s):

Lingling Gao ◽

Siying Wang ◽

Jianbo Xu ◽

Dan Lu ◽

Yugui Cui

Keyword(s):

Oocyte Maturation ◽

Follicular Development ◽

Control Group ◽

Transcription Control ◽

Chi Square ◽

Multifunctional Protein ◽

Pp2a Activity ◽

Chi Square Analysis

Abstract Background: SET is a multifunctional protein involved in a variety of molecular processes such as transcription control, chromatin remodeling, cell apoptosis, and cell-cycle regulation. In ovaries SET is predominantly expressed in theca cells and oocytes. And in PCOS patients the expression of SET was increased than normal people. The current study was designed to determine whether SET play a role in oocyte maturation and apoptosis, which may provide clues for the underlying pathological mechanism of follicular development in PCOS patients.Methods: Oocytes at GV stage were collected from 6-week-old female ICR mice. 20-25 oocytes per group were placed in M16 medium. Then these oocytes were randomly divided into control group or treatment group for further study. Normal distribution was assessed by the Shapiro-Wilk test, and the student t test was used to compare the mRNA and protein levels. Chi-square analysis was used to compare the ratios of oocytes.Results: SET overexpression improved oocyte maturation whereas SET knockdown inhibited oocyte maturation. Moreover, SET negatively regulated PP2A activity in oocytes. Treatment with PP2A inhibitor okadaic acid (OA) promoted oocyte maturation. Furthermore, PP2A knockdown confirmed the key role of PP2A in oocyte maturation, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition on oocyte maturation. The central role of PP2A in SET-mediated regulation of oocyte maturation was confirmed by the finding that SET increased the expression of BMP15 and GDF9 and PP2A inhibited their expression. Besides, SET inhibited oocyte apoptosis through decreased the expression of caspase 3 and caspases 8, while PP2A had no effect on oocyte apoptosis. Conclusions: SET promoted oocyte maturation by inhibiting PP2A activity and inhibited oocyte apoptosis in mouse in-vitro cultured oocytes, which may provide a pathologic pathway leading to oocyte development disorder in PCOS.

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The effect of bioidentical nanostructured progesterone in the in vitro culture of preantral follicles and oocyte maturation

Cell and Tissue Research ◽

10.1007/s00441-020-03233-6 ◽

2020 ◽

Vol 382 (3) ◽

pp. 657-664

Author(s):

Carlos Cordeiro Neto ◽

Kadja Lopes Soares ◽

Rodrigo Tenório Padilha ◽

Marco Antônio Botelho ◽

Dinalva Brito Queiroz ◽

...

Keyword(s):

In Vitro Culture ◽

Oocyte Maturation ◽

Preantral Follicles

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Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents

Reproduction ◽

10.1530/rep.1.00648 ◽

2005 ◽

Vol 130 (2) ◽

pp. 147-156 ◽

Cited By ~ 70

Author(s):

I Demeestere ◽

J Centner ◽

C Gervy ◽

Y Englert ◽

A Delbaere

Keyword(s):

Oocyte Maturation ◽

Culture Medium ◽

Follicular Growth ◽

Medium Effects ◽

Paracrine Factors ◽

Autocrine Factors ◽

Preantral Follicles ◽

Whole Process ◽

Complex Process

Folliculogenesis is a complex process regulated by various paracrine and autocrine factors. In vitro growth systems of primordial and preantral follicles have been developed for future use of immature oocytes, as sources of fertilizable oocytes and for studying follicular growth and oocyte maturation mechanisms. Rodents were often chosen for in vitro follicular culture research and a lot of factors implicated in folliculogenesis have been identified using this model. To date, the mouse is the only species in which the whole process of follicular growth, oocyte maturation, fertilization and embryo transfer into recipient females was successfully performed. However, the efficiency of in vitro culture systems must still be considerably improved. Within the follicle, numerous events affect cell proliferation and the acquisition of oocyte developmental competency in vitro, including interactions between the follicular cells and the oocyte, and the composition of the culture medium. Effects of the acting factors depend on the stage of follicle development, the culture system used and the species. This paper reviews the action of endocrine, paracrine factors and other components of culture medium on in vitro growth of preantral follicles in rodents.

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Growth Differentiation Factor-9 Mediates Follicle-Stimulating Hormone-Thyroid Hormone Interaction in the Regulation of Rat Preantral Follicular Development

Endocrinology ◽

10.1210/en.2009-0262 ◽

2009 ◽

Vol 150 (12) ◽

pp. 5566-5574 ◽

Cited By ~ 28

Author(s):

Noriko Kobayashi ◽

Makoto Orisaka ◽

Mingju Cao ◽

Fumikazu Kotsuji ◽

Arthur Leader ◽

...

Keyword(s):

Thyroid Hormone ◽

Direct Action ◽

Follicular Development ◽

Reproductive Function ◽

Normal Female ◽

Follicular Growth ◽

Preantral Follicles ◽

Differentiation Factor ◽

Growth Differentiation Factor 9

Abstract FSH regulates follicular growth in a stage-development fashion. Although preantral follicle stage is gonadotropin responsive, FSH is not required for preantral follicular growth. With the antrum, the follicles continue growing under the influence of FSH and become gonadotropin dependent. Although thyroid hormone is important for normal female reproductive function, its role and interaction with FSH in the regulation of preantral ovarian follicular growth is yet to be defined. In the present study, we have examined the action and interaction of FSH and T3 in the regulation of the growth of preantral follicles, especially in their transition from preantral to early antral stage, using an established follicle culture system and evaluated the involvement of growth differentiation factor-9 (GDF-9) in this process in vitro. We have demonstrated that although T3 alone had no effect on follicular development, it markedly enhanced FSH-induced preantral follicular growth. Although FSH alone significantly down-regulated FSH receptor (FSHR) mRNA abundance in the preantral follicles and T3 alone was ineffective, expression of the message was significantly increased in the presence of both hormones. In addition, intra-oocyte injection of GDF-9 antisense oligonucleotides (GDF-9 morpholino) induced follicular cell apoptosis and suppressed follicular growth induced by FSH and T3. These responses were attenuated by exogenous GDF-9. Our findings support the concept that thyroid hormone regulates ovarian follicular development through its direct action on the ovary and that promotes FSH-induced preantral follicular growth through up-regulation of FSHR, a mechanism dependent on the expression and action of oocyte-derived GDF-9.

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Role of nerve growth factor (NGF) and its receptors in folliculogenesis

Zygote ◽

10.1017/s0967199412000111 ◽

2012 ◽

Vol 21 (2) ◽

pp. 187-197 ◽

Cited By ~ 23

Author(s):

R.N. Chaves ◽

A.M.C.V. Alves ◽

L.F. Lima ◽

H.M.T. Matos ◽

A.P.R. Rodrigues ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Ovarian Follicle ◽

Follicular Development ◽

Nerve Growth ◽

Ovarian Follicle Development ◽

Ovarian Cells ◽

Affinity Receptor

SummaryNerve growth factor (NGF) is a prototype member of the neurotrophins family and has important functions in the maintenance of viability and proliferation of neuronal and non-neuronal cells, such as certain ovarian cells. The present review highlights the role of NGF and its receptors on ovarian follicle development. NGF initiates its multiple actions through binding to two classes of receptors: the high affinity receptor tyrosine kinase A (TrkA) and the low-affinity receptor p75. Different intracytoplasmic signalling pathways may be activated through binding to NGF due to variation in the receptors. The TrkA receptor activates predominantly phosphatidylinositol-3-kinase (PI3K) and mitogenic activated protein kinase (MAPK) to promote cell survival and proliferation. The activation of the phospholipase type Cγ (PLCγ) pathway, which results in the production of diacylglycerol (DAG) and inositol triphosphate (IP3), culminates in the release of calcium from the intracytoplasmic cellular stocks. However, the details of activation through p75 receptor are less well known. Expression of NGF and its receptors is localized in ovarian cells (oocyte, granulosa, theca and interstitial cells) from several species, which suggests that NGF and its receptors may regulate some ovarian functions such as follicular survival or development. Thus, the use of NGF in culture medium for ovarian follicles may be of critical importance for researchers who want to promote follicular developmentin vitroin the future.

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