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. 2017 Oct 25;32(12):2456–2464. doi: 10.1093/humrep/dex322

Direct actions of androgen, estrogen and anti-Müllerian hormone on primate secondary follicle development in the absence of FSH in vitro

T Baba 1,2, A Y Ting 1,*, O Tkachenko 1, J Xu 1,3, R L Stouffer 1,3
PMCID: PMC6075619  PMID: 29077845

Abstract

STUDY QUESTION

What are effects of androgen, estrogen and anti-Müllerian hormone (AMH), independent of FSH action, on the development and function of primate follicles from the preantral to small antral stage in vitro?

SUMMARY ANSWER

Androgen and estrogen, but not AMH, promote follicle survival and growth in vitro, in the absence of FSH. However, their growth-promoting effects are limited to the preantral to early antral stage.

WHAT IS KNOWN ALREADY

FSH supports primate preantral follicle development in vitro. Androgen and estrogen augment follicle survival and growth in the presence of FSH during culture.

STUDY DESIGN SIZE, DURATION

Nonhuman primate model; randomized, control versus treatment groups. Rhesus macaque (n = 6) secondary follicles (n = 24 per animal per treatment group) were cultured for 5 weeks.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Follicles were encapsulated in 0.25% (w/v) alginate and cultured individually in modified alpha minimum essential media with (i) FSH (1 ng/ml; control), (ii) no FSH, (iii) no FSH + estradiol (E2; 100 pg/ml)/dihydrotestosterone (DHT; 50 ng/ml) and (iv) no FSH + AMH (50 ng/ml). In a second experiment, follicles were cultured with (i) FSH (1 ng/ml), (ii) no FSH, (iii) no FSH + E2 (1 ng/ml), (iv) no FSH + DHT (50 ng/ml) and (v) no FSH + E2/DHT. Follicle survival, antrum formation and growth pattern were evaluated. Progesterone (P4), E2 and AMH concentrations in culture media were measured.

MAIN RESULTS AND THE ROLE OF CHANCE

In the first experiment, FSH deprivation significantly decreased (P < 0.05) follicle survival rates in the no FSH group (16 ± 5%), compared to CTRL (66 ± 9%). E2/DHT (49 ± 5%), but not AMH (27 ± 8%), restored follicle survival rate to the CTRL level. Similarly, antrum formation rates were higher (P < 0.05) in CTRL (56 ± 6%) and E2/DHT groups (54 ± 14%), compared to no FSH (0 ± 0%) and AMH (11 ± 11%) groups. However, follicle growth rate after antrum formation and follicle diameter at week 5 was reduced (P < 0.05) in the E2/DHT group (405 ± 25 μm), compared to CTRL (522 ± 29 μm). Indeed, the proportion of fast-grow follicles at week 5 was higher in CTRL (29% ± 5), compared to E2/DHT group (10 ± 3%). No fast-grow follicles were observed in no FSH and AMH groups. AMH levels at week 3 remained similar in all groups. However, media concentrations of P4 and E2 at week 5 were lower (P < 0.05, undetectable) in no FSH, E2/DHT and AMH groups, compared to CTRL (P4 = 93 ± 10 ng/ml; E2 = 4 ± 1 ng/ml). In the second experiment, FSH depletion diminished follicle survival rate (66 ± 8% in control versus 45 ± 9% in no FSH, P = 0.034). E2 plus DHT (31.5 ± 11%) or DHT alone (69 ± 9%) restored follicle survival rate to the control (FSH) level as expected. Also, E2 plus DHT or DHT alone improved antrum formation rate. However, in the absence of FSH, E2 plus DHT or DHT alone did not support growth, in terms of follicle diameter, or steroid (P4 or E2) production after the antral stage.

LIMITATIONS REASONS FOR CAUTION

This study is limited to in vitro effects of E2, DHT and AMH during the interval from the secondary to small antral stage of macaque follicular development. In addition, the primate follicle pool is heterogeneous and differs between animals; therefore, even though only secondary follicles were selected, follicle growth and developmental outcomes might differ from one animal to another.

WIDER IMPLICATIONS OF THE FINDINGS

This study provides novel information on the possible actions of estrogen and androgen during early follicular development in primates. Our results suggest that sequential exposure of preantral follicles to local factors, e.g. E2 and DHT, followed by gonadotropin once the follicle reaches the antral stage, may better mimic primate folliculogenesis in vivo.

STUDY FUNDING/COMPETING INTEREST(S)

Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Center for Translational Research on Reproduction and Infertility 5P50HD071836, and the NIH Primate Centers Program 8P510D011092. There are no conflicts of interest.

Keywords: androgen, anti-Müllerian hormone, estrogen, folliculogenesis, primate

Introduction

Women at menarche are estimated to have 300 000–400 000 primordial follicles, but only 500 of them will complete folliculogenesis and produce a mature, fertilizable oocyte (Broekmans et al., 2009). During each menstrual cycle, approximately a thousand primordial follicles exit the resting pool and start to grow, but most undergo atresia at various developmental stages prior to maturation. From those that reach the small antral stage during the early follicular phase, one will be selected to mature to the large preovulatory follicle and ovulate at midcycle (Gougeon, 1996; Knowlton et al., 2014). Considerable research on primate ovarian folliculogenesis has focused on the development of large follicles after antrum formation due to the demand for mature oocytes for assisted reproductive technologies and infertility treatments (Hamdine et al., 2014; Fadini et al., 2015). Fewer studies have investigated the characteristics and regulation of folliculogenesis from the early preantral to small antral stage, although these follicles are relevant to the etiology of follicular disorders, such as premature ovarian failure and polycystic ovary syndrome (Hsueh et al., 2015).

It is generally believed that follicular development in women is comprised of two phases: a ‘gonadotropin-independent’ phase for preantral to small antral follicles, and a ‘gonadotropin-dependent’ phase in larger antral to ovulatory follicles (Gougeon, 1996). This concept is supported by several clinical observations. For example, Kottler et al. (2010) reported that patients with primary amenorrhea and an inactivated mutation of the FSH beta chain have small antral follicles less than 3 mm diameter in their ovaries. In addition, women with hypogonadotropic amenorrhea often display an accumulation of small antral follicles in their ovaries (Schachter et al., 1996). These findings suggest that FSH is not essential until the follicle reaches the small antral stage. However, in vitro data indicate that human preantral follicles respond to FSH (Wright et al., 1999) and, in a defined culture system, FSH is required for survival and growth. Our group observed that macaque preantral follicles cultured in the absence of FSH, typically did not survive (Xu et al., 2010); FSH is consequently included in attempts to develop preantral follicles from women in vitro (Xiao et al., 2015). This evidence is contrary to the ‘gonadotropin-independent’ theory for early growing follicles in vivo. One can hypothesize that other hormones or local factors promote the survival and growth of preantral follicles; local factors arising from neighboring follicles being more likely since preantral follicles are in a relatively avascular milieu in vivo. Researchers have suggested that various growth factors and hormones stimulate early follicular development in several species (Zhao et al., 2001; Araujo et al., 2014; Silva et al., 2016). Recent studies by our group indicate that physiological levels of androgen plus estrogen, but not progesterone, can promote the in vitro development of preantral follicles from macaques to the small antral stage (Rodrigues et al., 2015; Ting et al., 2015). However, another group reported that supraphysiological levels of estrogen (either exogenous or endogenous) reduced the proportion of developing antral follicles with increased atretic follicles in monkeys in vivo (Koering et al., 1994). To date, steroid hormone actions on follicle growth have always been studied under the influence of FSH which in turn promotes steroidogenesis. The direct actions of estrogen and androgen on early folliculogenesis, in the absence of FSH, are not known.

Another local factor to consider is anti-Mullerian hormone (AMH), a member of the transforming growth factor β superfamily, which is produced by growing preantral and small antral follicles (Xu et al., 2010). However, its proposed roles in regulating follicle growth and differentiation, are controversial, and appear species-dependent (Xu et al., 2016b). AMH inhibits the recruitment and improves the survival of primordial follicles, and the growth of preantral follicles in vitro in rats (McGee et al., 2001; Schmidt et al., 2005). Also, the growth rate of preantral and early antral follicles from macaques is positively correlated to their AMH production in vitro (Xu et al., 2011). These results suggest that AMH is a positive regulator of early folliculogenesis. Conversely, other studies suggest that AMH inhibits the activation of primordial follicles and their subsequent growth in cultured ovarian cortical pieces in humans (Carlsson et al., 2006; Nilsson et al., 2011), and inhibits FSH action in cultured luteinized granulosa cells (Chang et al., 2013). The direct actions of AMH, in the absence of FSH, on primate follicle development are not known.

Our group developed a three-dimensional (3D) follicle culture system that allows the manipulation of individual preantral follicles from rhesus macaques, and investigation on their development, regulation and function (Xu et al., 2010). In the current study, effects of estrogen, androgen and AMH on early follicular development and function were examined in a FSH-depleted milieu to further evaluate the importance of gonadotropins versus local factors in primate folliculogenesis.

Materials and Methods

Animals and ovary collection

The general care and housing of rhesus macaques (Macaca mulatta) was provided by the Division of Comparative Medicine at the Oregon National Primate Research Center (ONPRC). Animals were typically pair-caged in a temperature-controlled (22°C) light-regulated (12 L:12D) room and fed with Purina monkey chow (Ralston-Purina, Richmond, IN, USA) twice a day and water ad libitum. Animals were treated according to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Protocols were approved by the ONPRC Institutional Animal Care and Use Committee.

Ovaries were collected from six adult female rhesus macaques (10.0 ± 3.1 years old). Four monkeys went to necropsy for reasons unrelated to reproductive function, and two monkeys were healthy and underwent laparoscopic oophorectomies. The average weight of these monkeys was 5.2 ± 0.3 kg (range: 4–6 kg). None of the animals were overweight or underweight. These were unassigned cycling animals with limited menses record (n = 5 nulliparous and n = 1 multiparous). Ovaries were placed in MOPS-buffered holding media (SAGE, Trumbull, CT, USA), kept at 37°C and immediately transferred to the laboratory.

Follicle isolation, encapsulation and culture

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Procedures of follicle isolation, encapsulation and culture were previously reported (Xu et al., 2009, 2013). Briefly, healthy secondary follicles (125–250 μm in diameter) were isolated mechanically in holding media using 30-gauge needles. Each follicle was placed individually into 5 μl of 0.25% (w/v) sterile sodium alginate (FMC BioPolymers, Philadelphia, PA, USA) in phosphate buffered saline (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, Invitrogen, Carlsbad, CA, USA). The alginate droplets were cross-linked in 50 mM CaCl2, 140 mM NaCl, 10 mM HEPES solution (pH 7.2) for 1 min. Each encapsulated follicle had an intact basement membrane, 2–4 layers of granulosa cells, and a healthy, centrally located oocyte. Encapsulated follicles were placed individually in a well of 48-well plates (ThermoFisher Scientific, Santa Clara, CA, USA) containing 300 μl of alpha minimum essential medium (Invitrogen, Carlsbad, CA, USA) supplemented with 0.3% (v/v) human Serum Protein Substitute (SAGE, Trumbull, CT, USA), 0.5 mg/ml bovine fetuin, 5 μg/ml transferrin, 0.5 μg/ml insulin and 5 ng/ml sodium selenite. Follicles were cultured at 37°C in 5% O2 and 6% CO2. Half of the culture media was exchanged with fresh media every other day, and stored at −20°C for subsequent hormone measurements. Culture was continued for 5 weeks and recombinant human chorionic gonadotropin (hCG; 100 ng/ml; Ovidrel, Serono, Geneva, Switzerland) was added to the media to initiate meiotic resumption in antral follicles that reached 500 μm in diameter.

Experiment 1: estrogen, androgen, and AMH replacement

To evaluate effects of local factors in FSH-depleted milieu on early folliculogenesis, either estradiol (E2) plus dihydrotestosterone (DHT) or AMH were added to culture media. Secondary follicles (24 follicles/monkey/group) from three rhesus macaques were randomly assigned to four groups: (i) CTRL group: basal media plus recombinant human (rh) FSH (1 ng/ml; NV Organon, Oss, the Netherlands) and vehicle (0.01% final ethanol concentration); (ii) no FSH group: basal media with vehicle; (iii) E2/DHT group: basal media plus E2 (100 pg/ml) and DHT (50 ng/ml) and (iv) AMH group: basal media plus rhAMH (50 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA) and vehicle.

The rhAMH used in this study contains only the mature region of AMH. The biological activity of this rhAMH Protein from the R&D Systems is measured by its ability to inhibit growth of OVCAR three human ovarian carcinoma cells according to the manufacture's data. In addition, the biological activity of this AMH product was also reported in our and others studies using rat ovary organ culture (Nilsson et al., 2011), monkey ovarian follicle culture (Xu et al., 2016a), and human endometrial stromal cell culture (Chen et al., 2014).

Experiment 2: estrogen and/or androgen replacement

To discern individual actions of estrogen and androgen, E2 and/or DHT were added to the culture media in the absence of FSH. Secondary follicles (24 follicles/monkey/group) from three rhesus macaques were randomly divided into five treatment groups: (i) CTRL group: basal media plus FSH and vehicle; (ii) no FSH group: basal media with vehicle; (iii) high E2 group: basal media plus E2 (1000 pg/ml; Ting et al., 2015); (iv) DHT group: basal media plus DHT (50 ng/ml) and (v) E2 + DHT group: basal media with E2 (1000 pg/ml; Ting et al., 2015) and DHT (50 ng/ml).

Exogenous steroid and AMH concentrations were equivalent to those tested previously in a FSH-replete milieu (Rodrigues et al., 2015; Ting et al., 2015; Xu et al., 2016b). While a 10-fold higher E2 concentration was used in Experiment 2 compared to Experiment 1, both doses are within physiological ranges (100 pg/ml used in Experiment 1 is equivalent to circulating E2 levels while 1000 pg/ml used in Experiment 2 is equivalent to E2 levels found in follicular fluid in the rhesus macaque). Previously, we have demonstrated that macaque secondary follicles treated with these E2 concentrations had similar survival and antrum formation rates (Ting et al., 2015).

Follicle survival, growth, antrum formation and classification of follicular growth

Follicle survival and growth were assessed weekly using an Olympus CK-40 inverted microscope and an Olympus digital camera (Olympus Imaging America, Inc., Center Valley, PA, USA). Follicles were considered to be degenerating if the oocyte became dark or no longer surrounded by 2–4 layers of granulosa cells, the granulosa cells were dark or lysed, or the diameter of the follicle decreased. The diameter of each follicle was determined by averaging two measurements, perpendicular to each other, using Image J 1.44 software (National Institute of Health, Bethesda, MD, USA).

Based on a prior study (Xu et al., 2011), surviving follicles were categorized into three types according to their sizes at week 5: (i) fast-grow follicles: diameters ≥500 μm; (ii) slow-grow follicles: diameters between 250 and 499 μm; and (iii) no-grow follicles: diameters ≤250 μm.

Oocyte retrieval and maturation

Oocyte retrieval and evaluation were performed at 37°C as previously described (Xu et al., 2011, 2013). Briefly, 36 h after addition of hCG to the culture media, follicles were punctured to release their cumulus-oocyte complex (COC) into Tyrode's albumin lactate pyruvate (TALP)-HEPES-bovine serum albumin (BSA) (0.3% w/v) medium. The COCs were then treated with 2 mg/ml hyaluronidase in TALP-HEPES-BSA for 30 s to dissociate cumulus cells and obtain denuded oocytes. Retrieved oocytes were transferred to TALP medium and photographed. Oocyte diameters (excluding the zona pellucida) and meiotic status were assessed using the same camera and software as described above.

Measurements of AMH, E2 and P4

Media concentrations of AMH known to peak at week 3 (Xu et al., 2010), plus E2 and P4 known to peak at week 5 (Xu et al., 2010) from fast-grow and slow-grow follicles were measured in conditioned culture media by the Endocrine Technology Support Core at ONPRC. AMH levels were analyzed by enzyme-linked immunosorbent assay using a commercial kit (AL-105; Ansh Labs, Webster, TX, USA) based on the manufacturer's instructions. An Immulite 2000 chemiluminescence-based automatic clinical platform (Siemens Healthcare Diagnostics, Deerfield, IL, USA) was used for E2 and P4 assays.

Data analysis

Data are presented as mean ± standard error of the mean with three animals per treatment group. Statistical significance was determined using two-way ANOVA and Student-Newman-Keuls post hoc analysis (SigmaPlot 11.0; Systat Software, Inc., San Jose, CA, USA) for data comparison among different treatment groups at a given time. One-way ANOVA with repeated measurements was performed for comparing data among different time points within the same group. Differences were considered significant at P ≤ 0.05.

Results

Experiment 1: estrogen, androgen and AMH replacement

Average follicle survival rates at week 5 were 65.9% in CTRL, 16.2% in no FSH, and 48.8% in E2/DHT and 26.6% in AMH groups (Fig. 1A). FSH deprivation decreased (P < 0.05) follicle survival rate compared to that of the CTRL. However, E2/DHT exposure restored follicle survival to the CTRL level. Follicle survival rate in the AMH group was not different from that of the no FSH group. Of the surviving follicles, 56% in CTRL, 0% in no FSH, 53.8% in E2/DHT and 11.1% in AMH groups reached the antral stage (Fig. 1B). Antrum formation was absent in the no FSH group and lower than that of the CTRL group (P < 0.05). E2 plus DHT restored the antrum formation rate to CTRL levels. AMH did not affect antrum formation rate in comparison to the no FSH group. CTRL follicles were comprised of 31.4% fast-grow, 54.3% slow-grow, and 14.3% no-grow follicles (Fig. 2A). FSH deprivation decreased (P < 0.05) the percentages of fast- and slow-grow follicles and increased (P < 0.05) the percent of no-grow follicles. E2/DHT exposure restored the proportion of slow-grow follicles, but the proportion of fast-grow follicles was reduced (P < 0.05) in comparison to the CTRL group. AMH treatment produced similar follicle growth pattern to the no FSH group. Diameters of fast-grow follicles were similar between CTRL and E2/DHT groups (only sources) at the beginning and after weeks in culture, but follicle diameters at week 5 (after antrum formation) were smaller (P < 0.05) in the E2/DHT group compared to CTRL (Fig. 2B).

Figure 1.

Figure 1

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Experiment 1: The effect of estrogen (E2)/androgen (DHT) and AMH on follicle survival (Panel A) and antrum formation rate (Panel B) of surviving follicles after 5 weeks of culture. CTRL, control; no FSH, media without FSH; E2/DHT, media without FSH plus E2 (100 pg/ml) and DHT (50 ng/ml); AMH, media without FSH plus AMH (50 ng/ml). Data are expressed as mean ± SEM with three animals per treatment group. Statistically significant differences among culture conditions are indicated by different letters. AMH, anti-Müllerian hormone.

Figure 2.

Figure 2

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Growth characteristics of surviving follicles (Panel A) and follicle diameters of fast-grow follicles at weeks 0, 3 and 5 (Panel B). CTRL, control; no FSH, media without FSH; E2/DHT, media without FSH plus estradiol (E2) (100 pg/ml) and DHT (50 ng/ml); AMH, media without FSH plus AMH (50 ng/ml). Different capital, lower case, Greek letters and asterisk represent significant differences between treatment groups. DHT, dihydrotestosterone.

Fast-grow follicles produced higher (P < 0.05) levels of P4 in comparison to slow-grow follicles in the CTRL group (Fig. 3A). In the absence of FSH, follicles did not produce any detectable P4 (Fig. 3A). With the addition of E2/DHT or AMH, only five follicles (out of 22) produced minimal levels of P4 while the rest did not produce any detectable P4 (Fig. 3A). E2 levels exhibited a similar pattern as P4 (Fig. 3B). In the media of the E2/DHT group, exogenous E2 was the sole source of assayed E2 detected. Fast-grow follicles also produced higher (P < 0.05) levels of AMH compared to slow-grow follicles in CTRL follicles (Fig. 4). However, AMH levels were not affected (P > 0.05) by any treatment.

Figure 3.

Figure 3

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Progesterone (P4, Panel A) and estradiol (E2, Panel B) concentrations in culture media of fast-grow and slow-grow follicles at week 5. CTRL, control; no FSH, media without FSH; E2/DHT, media without FSH plus estradiol (E2) (100 pg/ml) and DHT (50 ng/ml); AMH, media without FSH plus AMH (50 ng/ml). There was no fast-grow follicles in no FSH and AMH groups. Data are presented as mean ± SEM with three animals per treatment group. Different capital and lower case letters represent statistical differences in fast-grow and slow-grow follicle populations between treatment groups. An asterisk represents differences between fast-grow and slow-grow follicles within the same treatment group.

Figure 4.

Figure 4

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AMH concentrations in culture media of fast-grow and slow-grow follicles at week 3 (peak value). CTRL, control; no FSH, media without FSH; E2/DHT, media without FSH plus estradiol (E2) (100 pg/ml) and DHT (50 ng/ml); AMH, media without FSH plus AMH (50 ng/ml). Data are presented as mean ± SEM with three animals per treatment group. Different capital and lower case letters represent statistical differences in fast-grow and slow-grow follicle populations between treatment groups. An asterisk represents differences between fast-grow and slow-grow follicles within the same treatment group.

Meiotic maturation in response to hCG did not occur in any fast-grow follicles; only germinal vesicle (GV)-stage oocytes were retrieved. In the CTRL group, eight small antral follicles provided four healthy GV-stage oocytes (107 ± 5 μm in diameter). Three healthy GV-stage oocytes (118 ± 6 μm in diameter) were retrieved from the three follicles in E2/DHT group. There were no fast-grow follicles in no FSH and AMH groups.

Experiment 2: estrogen and/or androgen replacement

Follicle survival rates at week five were 65.5% in CTRL, 41.0% in no FSH, 49.3% in E2, 69.4% in DHT and 70.0% in E2/DHT groups (Fig. 5A). FSH deprivation decreased (P < 0.05) follicle survival rate compared to the CTRL group. While E2 supplementation did not affect follicle survival rate compared to the no FSH group, DHT and E2/DHT supplementation restored follicle survival to the CTRL level. Antrum formation rates were 60.0% in CTRL, 11.1% in no FSH, 22.1% in E2, 30.8% in DHT and 31.5% in E2/DHT group (Fig. 5B). FSH deprivation decreased (P < 0.05) antrum formation rates compared to CTRL. E2 and/or DHT supplementation increased the antrum formation rate to an intermediate level between CTRL and no FSH. Over 19% of CTRL follicles were fast-grow follicles (Fig. 6A). Deprivation of FSH eliminated fast-grow follicles (Fig. 6A). E2/DHT, and to a lesser extent E2 or DHT alone, resulted in fast-grow follicles, but their diameters were less than those of the CTRL (Fig. 6B).

Figure 5.

Figure 5

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Experiment 2: Estrogen and/or androgen replacement. Follicle survival (Panel A) and antrum formation of surviving follicles (Panel B) after 5 weeks of culture. CTRL, control; no FSH, media without FSH; E2, media without FSH plus E2 (1000 pg/ml); DHT, media without FSH plus DHT (50 ng/ml); E2/DHT, media without FSH plus estradiol (E2) (1000 pg/ml) and DHT (50 ng/ml). Data are presented as mean ± SEM with three animals per treatment group. Statistically significant differences among culture conditions are indicated by different letters.

Figure 6.

Figure 6

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Growth characteristics of surviving follicles (Panel A) and follicle diameters of fast-grow follicles at weeks 0, 3 and 5 (Panel B). CTRL, control; no FSH, media without FSH; E2, media without FSH plus estradiol (E2) (1000 pg/ml); DHT, media without FSH plus DHT (50 ng/ml); E2/DHT, media without FSH plus E2 (1000 pg/ml) and DHT (50 ng/ml). An asterisk represents significant differences between CTRL and all treatment groups.

Patterns of E2 and P4 production, as well as treatment effects, in Experiment 2 were similar to Experiment 1 (data not shown). Notably, steroid levels in the absence of FSH, or in the presence of DHT alone were negligible, low E2 levels in media from the E2 and E2/DHT groups could again be attributed to the exogenous estrogen added. As in Experiment 1, fast-grow follicles produced higher (P < 0.05) levels of AMH compared to slow-grow follicles in the CTRL group (data not shown). AMH levels produced by fast-grow or slow-grow follicles were similar in all treatment groups.

Similar to Experiment 1, fast-grow follicles did not produce any mature oocytes following hCG treatment. In the CTRL group, eight antral follicles yielded four healthy GV-stage oocytes (108 ± 6 μm in diameter). Only one healthy GV-stage oocyte (107 μm in diameter) was retrieved in the E2 group. No healthy oocytes were retrieved in either the DHT or E2/DHT groups. There were no fast-grow follicles, and thus no retrieved oocytes, in the no FSH group.

Discussion

It is well-established that gonadotropins, especially FSH, play a key role in regulating antral follicle growth and differentiation, including production of steroid hormones. As summarized by Hardy et al. (2016), depletion of FSH either genetically, surgically or pharmacologically results in arrest of follicle growth at the late preantral to early antral stage in animal models, e.g. mice or sheep. Clinical observations in women with mutations in FSH receptor also suggest that FSH is not crucial for follicle development until after antrum formation (Kottler et al., 2010). However, in vitro studies indicate that preantral follicles can express FSH receptors and respond to this gonadotropin, such that FSH either promotes (mouse and goat; Kreeger et al., 2005; Saraiva et al., 2011; Laird et al., 2017) or is required (nonhuman primate; Xu et al., 2011) for their survival and growth. Thus, FSH may have a direct physiological action on preantral follicles (Hardy et al., 2016) and/or other local factors (either regulated or independent of FSH) could serve to promote follicle development to the antral stage. In the current study, we demonstrated that macaque preantral follicles can survive and grow independent of FSH in vitro with support from local ovarian steroid hormones, specifically E2 and DHT, but not from AMH.

Similar to our previous report (Xu et al., 2010), we observed that FSH deprivation diminished survival and inhibited antrum formation in macaque preantral follicles cultured from the secondary stage. One study in mice discovered that preantral follicles, with two layers of granulosa cells or more, grew in the presence or absence of FSH (Hardy et al., 2016) although the culture interval was only 4 days. However, the effect of FSH appeared to vary between mouse secondary follicles as a function of follicle diameter. Follicles of <130 μm diameter grew in the absence of FSH, whereas those greater than 130 μm did not. Likewise, Kreeger et al (2005) reported that survival of multilayered but not two-layered secondary follicles from mice was significantly less in the absence of FSH. Our in vitro data are consistent with the mouse model that FSH can be a trophic hormone for more advanced secondary follicles. However, it is unknown if FSH provides support in vivo, especially considering the relative avascularity of follicles at this stage.

Interestingly, combined E2 and DHT supplementation was able to restore preantral follicle development to the antral stage in the absence of FSH. But after reaching the antral stage, follicles cultured with E2 and DHT in a FSH-depleted milieu grew slower, in comparison to CTRL follicles cultured in the presence of FSH. In addition, follicles treated with E2 and DHT, while producing similar levels of AMH at week 3, did not produce detectable amounts of E2 or P4 at week 5. Thus, E2 plus DHT displayed the ability to promote preantral growth and differentiation, as judged by timely AMH production (Xu et al., 2010) until antrum formation (week 3; AMH measured). But while supporting preantral follicle development, exogenous E2 and DHT cannot replace FSH action to promote further follicular development and steroid production in the antral stage. Our results suggest that the local actions of E2/DHT on primate follicular development are stage-specific, and are more important during the FSH-sensitive preantral stage than the FSH-dependent antral stage.

In our previous reports, E2 alone (Ting et al., 2015) was as effective as DHT (Rodrigues et al., 2015) in promoting survival and growth of macaque preantral follicles in vitro in the presence of FSH. In the current report, the pro-survival and growth effect of E2 effects appeared somewhat less than those of DHT alone, with the latter's effects comparable to E2 plus DHT (% survival, antrum formation) and/or controls (% survival). Perhaps, E2 actions are more dependent on FSH; this concept is supported by extensive evidence from non-primate species that FSH and E2 synergize to promote preantral follicle growth (Richards, 1980; Gore-Langton and Daniel, 1990). For example, E2 promotes the expression of FSH receptors on granulosa cells (Cai et al., 2015) and augments FSH-stimulated antrum formation in rats (Gore-Langton and Daniel, 1990). However, the combination of E2 and DHT may be important for primate follicular development, since DHT alone can inhibit follicular differentiation either at the preantral stage (AMH production) or antral stage (estrogen production), based on our in vitro studies (Rodrigues et al., 2015). The later evidence supports the concept that androgens can have dose- and stage-dependent effects in the primate follicle to support early growth, but suppress later differentiation. High levels of androgen in developing follicles could initially promote preantral follicle growth, but later suppress antral follicle growth and E2 production. Androgens are also reported to cause follicular atresia in large antral follicles, including apoptosis of granulosa cells, in both in vivo and in vitro systems (Hillier and Tetsuka, 1997; Drummond, 2006; Gleicher et al., 2011). The follicle survival data from our studies do not support this androgen action, and other reports demonstrated that androgen inhibits atresia of antral follicles in mouse, pig, and monkey (Drummond, 2006; Chen et al., 2015). Our model is consistent with the hyperandrogenemia associated with altered follicular dynamics (both preantral and antral cohorts) and reduced estrogen production in women with polycystic ovary syndrome (Franks et al., 2008; Franks, 2008).

The current study provides no evidence that AMH influences the growth of primate secondary to small antral follicles in the absence of FSH in vitro. However, exogenous AMH promotes the growth of macaque preantral follicles in the presence of FSH (Xu et al., 2016b). Therefore, FSH action may be required for the trophic effects of AMH, just as it promotes AMH production in follicles at the preantral stage. Indeed, our laboratory demonstrated that AMH production by preantral follicles in the presence of FSH serves as a predictor for follicle growth to the antral stage; higher AMH levels correlated with increased follicle diameter (Xu et al., 2011, 2016a, 2016b). FSH may also promote AMH receptor (Ilha et al., 2016) but this effect in our culture system is unclear.

In this study, macaque secondary follicles (125–250 μm) grew to a maximum diameter of 500–1000 μm, representative of the small antral stage, after five weeks in culture. However, no oocyte matured to the MII stage following hCG treatment in these fast-grow follicles, possibly due to the small number of fast-grow follicles achieved in the current study. In addition, these small antral follicles may require further growth/maturation for their oocytes to resume meiotic maturation in response to an ovulatory stimulus. Nevertheless, healthy GV-intact oocytes can be obtained from some hCG-exposed follicles. Although the number of GV oocytes did not differ significantly among treatment groups, greater numbers are required to assess effects of steroids or AMH on oocyte parameters in the absence of FSH.

In summary, FSH deprivation decreased survival rate in macaque preantral follicles. E2 plus DHT or DHT alone, and to a lesser extent E2 alone, rescued follicle survival and antrum formation in FSH-deprived milieu. However, the growth-promoting effects of E2 and DHT did not continue after antrum formation; DHT/E2 did not compensate for FSH action with regard to the production of E2 and P4. Limitations of this study include the following. At this time, we cannot state whether E2 and DHT actions are mediated through their receptors. In addition, it is possible that DHT can be converted to 5α-androstane-3β,17β-diol which can activate ERβ (Handa et al., 2009). Localization of receptors for E2 and DHT, as well as blocking their actions using receptor antagonists will be examined in future studies. Furthermore, the primate follicle pool is heterogeneous and differs between animals; therefore, even though only secondary follicles were selected, follicle growth and developmental outcomes might differ from one study to another. To circumvent this limitation, all studies are performed with a control group and follicles from the same animals are randomly distributed among treatment groups. Together, our findings suggest that DHT and E2 or DHT alone are important for primate follicle survival and growth during the preantral stage. After antrum formation, FSH is required for follicle growth as well as steroid production. Therefore, sequential exposure of preantral follicles in vitro to local factors, such as estrogen and androgen, followed by exposure FSH/LH at the antral stage, may better mimic primate folliculogenesis in vivo, including steroidogenesis and gametogenesis. This information could be valuable for understanding the mechanisms underlying abnormal folliculogenesis (i.e. PCOS). Also, it may advance our current method of in vitro culture of primate follicles to produce mature oocytes for fertility preservation in cancer patients.

Acknowledgements

We are grateful to the members of the ONPRC Surgical Services Unit and Division of Comparative Medicine for surgical and animal care, the ART Core for providing oocyte culture media, the Endocrine Technology Support Core for hormone assay, and Dr Mary Zelinski and Ms. Maralee Lawson for assistance with follicle isolation.

Authors’ roles

T.B. provided contributions to (i) follicle isolation and culture, (ii) data analysis and interpretation and (iii) manuscript drafting and critical revising. A.Y.T. provided contributions to (i) experimental design, (ii) follicle isolation and culture (iii) data analysis and interpretation and (iv) critical manuscript revising for important intellectual content. O.T. and J.X. provided contributions to (i) follicle isolation and culture and (ii) data analysis. R.L.S. provided contributions to (i) experimental design, (ii) data analysis and interpretation and (iii) critical manuscript revising for important intellectual content. All authors have approved the final version and submission of this manuscript.

Funding

Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Center for Translational Research on Reproduction and Infertility 5P50HD071836, and the NIH Primate Centers Program 8P510D011092. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Conflict of interest

None declared.

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