Stage-dependent actions of antimüllerian hormone in regulating granulosa cell proliferation and follicular function in the primate ovary

Abstract

Objective:

To study the direct action and physiological role of antimüllerian hormone (AMH) in regulating ovarian follicular development and function in vivo in primates.

Design:

Animals were assigned to six treatment sequences in a crossover design study. Intraovarian infusion was performed during the follicular phase of the menstrual cycle with agents including: control vehicle; recombinant human AMH (rhAMH); and neutralizing anti–human AMH antibody (AMHAb). Before ovariectomy after the final treatment, the animals received intravenous injections of bromodeoxyuridine (BrdU).

Setting:

National primate research center.

Animal(s):

Adult female rhesus macaques (Macaca mulatta).

Intervention(s):

None.

Main Outcome Measure(s):

Cycle length, follicle cohorts, and serum steroid levels were assessed. Ovarian histology, as well as granulosa cell (GC) proliferation and oocyte viability, were evaluated.

Result(s):

In vehicle-infused ovaries, a dominant follicle was observed at midcycle E₂ peak. However, rhAMH-treated ovaries exhibited an increased number of small antral follicles, whereas AMHAb-treated ovaries developed multiple large antral follicles. Serum E₂ levels in the follicular phase decreased after rhAMH infusion and increased after AMHAb infusion. The rhAMH infusion increased serum T levels. Whereas early-growing follicles of rhAMH-treated ovaries contained BrdU-positive GCs, antral follicles containing BrdU-positive GCs were identified in AMHAb-treated ovaries. Autophagy was observed in oocytes of early-growing and antral follicles exposed to AMHAb and rhAMH, respectively.

Conclusion(s):

AMH enhanced early-stage follicle growth, but prevented antral follicle development and function via its stage-dependent regulation of GC proliferation and oocyte viability. This study provides information relevant to the pathophysiology of ovarian dysfunction and the treatment of infertility.

Antimüllerian hormone (AMH), also known as müllerian inhibitory substance, is a member of the transforming growth factor beta family that regulates cell proliferation and differentiation. In female mammals, circulating AMH is secreted by granulosa cells (GCs) of growing follicles in the postnatal ovary (1). During follicular development, although the dormant primordial follicles do not produce AMH, AMH expression begins in early-growing follicles, peaks in small antral follicles, and diminishes in large antral follicles. Similar patterns of AMH expression are observed in the ovaries of rodents (1, 2), domestic animals (3, 4), nonhuman primates (5, 6), and humans (7). Because AMH-specific type II receptor is coexpressed with AMH in GCs (2, 6), AMH can serve as an autocrine or paracrine factor to regulate ovarian follicular development. AMH actions could be associated with its dynamic production by growing follicles.

The direct action of AMH during follicular development has been investigated in vitro in various species (8–11). Findings from rat, goat, and rhesus macaque follicle cultures, as well as human ovarian cortex cultures, demonstrated that AMH protein supplementation promoted early-stage follicle growth to the antral stage (8–11) but inhibited subsequent antral follicle growth and maturation in the presence of FSH (9, 10). However, data from in vivo studies are limited and correlative. Although AMH protein administration was performed in mice, prolonged systemic treatment (subcutaneous injections every 12 hours for 40 days) resulted in supraphysiologic serum levels of AMH, i.e., ~1,000 ng/mL based on pharmacokinetic data for subcutaneous AMH injection in mice, relative to a physiologic level of <50 ng/mL (12–15). Consequently, follicular development was completely suppressed from the early-growing to antral stage and the estrous cycles ceased (12). A negative correlation was observed between AMH expression, at mRNA and protein levels, in GCs and antral follicle sizes in rats (2) and sheep (16). Similarly, AMH levels in GCs and follicular fluid decreased with increasing antral follicle diameters in humans, suggesting that AMH action could be nonessential or attenuative during antral follicle development (7, 17).

GC culture has been used to examine AMH action on cell viability and proliferation, although a limitation is that GCs are obtained only from antral follicles. Similar results were found in immature GCs from bovine small antral follicles before dominant follicle selection and human KGN-GCs (a cell line corresponding to immature GCs from small antral follicles), wherein AMH treatment decreased GC proliferation without affecting cell viability (18, 19). Antral follicles and cumulus-oocyte complexes obtained from antral follicles have been treated with AMH in vitro to determine AMH effects on oocytes, with data being inconclusive. Although AMH supplementation improved mouse oocyte developmental competence during in vitro maturation (20), it did not alter oocyte competence during in vitro maturation in cows or oocyte maturation rates in cultured goat antral follicles (21, 9). Differently from in vitro studies, in vivo observations in women undergoing infertility treatment suggested a negative impact of AMH on oocyte viability and maturation, because cumulus cell AMH gene expression and follicular fluid AMH protein levels were low in antral follicles yielding mature oocytes, but were high in those containing premature or degenerating oocytes (22, 23).

Given these contrasting lines of evidence, we performed studies to elucidate AMH effects on follicular development and function in vivo during the follicular phase of the spontaneous menstrual cycle in rhesus macaques, an adequate nonhuman primate model for women’s reproductive health. Both GC and oocyte responses to AMH modulation were evaluated in developing early-stage and antral follicles.

MATERIALS AND METHODS

Ethical Approval for Animal Use

The general care and housing of rhesus macaques (Macaca mulatta) were provided by the Division of Comparative Medicine at the Oregon National Primate Research Center (ONPRC), Oregon Health & Science University, as previously described (10). Briefly, animals were pair-caged in a temperature-controlled (22°C), light-regulated (12 h light/12 h dark) room. Diet consisted of Purina monkey chow (Ralston-Purina) twice a day, supplemented with fresh fruit or vegetables once 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.

Intraovarian Infusion

Twelve adult female animals (5–9 years old; puberty occurs at 3.5 years of age and menopause at 25 years in macaques) exhibiting regular menstrual cycles of ~28 days were monitored daily for menstruation, with the first day of menses termed day 1 of the cycle. Unilateral ovariectomy was performed at the early follicular phase (cycle day 1–2) on anesthetized animals via laparoscopy by the Surgical Services Unit at ONPRC, as previously described (24). Removing one ovary in macaques does not cause aberrant menstrual cycles or compensatory ovarian hypertrophy, but forces the follicle growth and dominant follicle selection to occur in the remaining ovary (10). The procedure is done to avoid local distinction between two ovaries at the end of the menstrual cycle, i.e., one bearing the regressing corpus luteum and the contralateral ovary, which could influence the growth of remaining follicles or selection of the next dominant follicle. Blood samples were collected before the surgery to obtain baseline serum E₂, P, and T levels with the use of a chemiluminescence assay (Roche Diagnostics), as well as baseline LH levels with the use of radioimmunoassay, by the Endocrine Technologies Core at ONPRC (10).

Hemiovariectomized animals (n = 12) were assigned randomly to six treatment sequences, with two animals per sequence in a crossover design, so that animals served as their own control to increase the power of the study (Fig. 1). As illustrated in the protocol timeline (Fig. 1), intraovarian treatments were administered during menstrual cycles 1, 3, and 5, with one recuperation cycle (cycles 2 and 4) between treatment cycles to prevent carryover effects. Treatments included: 1) 5 μL/h control vehicle (phosphate-buffered saline solution [PBS]); 2) 25 ng/h recombinant human AMH protein (rhAMH; 1737-MS; R&D Systems); and 3) 500 ng/h neutralizing anti–human AMH antibody (AMHAb; MAB1737; R&D Systems). Intraovarian infusion procedures were performed as previously described (10). Briefly, treatment agents were constantly infused into the ovary during the follicular phase of the menstrual cycle via an intraovarian catheter implanted by laparotomy. Because the highest serum E₂ level during the menstrual cycle appears in 2–5 days after E₂ is > 100 pg/mL in macaques, treatment agents were delivered from cycle day 1–2 (early follicular phase) to the second day after serum E₂ was > 100 pg/mL (midcycle E₂ peak). The catheter was connected to an Alzet osmotic pump (model 2ML2; Durect Corp.) placed subcutaneously in the abdomen. Saline solution was infused at 0.11 μL/h (pump model 1004) during the recuperation cycles to keep the catheter patent; thus, only pumps were exchanged for subsequent treatment.

FIGURE 1.

A crossover design study with six treatment sequences for three treatment agents and a flowchart of protocol timeline for each individual animal. AMHAb = neutralizing anti–human antimüllerian hormone antibody infusion; BrdU = bromodeoxyuridine; rhAMH = recombinant human antimüllerian hormone protein infusion.

After a recuperation cycle 6, the 12 animals were randomly assigned to three groups for the final intraovarian infusion treatment of: 1) control vehicle (n = 4); 2) rhAMH (n = 4); and 3) AMHAb (n = 4) during cycle 7, as described above (Fig. 1). Ovaries were removed at the midcycle E₂ peak via laparoscopy. One hour before the ovariectomy, animals received an intravenous injection of 20 mg bromodeoxyuridine (BrdU; Millipore Sigma) in 10 mL Hank balanced salt solution to label proliferating cells, as previously described (25).

Serum Hormone Assays

Daily blood samples were collected from treatment cycle 1, 3, 5 (the crossover design study), and 7 (the final treatment) for steroid hormone assays to determine menstrual cycle stages and ovarian endocrine function. Serum E₂, P, and T (the crossover design study only) concentrations were measured, as described above. Blood samples from the midcycle E₂ peak (the crossover design study) were assayed for LH, as described above, to assess ovulation.

Ultrasound Imaging

To evaluate antral follicle growth during the crossover design study, ovarian imaging was performed on sedated animals at the midcycle E₂ peak of treatment cycles 1, 3 and 5 with the use of a GE Voluson 730 Expert Ultrasound Machine with 4D (3.3–9.1 MHz) transabdominal probes (General Electric Co.), as previously described (26). Briefly, two images per ovary (x and y planes) were analyzed to measure antral follicle diameters. The number of small (diameter <4 mm) and large (diameter >4 mm) antral follicles were counted. To avoid bias and decrease interobserver variation, image analyses were performed blindly by one investigator (C.V.B.) for all animals and treatment cycles.

Ovarian Morphology and Histology

Ovaries collected at the end of the final intraovarian infusion treatment during cycle 7 were fixed in 4% paraformaldehyde-PBS solution and embedded in paraffin. The 5-μm serial sections were obtained from each ovary with every tenth section stained with hematoxylin and eosin. Section imaging was performed with the use of an Aperio AT2 Scanner (Leica Biosystems). Primordial and early-growing (intermediary, primary, secondary, and preantral) follicles were counted manually by microscopy.

To determine follicular steroidogenic activity, selected ovarian sections were assessed for aromatase expression with the use of immunohistochemistry (6). Briefly, sections were incubated with goat anti–human CYP19A1 antibody (1:100; sc-14245; Santa Cruz Biotechnology). Slides were then incubated with biotinylated secondary antibodies and processed with the use of Vectastain Elite ABC Kits (Vector Laboratories). After incubation with 3,3’-diaminobenzidine, sections were counterstained with hematoxylin. Images were captured with the use of an Olympus BX40 inverted microscope and an Olympus DP72 digital camera (Olympus Imaging America). Sections with primary antibody omission served as negative control.

To assess GC proliferation, immunohistochemistry was performed on selected ovarian sections with the use of mouse anti–human BrdU antibody (1:400; SKU 0811200; MP Biomedicals) and rabbit anti-human cyclin-dependent kinase inhibitor 1A (p21) antibody (1:100; 2947; Cell Signaling Technology), as previously described (27, 28). The sections with primary antibody omission served as negative control.

Selected ovarian sections were also evaluated for GC and oocyte viability by means of immunohistochemistry using a rabbit anti-human antibody to detect autophagy-associated protein beclin- 1 (1:2,000; ab62557; Abcam), as described above. The sections with primary antibody omission served as negative control. Apoptotic cells were determined with the use of a terminal deoxynucleotide transferase–mediated dUTP nick-end labeling (TUNEL) Assay Kit (HRP-DAB; ab206386; Abcam) according to the manufacturers’ instructions. The negative control sections were incubated with the label solution only (without terminal transferase) instead of the TUNEL reaction mixture.

Statistical Analyses

Statistical analysis was performed with the use of SAS version 9.4 software (SAS Institute). Mixed-effect models were used to evaluate treatment effects that accounted for the nature of the crossover design study, such as carryover, period, and sequence effects (29). A likelihood ratio was constructed to test the overall significance of carryover effects. If there were carryover effects, carryover covariates were dropped from the full model. One-way analysis of variance followed by the Student-Newman-Keuls post hoc test was performed to analyze histologic follicle count data. Differences were considered to be significant at P< .05, and values are presented as mean ± SEM.

RESULTS

Menstrual Cycle Length

Menstrual cycle stages were determined during treatment cycles based on menses evaluation and serum E₂ levels. When data from the crossover design study were analyzed, intraovarian infusion of either rhAMH or AMHAb during the follicular phase did not alter the length of follicular phase (the first day of menses to midcycle E₂ peak), luteal phase (the day after midcycle E₂ peak to the day before the subsequent menses), or the entire menstrual cycle period (the first day of menses to the day before the subsequent menses), compared with the control vehicle infusion cycles (Table 1). Based on information from the Primate Records and Information Management at ONPRC, animal body weight remained stable from the beginning (before the first unilateral ovariectomy) to the end (after the second unilateral ovariectomy) of the treatment protocol (6.3 ± 0.5 vs. 6.6 ± 0.5 kg, respectively).

TABLE 1.

Menstrual cycle parameters following intraovarian infusion in the crossover design study.

Parameters	CTRL	rhAMH	AMHAb
Follicular phase (d)	12.3 ± 1.5	13.2 ± 1.0	11.8 ± 1.1
Luteal phase (d)	16.7 ± 0.4	17.0 ± 0.5	17.2 ± 0.5
Cycle length (d)	29.0 ± 1.2	30.2 ± 0.9	29.0 ± 0.7

Note: Values are presented as mean ± SEM, with each animal as an individual data point. AMHAb = neutralizing anti–human antimüllerian hormone antibody infusion; CTRL = control vehicle infusion; rhAMH = recombinant human antimüllerian hormone protein infusion.

Follicular Development

According to ultrasound imaging analysis during the crossover design study, one dominant follicle (diameter 5.4–8.8 mm) was observed in the ovary for all 12 animals at the midcycle E₂ peak in the control vehicle infusion cycles (Fig. 2A), whereas in the rhAMH infusion cycles, six of the 12 animals failed to form a large antral follicle (Fig. 2B). For animals developing a dominant follicle, follicle diameters in rhAMH infusion cycles (n = 6) were less (P< .05) than those in the control vehicle infusion cycles (n = 12) (Fig. 3A; Supplemental Fig. 1A [available online at www.fertstert.org]). In contrast, in the AMHAb infusion cycles, ten of the 12 animals exhibited multiple (n = 2–7) large antral follicles (diameters 4.6–8.7 mm) in the ovary (Fig. 2C), and the other two animals developed a single dominant follicle (diameters 8.1 and 9.2 mm; Fig. 3A; Supplemental Fig. 1A). There were more (P< .05) large antral follicles in the ovary in AMHAb infusion cycles compared with the control vehicle and rhAMH infusion cycles, whereas the number of small antral follicles (diameters 0.7–3.9 mm) was greater (P< .05) in rhAMH infusion cycles compared with the control vehicle and AMHAb infusion cycles (Table 2).

FIGURE 2.

The effects of intraovarian antimüllerian hormone (AMH) modulation during the follicular phase of the menstrual cycle on ovarian morphology in rhesus macaques. Representative ultrasound images were from ovaries (yellow circles) at midcycle E₂ peak after receiving the infusion of (A) control vehicle (CTRL), (B) recombinant human AMH protein (rhAMH), and (C) neutralizing anti–human AMH antibody (AMHAb) during the crossover design study. Representative hematoxylin and eosin-stained sections were from ovaries after receiving the final intraovarian infusion of (D) CTRL, (E) rhAMH, and (F) AMHAb. D = dominant follicle; L = large antral follicle. * = small antral follicle. Scale bar = 5 mm.

FIGURE 3.

The effects of intraovarian antimüllerian hormone (AMH) modulation during the follicular phase of the menstrual cycle on antral follicle growth and function in rhesus macaques. Recombinant human AMH protein (rhAMH) and neutralizing anti–human AMH antibody (AMHAb) infusion altered (A) the dominant follicle diameters, (B) the average serum T concentrations during the menstrual cycle (total T of daily blood samples/days of the menstrual cycle), (C) the average serum E₂ concentrations during the follicular phase (total E₂ of daily blood samples/days of the follicular phase), and (D) the average serum P concentrations during the luteal phase (total P of daily blood samples/days of the luteal phase), compared with control vehicle infusion (CTRL). Data are presented as mean ± SEM with 12 animals in the crossover design study. *P<.05.

TABLE 2.

Follicle counts after intraovarian infusion.

Counting method	Follicle category	CTRL	rhAMH	AMHAb
Microscopy (final intraovarian infusion)	Primordial, %	72.6 ± 10.0	74.1 ± 8.7	73.4 ± 7.8
	Early-growing, %	27.4 ± 6.4	25.9 ± 7.9	26.6 ± 7.1
Ultrasound (crossover design study)	Small antral (<4 mm), n	8 ± 3	15 ± 3a	7 ± 3
	Large antral (>4 mm, n	1 ± 0	0.5 ± 0.2	3 ± 1a

Note: Values are presented as mean ± SEM, with each animal as an individual data point. AMHAb = neutralizing anti–human antimüllerian hormone antibody infusion; CTRL = control vehicle infusion; rhAMH = recombinant human antimüllerian hormone protein infusion.

^aP<.05.

Similar patterns of antral follicle development were also observed in ovaries receiving the final intraovarian infusion treatment, as demonstrated by histologic staining. Whereas ovaries receiving the control vehicle infusion developed a single dominant follicle at the midcycle E₂ peak (Fig. 2D), rhAMH-infused ovaries exhibited multiple peripherally distributed small antral follicles (Fig. 2E) and AMHAb-infused ovaries contained several large antral follicles (Fig. 2F). Unlike antral follicles, when primordial and early-growing follicles were counted on serial ovarian sections (total follicles counted: 9,048 ± 3,273 with control vehicle, 8,110 ± 2,135 with rhAMH, and 8,737 ± 1,843 with AMHAb infusion), the percentages of follicles in each cohort were not changed by either rhAMH or AMHAb infusion (Table 2).

Hormone Production

The baseline serum E₂, P, T, and LH levels at the early follicular phase were 69.3 ± 5.5 pg/mL, 0.10 ± 0.02 ng/mL, 47.8 ± 9.5 pg/mL, and 0.53 ± 0.07 ng/mL, respectively. For the crossover design study, the average serum T concentrations during the menstrual cycle (total T of daily blood samples/days of the menstrual cycle) were higher (P< .05) in the cycles with rhAMH infusion, but not in the cycles with AMHAb infusion, than in the control vehicle infusion cycles (Fig. 3B; daily levels shown in Supplemental Fig. 1B). While the average serum E₂ concentrations during the follicular phase (total E₂ of daily blood samples/days of the follicular phase) tended to decrease (P= .06) in rhAMH infusion cycles, they increased (P< .05) in AMHAb infusion cycles, compared with control vehicle infusion cycles (Fig. 3C; daily levels shown in Supplemental Fig. 1C). The average serum P concentrations during the luteal phase (total P of daily blood samples/days of the luteal phase) were reduced (P< .05) in cycles with rhAMH infusion compared with control vehicle infusion cycles, whereas the levels were similar between AMHAb and control vehicle infusion cycles (Fig. 3D; daily levels shown in Supplemental Fig. 1D). Serum LH levels at the midcycle E₂ peak were not different between the control vehicle, rhAMH, and AMHAb infusion cycles (6.1 ± 1.4, 3.8 ± 2.9, and 5.8 ± 3.1 ng/mL, respectively).

Positive CPY19A1 staining was detected in GCs of antral follicles in ovaries receiving the final intraovarian control vehicle infusion (Supplemental Fig. 2A, available online at www.fertstert.org). Although antral follicle CPY19A1 immunostaining in rhAMH-infused ovaries was diminished (Supplemental Fig. 2B), it remained intensive in AMHAb-infused ovaries (Supplemental Fig. 2C). CYP19A1 staining was absent in the negative control ovarian section (Supplemental Fig. 2A inserts).

GC Proliferation and GC/Oocyte Viability

Ovaries were analyzed with the use of immunohistochemistry after receiving the final intraovarian infusion treatment. Positive staining of BrdU (brown) was detected in the nucleus (DNA-incorporated) of GCs in early-growing follicles, as well as in mural GCs and cumulus cells in antral follicles, in ovaries receiving the control vehicle infusion, indicating active cell division with DNA replication (Fig. 4A). In rhAMH-infused ovaries, multiple BrdU-positive GCs were observed in early-growing follicles, but BrdU-positive GCs were relatively scarce in antral follicles (Fig. 4C). In contrast, in AMHAb-infused ovaries, very few early-growing follicles contained BrdU-positive GCs whereas GCs and some theca cells were often BrdU positive in antral follicles (Fig. 4D). Primordial follicles did not contain any BrdU-positive pre-GCs despite infusion treatment agents (Figs. 4C and 4D). Immunostaining of BrdU was absent in the negative control ovarian sections (Fig. 4B).

FIGURE 4.

The effects of intraovarian antimüllerian hormone (AMH) modulation during the follicular phase of the menstrual cycle on granulosa cell proliferation in rhesus macaques. One hour before ovariectomy after the final intraovarian infusion treatment, the animals received an intravenous injection of bromodeoxyuridine (BrdU) to label proliferating cells. BrdU uptake during cell division was determined with the use of immunohistochemistry. Representative sections with positive BrdU nuclear staining (brown) were from ovaries after receiving the infusion of (A) control vehicle (CTRL), (C) recombinant human AMH protein (rhAMH), and (D) neutralizing anti–human AMH antibody (AMHAb). (B) The section with primary antibody omission served as negative control (Neg). A = antral follicle; E = early-growing follicle. Arrow: primordial follicle. Scale bar = 100 μm.

In addition, positive immunostaining of p21 (brown) was detected in the nucleus of mural GCs and cumulus cells in antral follicles in ovaries receiving the control vehicle infusion, indicating cell cycle arrest (Fig. 5A). In rhAMH-infused ovaries, very few early-growing follicles contained p21-positive GCs, whereas mural GCs and cumulus cells in antral follicles were often p21 positive (Fig. 5C). In contrast, in AMHAb-infused ovaries, p21-positive GCs were often observed in early-growing follicles, but were were relatively scarce in antral follicles (Fig. 5D). Primordial follicles did not contain p21-positive pre-GCs despite infusion treatment agents (Figs. 5C and 5D). Immunostaining of p21 was absent in the negative control ovarian sections (Fig. 5B).

FIGURE 5.

The effects of intraovarian antimüllerian hormone (AMH) modulation during the follicular phase of the menstrual cycle on granulosa cell proliferation in rhesus macaques. Cell cycle arrest was determined by assessing cyclin-dependent kinase inhibitor 1A (p21) expression using immunohistochemistry. Representative sections with positive p21 nuclear staining (brown) were from ovaries after receiving the final intraovarian infusion of (A) control vehicle (CTRL), (C) recombinant human AMH protein (rhAMH), and (D) neutralizing anti–human AMH antibody (AMHAb). (B) The section with primary antibody omission served as negative control (Neg). A = antral follicle; E = early-growing follicle. Arrow: primordial follicle. Scale bar = 100 μm.

GC and oocyte viability were further analyzed by means of TUNEL staining and beclin-1 immunostaining. Positive TUNEL staining (brown) was detected in the nucleus of GCs in atrectic follicles of all ovaries collected, indicating apoptotic cell death with DNA fragmentation (Supplemental Fig. 3A, available online at www.fertstert.org). However, morphologically healthy primordial, early-growing, and antral follicles did not contain any TUNEL-positive cells despite infusion treatment agents (Supplemental Figs. 3C and 3D). Positive TUNEL staining was absent in the negative control ovarian sections (Supplemental Fig. 3B). Immunostaining of beclin-1 was not evident in ovaries receiving the control vehicle infusion (Supplemental Fig. 3E). However, whereas early-growing follicles were beclin-1 negative, the cytoplasm of oocytes, but not GCs, in antral follicles exhibited beclin-1–positive staining in rhAMH-infused ovaries (Supplemental Fig. 3G). In contrast, in AMHAb-infused ovaries, the cytoplasm of oocytes, but not GCs, in early-growing follicles were beclin-1 positive, whereas beclin-1 staining was not detected in antral follicles (Supplemental Fig. 3H). Primordial follicles did not contain beclin-1–positive pre-GCs or oocytes despite infusion treatment agents (Supplemental Figs. 3G and 3H). Immunostaining of beclin-1 was absent in the negative control ovarian sections (Supplemental Fig. 3F).

DISCUSSION

Using the intraovarian infusion technique, the present study investigated the direct effects and physiologic role of AMH during follicular development in vivo in nonhuman primates. Although the impact of local AMH depletion on antral follicles in macaques was explored in a longitudinal design study (10), the present crossover design study examined effects of both AMH supplementation and depletion on follicles from the primordial to the antral stage with histologic assessment of treated ovaries. The short-term local AMH modulation during the follicular phase of the spontaneous menstrual cycle did not cause adverse systemic effects, e.g., cessation of menstruation or variation of cycle length, in rhesus macaques. Instead, follicle growth patterns and corresponding steroid hormone production were altered by AMH protein supplementation or blocking endogenous AMH action. Both GCs and the oocyte in follicles at the different stages of folliculogenesis, i.e., the primordial, early-growing, and antral stages, appeared to respond differently to AMH in the primate ovary.

Follicular development with in vivo AMH modulation was assessed at the end of the follicular phase of the menstrual cycle, i.e., midcycle E₂ peak. The histologic evaluation suggested that proportions of primordial and early-growing follicles were not changed despite alterations in local AMH ligand availability. The absence of AMH effect on primordial follicle activation is consistent with the previous in vivo observation that primordial follicle recruitment was not affected in sheep with AMH bioactivity knockdown by active immunization (16). However, the prolonged supraphysiologic systemic AMH protein administration was reported to inhibit primordial follicle activation in juvenile mice (13), which could be due to differences in treatment dose and interval or in animal species and age. Because it takes months for early-stage follicles to grow to the antral stage in vivo in primate species (30), changes in early-growing follicle numbers were not evident with the short-term (follicular phase) AMH modulation in the present study. However, antral follicle cohorts exhibited significant differences between treatment cycles, as revealed by ultrasound and histologic examination. AMH protein supplementation inhibited antral follicle growth, leading to an increased number of small antral follicles and a reduced size of large antral follicles. In contrast, blocking endogenous AMH action promoted antral follicle development and overruled follicle selection process resulting in multiple large antral follicles in the ovary. These data are consistent with a previous study in sheep indicating that systemically knocking down AMH bioactivity increased the number of large antral follicles (16). Therefore, AMH appears to prevent antral follicle maturation, which supports the postulated role of AMH in the dominant follicle selection process.

To determine changes to follicular function by intraovarian AMH modulation, serum steroid hormone levels were measured during the menstrual cycle. With limited antral follicle growth, circulating E₂ levels during the follicular phase were relatively low with AMH protein supplementation, followed by decreased serum P levels, indicating the limited corpus luteum function during the luteal phase. Conversely, blocking endogenous AMH action caused the elevation of circulating E₂ levels during the follicular phase, which correlated with the increased number and size of large antral follicles in the ovary. In addition, serum E₂ level variations due to AMH modulation could also result from altered follicular E₂ production because of an inhibitory action of AMH on FSH-induced aromatase expression and E₂ biosynthesis, as suggested by in vitro evidence from cultured GCs of domestic animals (16) and humans (31). Furthermore, AMH protein supplementation increased circulating T levels. During steroidogenesis in the ovary, androstenedione produced in theca cells can be converted to T or transferred to GCs. In GCs, androstenedione can also be converted to T or aromatized to E₂ (32). Therefore, if E₂ biosynthesis is blocked by AMH, it would be postulated that T produced by theca cells and GCs accumulates and enters the blood stream, resulting in elevated T levels in the circulation. Interestingly, the present study showed that macaque ovaries infused with AMH protein exhibited multiple peripherally distributed small antral follicles, resembling a polycystic ovarian morphology (PCOM) (33). It is known that clinical phenotypes of polycystic ovary syndrome (PCOS) include hyperandrogenism and PCOM. Notably, patients with PCOS also have elevated serum AMH levels (33). Thus, dysregulated AMH production may contribute, at least in part, to circulating T elevation and PCOS phenotypes or etiology in women.

AMH-regulated cellular activities associated with GC proliferation and follicle growth were further investigated in ovaries after the final intraovarian infusion treatment and analyzed at different stages of follicular development with the use of histologic evaluation. Pre-GCs in primordial follicles remained quiescent without showing any response to local AMH modulation during the follicular phase. However, AMH protein supplementation enhanced GC proliferation in early-growing follicles, as indicated by active cell division and the limited expression of a cell cycle inhibitor. In contrast, antral follicles exposed to exogenous AMH protein exhibited reduced GC proliferation, as shown by cell cycle arrest with extensive cell cycle inhibitor expression. Blocking endogenous AMH action generated opposite effects in follicles at the early-growing and antral stages. Previous in vitro studies suggested that AMH protein supplementation promoted early-stage follicle growth and inhibited antral follicle maturation in cultured goat and macaque follicles (9, 10). Although the mechanism of AMH in enhancing GC proliferation in early-growing follicles requires further investigation, a stimulatory effect of AMH on cultured Sertoli cells was reported in mice via an increase in expression of stem cell factor/c-kit ligand (34), a trophic cytokine that is also expressed by GCs. The restriction by AMH of cell cycle progression in GCs of antral follicles could be due to insufficient E₂ production, as described above, because E₂ is known to be critical for GC proliferation. Therefore, the present histologic data complement the observations in the longitudinal study supporting stage-dependent AMH actions during follicular development.

In addition, GC and oocyte viability in response to AMH modulation were determined in presumably healthy follicles at different developmental stages. Apoptotic cell death was not evident in the oocytes or GCs of morphologically healthy follicles following the intraovarian AMH modulation in the follicular phase. However, autophagy activity was detected in the oocytes, but not GCs, of antral follicles receiving AMH protein supplementation and of early-growing follicles with endogenous AMH action blocked. Previous research in immature mice and rats demonstrated that the activation of autophagy could serve as a cell survival mechanism to maintain the oocyte integrity (35, 36). When growth factors were insufficient, protective autophagy could be activated by the oocyte to prevent oocyte metamorphosis and follicle death (36). Thus, it is possible that autophagy is initiated in the oocyte when antral follicles face an E₂ shortage due to AMH treatment and when early-growing follicles experience a lack of stimulatory AMH bioactivity during AMH neutralization. It may be that moderate autophagy could sustain oocyte viability and follicle survival under conditions of tolerable stress without inducing apoptosis or follicular atresia. The effects of AMH-associated autophagy on oocyte quality and developmental competence remain to be determined.

In summary, this in vivo study demonstrated that AMH enhances early-stage follicle growth, but conversely interferes with antral follicle maturation and function in the primate ovary, via stage-dependent regulation of GC proliferation and oocyte viability. The dynamic AMH production by growing follicles is therefore essential for its autocrine/paracrine actions in coordinating follicular development and dominant follicle selection. The findings provide valuable information relevant to the pathophysiology of ovarian dysfunction, as well as the treatment of endocrine disorders and infertility, such as PCOS.