Human Reproduction, Vol.31, No.7 pp. 1522 –1530, 2016 Advanced Access publication on May 9, 2016 doi:10.1093/humrep/dew100

ORIGINAL ARTICLE Reproductive biology

Anti-Mu¨llerian hormone promotes pre-antral follicle growth, but inhibits antral follicle maturation and dominant follicle selection in primates J. Xu 1,*, C.V. Bishop 1, M.S. Lawson 1, B.S. Park 2, and F. Xu 1 1 Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006, USA 2OHSU-PSU School of Public Health, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA

*Correspondence address. E-mail: [email protected]

Submitted on January 11, 2016; resubmitted on February 18, 2016; accepted on March 18, 2016

study question: What are the direct effects and physiological role of anti-Mu¨llerian hormone (AMH) during primate follicular development and function at specific stages of folliculogenesis? summary answer: AMH actions in the primate ovary may be stage-dependent, directly promoting pre-antral follicle growth while inhibiting antral follicle maturation and dominant follicle selection. what is known already: AMH is expressed in the adult ovary, particularly in developing follicles. Studies in mice suggest that AMH suppresses pre-antral follicle growth in vitro, and inhibits primordial follicle recruitment and FSH-stimulated antral follicle steroidogenesis. study design, size, duration: For in vitro study, secondary follicles were isolated from ovaries of 12 rhesus macaques and cultured for 5 weeks. For in vivo study, intraovarian infusion was conducted on five monkeys for the entire follicular phase during two spontaneous menstrual cycles.

participants/materials, setting, methods: For in vitro study, individual follicles were cultured in a 5% O2 environment, in alpha minimum essential medium supplemented with recombinant human FSH. Follicles were randomly assigned to treatments of recombinant human AMH protein or neutralizing anti-human AMH antibody (AMH-Ab). Follicle survival, growth, steroid production, steroidogenic enzyme expression, and oocyte maturation were assessed. For in vivo study, ovaries were infused with control vehicle or AMH-Ab during the follicular phase of the menstrual cycle. Cycle length, serum steroid levels, and antral follicle growth were evaluated. main results and the role of chance: AMH exposure during culture weeks 0 –3 (pre-antral stage) promoted, while AMH-Ab delayed, antrum formation of growing follicles compared with controls. AMH treatment during culture weeks 3–5 (antral stage) decreased (P , 0.05) estradiol (E2) production, as well as the mRNA expression of cytochrome P450 family 19 subfamily A polypeptide 1, by antral follicles relative to controls, whereas AMH-Ab increased (P , 0.05) follicular mRNA levels of the enzyme. Intraovarian infusion of AMH-Ab during the follicular phase of the menstrual cycle increased (P , 0.05) the average levels of serum E2 compared with those of the control cycles. Three of the five AMH-Ab-treated ovaries displayed multiple (n ¼ 2–9) medium-to-large (2– 8 mm) antral follicles at the mid-cycle E2 peak, whereas only one large (4 –7 mm) antral follicle was observed in all monkeys during their control cycles. The average levels of serum progesterone were higher (P , 0.05) during the luteal phase of cycles following the AMH-Ab infusion relative to the vehicle infusion.

limitations, reasons for caution: The in vitro study of AMH actions on cultured individual macaque follicles was limited to the interval from the secondary to small antral stage. A sequential study design was used for in vivo experiments, which may limit the power of the study.

wider implications of the findings: The current study provides novel information on direct actions and role of AMH during primate follicular development, and selection of a dominant follicle by the late follicular phase of the menstrual cycle. We hypothesize that AMH acts positively on follicular growth during the pre-antral stage in primates, but negatively impacts antral follicle maturation, which is different from what is reported in the mouse model.

study funding/competing interest(s): NIH NICHD R01HD082208, NIH ORWH/NICHD K12HD043488 (BIRCWH), NIH OD P51OD011092 (ONPRC), Collins Medical Trust. There are no conflicts of interest. & The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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AMH regulates primate follicle development

trial registration number: Not applicable. Key words: anti-Mu¨llerian hormone / follicle culture / intraovarian infusion / pre-antral follicle / antral follicle

Introduction The growth and maturation of ovarian follicles and their enclosed oocyte during the menstrual cycle is regulated by gonadotropic hormones and local paracrine/autocrine factors in a stage-dependent manner (Gougeon, 1996). Members of the transforming growth factor beta (TGFb) superfamily are under scrutiny for their roles in controlling follicular development in various species (Knight and Glister, 2006). One particular intraovarian peptide, anti-Mu¨llerian hormone (AMH), has received considerable attention since its discovery in the post-natal sheep ovary (Be´zard et al., 1987). Besides its role as a fetal hormone regulating sexual differentiation (Mu¨nsterberg and Lovell-Badge, 1991), the local actions of AMH in the ovary are being investigated as its expression is apparent in ovarian follicles of adult female mammals. In rodents, AMH expression starts in pre-antral follicles (early primary follicles), peaks in antral follicles, and diminishes afterwards till becoming absent in pre-ovulatory follicles (Durlinger et al., 2002). Similar patterns of AMH protein expression were observed in nonhuman primates and humans. Quantitative data indicated that AMH production by macaque follicles was detectable at the primary stage, increased at the secondary stage, peaked following the antrum formation, and plateaued or declined thereafter (Xu et al., 2013a). AMH levels in follicular fluid decreased with increasing antral follicle diameters in women (Andersen et al., 2010). In addition, the potential for AMH production varies among pre-antral follicles in primates, which is not reported in other species. Consistent with immunohistochemical observations (Thomas et al., 2007), individually cultured macaque pre-antral follicles secreted different amounts of AMH, which correlated positively with follicle growth rates in vitro (Xu et al., 2013a). Thus, AMH actions as a local factor may be associated with its dynamic and heterogeneous production by growing follicles. A model of ovarian AMH actions, based primarily on in vivo and in vitro evidence in mice (Durlinger et al., 2001), suggests that AMH negatively impacts the development and function of both the pre-antral and antral follicles (Visser and Themmen, 2005). While data are consistent regarding the inhibitory effect of AMH on antral follicle maturation, findings of AMH actions on pre-antral follicle growth vary among species. A study conducted in rats indicated that AMH promoted secondary follicle growth in vitro (McGee et al., 2001). Furthermore, female sheep actively immunized to AMH, which reduced AMH bioactivity, exhibited a decline in pre-antral follicle populations (Campbell et al., 2012). Nevertheless, the direct actions and physiological role of AMH in the primate follicle remain unsubstantiated due to the lack of an adequate model in vitro or in vivo. Advances in follicle culture techniques allow individual primate preantral follicles to grow to the small antral stage in vitro, achieving follicular function in steroidogenesis, paracrine/autocrine factor production, and oocyte maturation (Xu et al., 2013b). Therefore, the present study was designed to examine the direct actions of AMH on development and function of primate secondary and small antral follicles in vitro during follicle culture. Experiments were also performed using intraovarian infusion to assess the role of AMH in primate antral follicle growth and

dominant follicle selection in vivo during the spontaneous menstrual cycle.

Materials and Methods Animal use The Division of Comparative Medicine provided the general care and housing of rhesus macaques (Macaca mulatta) at the Oregon National Primate Research Center (ONPRC), Oregon Health & Science University as previously described (Xu et al., 2013a).

Ethical approval Animal care and experimental protocols followed the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals policy, and were approved by the ONPRC Institutional Animal Care and Use Committee (Xu et al., 2013a).

In vitro study Ovary collection Twelve adult, female macaques provided ovarian tissue. Five monkeys that exhibited regular menstrual cycles were assigned to the study. Hemiovariectomies were conducted by laparoscopy at early follicular phase (Day 1 – 4 of the menstrual cycle) as previously described (Duffy and Stouffer, 2002). Seven additional animals provided ovaries at necropsy by the Pathology Services Unit, via the ONPRC Tissue Distribution Program, due to reasons unrelated to reproductive health. Ovaries were maintained in the HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-buffered holding media (Cooper Surgical, Inc., Trumbull, CT, USA) at 378C for the subsequent follicle isolation (Rodrigues et al., 2015).

Follicle isolation and culture The process of follicle isolation and culture was reported previously (Xu et al., 2013a). The ovarian cortex was cut into 1 × 1×1 mm pieces. Healthy secondary follicles (diameter 125 – 225 mm) with 2 – 4 layers of granulosa cells were isolated using 30-gauge needles. Follicles were divided among the treatment groups with 6 – 24 follicles from each monkey per group. Individual follicles were transferred into 5 ml 0.25% (w/v) sodium alginate (FMC BioPolymers, Philadelphia, PA, USA)-PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl; Thermo Fisher Scientific Inc., Grand Island, NY, USA). The droplets were cross-linked in 50 mM CaCl2, 140 mM NaCl, 10 mM HEPES solution (pH 7.2). Each encapsulated follicle was placed in individual wells of 48-well plates containing 300 ml alpha minimum essential medium (Thermo Fisher Scientific Inc.) supplemented with 1 ng/ml recombinant human FSH (NV Organon, Oss, Netherlands), 6% (v/v) human serum protein supplement (SPS; Cooper Surgical, Inc.), 0.5 mg/ml bovine fetuin, 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml sodium selenite, and 10 mg/ ml gentamicin (Sigma-Aldrich, St Louis, MO, USA) (Xu et al., 2013a). Follicles were cultured at 378C in a 5% O2 environment (in 6% CO2/89% N2) for 40 days. Media (150 ml) was collected and replaced every other day, and stored at 2208C for analysis of steroid hormone concentrations (Xu et al., 2013a).

Experiment 1: AMH addition. To examine AMH effects during follicular development, recombinant human AMH protein (catalog number: 1737-MS,

1524 Ala453-Arg560 disulfide-linked homodimer; R&D Systems, Inc., Minneapolis, MN, USA) was added to the culture media at a dose of 60 ng/ml (based on a preliminary dose – response study using 15, 30, and 60 ng/ml AMH; unpublished data). Macaque follicles that develop under control conditions generally form an antrum at Week 3 in vitro (Xu et al., 2010). Therefore, secondary follicles from 5 animals were randomly assigned to 3 groups with 93 follicles/group (2 – 4% small secondary follicles with 125 – 149 mm in diameter and 96 – 98% large secondary follicles with 150 – 225 mm in diameter per group): (i) control media; (ii) exogenous AMH during Weeks 0 – 3; and (iii) exogenous AMH during Weeks 3 – 5.

Experiment 2: AMH ablation. To evaluate the role of AMH in primate folliculogenesis, a neutralizing anti-human AMH antibody (AMH-Ab; catalog number: MAB1737; R&D Systems, Inc.) was added to the culture media at a dose of 120 ng/ml (based on a preliminary dose – response study; unpublished data). Secondary follicles from 7 animals were randomly assigned to 3 groups with 80 follicles/group (5– 9% small secondary follicles with 125 – 149 mm in diameter and 91 – 95% large secondary follicles with 150 – 225 mm in diameter per group): (i) control media; (ii) AMH-Ab during Weeks 0 –3; and (iii) AMH-Ab during Weeks 3 – 5. A separate follicle culture study using a control antibody (catalog number: MAB002; R&D Systems, Inc.) indicated that species-matched non-immune IgG did not affect macaque follicular development in vitro (unpublished data). Follicle survival and growth Follicle survival and growth were evaluated every week using an Olympus CK-40 inverted microscope and an Olympus DP11 digital camera (Olympus Imaging America Inc., Center Valley, PA, USA) as described previously (Xu et al., 2013a). The distance from the outer layer of the follicle at the widest diameter was measured using Image J 1.48 software (National Institutes of Health, Bethesda, MD, USA), so was the diameter perpendicular to the first measurement. The mean value of the two measurements was considered the follicle’s diameter. Follicles were considered atretic if the oocyte became denuded, the granulosa cells were fragmented, or the follicle diameter reduced.

Ovarian steroid assays Media samples were analyzed weekly for progesterone (P4), androstenedione (A4), and estradiol (E2) concentrations by the Endocrine Technology Support Core at ONPRC. P4 and E2 were measured using an immunochemiluminescence assay (Xu et al., 2010). A4 concentrations were measured by ELISA using an AA E-1000 kit (Rocky Mountain Diagnostics, Inc., Colorado Springs, CO, USA) based on the manufacturer’s instruction.

Follicular steroidogenic enzyme expression At the end of culture, selected healthy, in vitro-developed antral follicles devoid of dark oocytes or granulosa cells were analyzed for mRNA expression of cytochrome P450 family 17 subfamily A polypeptide 1 (CYP17A1) and cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1). Antral follicles were pooled by monkey and treatment (four follicles/monkey/treatment group), ruptured using 30-gauge needles, and entire cellular contents (follicle wall and cumulous-oocyte complex) transferred into the lysis buffer of an Absolutely RNA Nanoprep Kit (Agilent Technologies, Santa Clara, CA, USA) for subsequent RNA isolation based on the manufacturer’s instruction. RNA was reverse-transcribed into cDNA using a GoScriptTM Reverse Transcription System (Promega Corporation, Madison, WI, USA) based on the manufacturer’s instruction. Quantitative real-time PCR was performed using TaqManw Gene Expression Assays (Thermo Fisher Scientific Inc.; CYP17A1 Assay ID: Hs01124136_m1, CYP19A1 Assay ID: Hs00903413_m1) and Applied Biosystems 7900HT Fast Real-time PCR System (Thermo Fisher Scientific Inc.) as previously described (Xu et al., 2011).

Xu et al.

Oocyte retrieval, maturation and fertilization The remaining healthy antral follicles were treated with 100 ng/ml recombinant human chorionic gonadotrophin (hCG; Merck Serono, Geneva, Switzerland). Oocytes were retrieved 34 h post-hCG and evaluated as previously described (Xu et al., 2013a). Cumulus-oocyte complexes were treated with 2 mg/ml hyaluronidase (Sigma-Aldrich) in Tyrode’s albumin lactate pyruvate (TALP)-HEPES-BSA (Bovine serum albumin; 0.3% v/v) medium for 30 s. Denuded oocytes were transferred to TALP medium and photographed. Oocyte cytoplasm diameters and meiotic status were assessed using the same camera and software as described above. In vitro fertilization (IVF) was performed for metaphase II (MII) oocytes in TALP medium at 378C in 20% O2/5% CO2/75% N2 within 3 h of oocyte retrieval as previously reported (Wolf et al., 1989). Macaque semen samples were collected by the Assisted Reproductive Technologies (ART) Support Core at ONPRC as previously described (Lanzendorf et al., 1990). The resulting zygotes were cultured in 100 ml Globalw Medium (LifeGlobalw Group, IVFonline.comw) at 378C in 5% O2/6% CO2/89% N2. Embryos were photographed daily to document development. Reagents and protocols for embryo culture were provided by the ART Support Core.

In vivo study Intraovarian infusion Intraovarian infusion procedures were performed by the Surgical Services Unit, ONPRC as previously reported (Zelinski-Wooten and Stouffer, 1990). Briefly, hemi-ovariectomized monkeys (n ¼ 5) first received control vehicle (PBS) constantly infused into the ovary at 5 ml/h from Day 1–4 to the mid-cycle E2 peak (follicular phase) of the control cycle via an intraovarian catheter placed by laparotomy. The catheter was connected to an ALZET osmotic pump (Model 2ML2; Durect Corporation, Cupertino, CA, USA) placed subcutaneously in the abdomen. The catheters remain patent for up to 5 months (unpublished data), thus the pumps were exchanged for subsequent treatment. After a recuperation cycle, the same ovary of each monkey (n ¼ 5) was infused constantly with AMH-Ab at 500 ng/h from Day 1–4 to the midcycle E2 peak of the treatment cycle. A separate study comparing intraovarian infusions of the vehicle and control antibody (catalog number: MAB002; R&D Systems, Inc.) indicated that species-matched non-immune IgG did not affect monkey follicular development in vivo (unpublished data).

Steroid hormone assays Daily blood samples were collected from each cycle for assays of serum E2 and P4 concentrations by the Endocrine Technology Support Core, ONPRC as described above.

Ultrasound imaging Ovarian imaging was performed on sedated (10 mg/kg Ketamine HCl; KetaVedw, Vedco Inc., Saint Joseph, MO, USA) monkeys at the mid-cycle E2 peak using a GE Medical Systems Voluson& 730 Expert Doppler ultrasound instrument (General Electric Company, Waukesha, WI, USA) with 4D (3.3 –9.1 MHz) transabdominal probes as demonstrated in a previous report (Bishop et al., 2009). Two images per ovary (the X and Y planes) ‘were analyzed, and the number of medium-size (diameter ≥2 but ,4 mm) and large pre-ovulatory (diameter ≥4 mm) antral follicles on each image averaged, respectively. To avoid bias and decrease inter-observer variation, blind analyses were performed by only one investigator (C.V.B.) for antral follicle count and measurement from all monkeys in each cycle.

Statistical analysis Statistical analysis was performed using SAS V9.4 software (SAS Institute Inc., Cary, NC, USA). Data represent five individual animals in in vitro Experiment 1, seven individual animals in in vitro Experiment 2, and five individual animals

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in the in vivo experiment. Due to the limited sample size and uncertainty of distribution for formal statistical hypothesis testing, a permutation test (randomization test) was used to compare follicle survival rates, growing/antrum formation rates, steroidogenic enzyme expression, cycle lengths, and serum steroid concentrations between groups. A permutation test is a tool that provides statistical inferences tied to chance mechanism in random assignment. Depending on the number of classified treatment groups, one-way ANOVAs for the permuted group memberships followed by pair-wise comparison between groups or two-sample t-tests for permuted groups were performed to generate pseudo distributions of group differences. Follicle diameters, media steroid concentrations, and oocyte sizes were analyzed for each individual follicle or oocyte. Kruskal– Wallis tests followed by pair-wise mean rank tests, with each follicle/oocyte as an experimental unit, were performed to evaluate treatment effects. Due to the skewed distributions and outliers/ influential observations, as well as zero values, nonparametric Kruskal – Wallis was applied. Differences were considered significant at P , 0.05 and values are presented as the mean + SEM.

Results In vitro study Experiment 1: AMH addition AMH addition during Weeks 0– 3 did not alter the survival rates (surviving follicles/total cultured follicles) of cultured secondary follicles at the end of the first week (pre-antral stage; Table I). Macaque follicles that survived in vitro could be divided into distinct cohorts based on their growth rates by Week 5 (Xu et al., 2010). While non-growing follicles remained at the secondary stage throughout 5 weeks of culture, growing follicles formed an antrum during Weeks 3–4 under control conditions (Fig. 1A). Different from the control group, the majority of the growing follicles treated with AMH during Weeks 0–3 entered the antral stage earlier at Week 2 (Fig. 1A). However, the percentages of growing follicles (growing follicles/total surviving follicles) at Week 5 were not changed by AMH treatment during Weeks 0–3 relative to those of the control group (Table I). For AMH treatment during Weeks 3–5, diameters of growing follicles were comparable between the control and AMH groups at the end of Week 5 (Table I). However, E2 concentrations produced by the

Table I Characteristics of follicles during culture. Culture conditions

Week 1 survival (%)

Growing follicle percentage (%)

Week 5 diameter (mm)

........................................................................................ Experiment 1 CTRL

69 + 12

70 + 14

648 + 45

AMH week 0 –3

76 + 6

85 + 5

768 + 47

AMH week 3 –5

79 + 5

89 + 5

664 + 35

67 + 8

90 + 5

705 + 40

Experiment 2 CTRL AMH-Ab week 0 –3

62 + 8

90 + 5

623 + 31

AMH-Ab week 3 –5

74 + 9

85 + 8

752 + 60

A total of 93 follicles/group for Experiment 1 and 80 follicles/group for Experiment 2. Values are the mean + SEM with each follicle as an individual data point. CTRL, control; AMH, anti-Mu¨llerian hormone; AMH-Ab, AMH antibody.

Figure 1 The effects of anti-Mu¨llerian hormone (AMH; A) and AMH antibody (AMH-Ab; B) treatment during culture weeks 0 – 3 on follicle antrum formation in vitro (percentage of antral follicles/total growing follicles). Data are presented as the mean + SEM with five (A) or seven (B) animals per group. CTRL, control; N, none of the follicles formed an antrum; *, significant difference with P , 0.05.

growing follicles at Week 5 were markedly lower (P , 0.05, Kruskal – Wallis test) in the AMH group compared with the control group (Fig. 2A). Correspondingly, AMH addition during Weeks 3–5 decreased (P , 0.05, permutation test) CYP19A1 mRNA levels of cultured antral follicles harvested at Week 5 (Fig. 2A). There were no differences in media A4 concentrations or CYP17A1 mRNA expression of the growing follicles between the control and AMH groups at Week 5 (Fig. 2B). Media P4 concentrations were also comparable between groups (control versus AMH group ¼ 3.7 + 1.4 versus 1.1 + 0.2 ng/ ml). Healthy, germinal vesicle (GV)-stage oocytes were retrieved from the control and AMH groups after 34-hour hCG exposure. The oocyte diameters were comparable between the control and AMHtreated follicles (Table II). One oocyte from the control group reinitiated meiotic maturation to the metaphase I (MI) stage.

Experiment 2: AMH ablation AMH-Ab addition during Weeks 0–3 did not alter the survival rates of cultured secondary follicles at the end of the first week (pre-antral

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Figure 2 The effects of anti-Mu¨llerian hormone (AMH; A and B) and AMH antibody (AMH-Ab; C and D) treatment during culture weeks 3 – 5 on follicle steroid production and steroidogenic enzyme mRNA expression in vitro. Data are presented as the mean + SEM with 14 follicles per group. CTRL, control; CYP19A1, cytochrome P450 family 19 subfamily A polypeptide 1; CYP17A1, cytochrome P450 family 17 subfamily A polypeptide 1; *, significant difference with P , 0.05.

Table II Characteristics of oocytes retrieved from antral follicles at Week 5 (34 h after addition of recombinant human chorionic gonadotrophin). Culture conditions

Number (n) of

...........................................................................................

Follicles harvested

Oocytes retrieved

Degenerate oocytes

Healthy oocytes

..........................

GV

MI

Diameter (mm)

............................................................

GV oocytes

MI oocytes

MII oocytes

MII

............................................................................................................................................................................................. Experiment 1 CTRL

17

17

4

12

1

0

109 + 3

135

–

AMH week 0– 3

11

11

4

7

0

0

109 + 2

–

–

AMH week 3– 5

7

7

3

4

0

0

110 + 3

–

–

Experiment 2 18

18

4

13

1

0

105 + 2

111

–

AMH-Ab week 0– 3

5

5

4

1

0

0

109 + 14

–

–

AMH-Ab week 3– 5

10

10

4

5

0

1

108 + 4

–

121

CTRL

Values are the mean + SEM with each oocyte as an individual data point. CTRL, control; AMH, anti-Mu¨llerian hormone; AMH-Ab, AMH antibody; GV, germinal vesicle; MI, metaphase I; MII, metaphase II.

stage; Table I). While most of the growing follicles formed an antrum during Week 3 under control conditions, the majority of the growing follicles treated with AMH-Ab during Weeks 0–3 delayed their antrum

formation to Week 4 (Fig. 1B). However, the percentages of growing follicles at Week 5 were not changed by AMH-Ab treatment during Weeks 0–3 relative to those of the control group (Table I).

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AMH regulates primate follicle development

For AMH-Ab treatment during Weeks 3–5, diameters of growing follicles were comparable between the control and AMH-Ab groups at the end of Week 5 (Table I). The average E2 concentrations produced by the growing follicles at Week 5 were .3.6-fold higher following AMH-Ab treatment than those of the control group, though there were no statistical differences between groups (Fig. 2C). AMH-Ab addition during Weeks 3–5 increased (P , 0.05, permutation test) CYP19A1 mRNA levels of cultured antral follicles harvested at Week 5 (Fig. 2C). There were no differences in media A4 concentrations or CYP17A1 mRNA expression of the growing follicles between the control and AMH-Ab groups at Week 5 (Fig. 2D). Media P4 concentrations were also comparable between groups (control versus AMH-Ab group ¼ 5.1 + 1.3 versus 6.8 + 2.6 ng/ml). Healthy, GV-stage oocytes were retrieved from the control and AMH-Ab groups after 34-h hCG exposure. The oocyte diameters were comparable between the control and AMH-Ab-treated follicles (Table II). One oocyte from the control group reinitiated meiotic maturation to the MI stage. One oocyte from the AMH-Ab group matured to the metaphase II (MII) stage. Following IVF, the zygote cleaved to the 8-cell stage.

In vivo study Compared with the control, intraovarian infusion of AMH-Ab did not alter the length of the menstrual cycle, the follicular phase, or the luteal phase (Table III). In all five ovaries during the control cycles, only one large antral follicle (diameter ¼ 4–7 mm) was observed at the mid-

Table III Menstrual cycle parameters following intraovarian infusion. Treatment

Cycle length (days)

Follicular phase (days)

Luteal phase (days)

........................................................................................ CTRL

29.6 + 0.5

11.2 + 0.6

18.4 + 0.4

AMH-Ab

28.0 + 0.7

10.0 + 0.6

18.0 + 0.3

Values are the mean + SEM with each animal as an individual data point. CTRL, control (PBS); AMH-Ab, anti-Mu¨llerian hormone antibody.

cycle E2 peak (Fig. 3A). In contrast, three of the five ovaries receiving the AMH-Ab infusion displayed multiple (n ¼ 2–9) medium-to-large antral follicles (diameter ¼ 2– 8 mm) (Fig. 3B), while the other two developed a single large antral follicle (diameter ¼ 6 and 8 mm, respectively). While serum E2 levels were comparable at the day of pump placement, the average levels of E2 during the follicular phase (total E2/days of the follicular phase) were increased (P , 0.05, permutation test) following AMH-Ab infusion relative to those of the control cycles (Fig. 4A). There were no differences in peak levels of serum E2 between the control and AMH-Ab treatment cycles (Fig. 4A). While serum P4 levels were comparable at the day after the E2 peak, the average levels of P4 during the luteal phase (total P4/days of the luteal phase) were increased (P , 0.05, permutation test) in cycles with AMH-Ab treatment relative to those of the control cycles (Fig. 4B).

Discussion Using follicle culture and intraovarian infusion approaches, the current study provides the first in vitro and in vivo evidence supporting direct actions and physiological roles for AMH during folliculogenesis in primates. The data suggest that the local actions of AMH on follicular development are stage-dependent, particularly in the switch from FSHindependent pre-antral stage to FSH-dependent antral stage, which is different from findings in mice where AMH appears inhibitory to both the pre-antral follicle growth and antral follicle maturation (Durlinger et al., 2001). The stage-dependent effect on follicular development has been noted for other TGFb superfamily members as well (Knight and Glister, 2006). In addition, individual primate pre-antral follicles act differently in responding to AMH, which is not reported in other species. Thus, AMH actions on folliculogenesis may be species- and follicle-specific. Initially, AMH appears to promote pre-antral follicle growth to the antral stage in primates. Exogenous AMH expedited antrum formation of cultured macaque follicles, whereas blocking endogenous AMH actions by AMH-Ab delayed the preantral-to-antral follicle transition in vitro. The data are consistent with our report that AMH production by macaque secondary follicles during culture correlated positively with

Figure 3 Ultrasound images of macaque ovaries at mid-cycle estradiol peak following intraovarian infusion beginning day 1 – 4 of the menstrual cycle. The orange circle indicates the boundary of the ovary. The widest diameters of follicles are indicated with white dash lines. (A) One large antral follicle (diameter ¼ 6.1 mm) in the ovary following the control vehicle infusion. (B) Five medium-to-large antral follicles in the ovary following the anti-Mu¨llerian hormone antibody infusion. *, a large antral follicle (diameter ¼ 4.3 mm).

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Figure 4 Serum estradiol levels during the follicular phase (A) and serum progesterone levels during the luteal phase (B) following intraovarian infusion of the control vehicle (CTRL) and anti-Mu¨llerian hormone antibody (AMH-Ab) beginning day 1 – 4 of the menstrual cycle. Data are presented as the mean + SEM with five animals per group. *, significant difference with P , 0.05.

follicle growth rates in vitro (Xu et al., 2013a). This positive effect of AMH is also supported by results from cultured human ovarian cortex in which AMH addition enhanced pre-antral follicle growth to greater sizes (diameters) in vitro (Schmidt et al., 2005). Therefore, endogenous AMH may perform a stimulatory autocrine function in primate pre-antral follicles. Contrary to nonhuman primate and human studies, exogenous AMH attenuated secondary follicle growth in vitro during mouse follicle culture (Durlinger et al., 2001). However, the same study also indicated that more pre-antral follicles were observed in vivo in AMH-knockout mice, which could be due to the lack of pre-antral follicle growth to the antral stage in the absence of AMH. Interestingly, the potential for growth in response to AMH stimulation appears to vary among pre-antral follicles in primates. Despite the AMH treatment, some macaque follicles remained at the secondary stage without growth throughout culture as observed in the control group. Our previous study indicated that, though similar in size, macaque secondary follicles had different abilities to produce AMH during culture (Xu et al., 2013a). This heterogeneous production of AMH protein by pre-antral follicles was also observed in marmosets (Thomas et al.,

Xu et al.

2007). Although data are not available in nonhuman primates or women, a study in rats observed colocalization of AMH and AMH receptor II mRNAs in granulosa cells of pre-antral antral follicles (Baarends et al., 1995). Therefore, the lack of response to the AMH by non-growing follicles may be due to the insufficient expression or absence of AMH receptor II in a subgroup of the secondary follicle population in primates. Subsequently, AMH appears to suppress the maturation of primate antral follicles by limiting follicular function including steroidogenesis. In the presence of FSH, AMH treatment during the antral stage reduced E2 production by cultured macaque follicles compared with controls; notably these follicles were similar in size. As judged by mRNA data, this likely resulted from the decreased expression of the steroidogenic enzyme CYP19A1, also known as aromatase which is responsible for E2 biosynthesis, by AMH directly or AMH-mediated reduction of granulosa cell FSH-sensitivity. In contrast, neutralizing AMH-Ab treatment increased the average E2 levels .3.6-fold relative to those of the controls, though there were no statistical differences. Correspondingly, AMH-Ab addition increased aromatase mRNA expression by macaque antral follicles during culture. The data are consistent with our previous report that AMH production plateaued or decreased after the antrum formation in in vitro-developed macaque follicles when E2 production increased (Xu et al., 2013a). This detrimental effect of AMH is also supported by results from clinical studies which indicated the negative correlation between AMH concentrations in the follicular fluid and aromatase mRNA levels in granulosa cells of human antral follicles (Eilsø Nielsen et al., 2010; Jeppesen et al., 2013). Thus, the reduction of endogenous AMH may be critical for sufficient E2 production and subsequent antral follicle maturation. Additional evidence from in vitro studies using granulosa cell cultures also suggested an inhibitory action of AMH on FSH-induced aromatase expression and E2 production by antral follicles in rodents (di Clemente et al., 1994), domestic animals (Campbell et al., 2012), and women (Pellatt et al., 2011). It is noted that AMH may have negligible effects on androgen and progesterone biosynthesis by antral follicles in primates. Although E2 production and aromatase mRNA expression by cultured macaque follicles were altered by AMH and AMH-Ab addition, the treatment did not change A4/P4 production or CYP17A1 mRNA expression. Our previous study indicated that cultured macaque follicles developed granulosa and theca layers with cell-specific protein expression of aromatase and CYP17A1, respectively (Xu et al., 2013a). According to the 2-cell, 2-gonadotrophin model, A4 produced from P4 precursor by theca cells allows steroidogenic maturation of follicles by providing substrate for E2 biosynthesis in granulosa cells (McNatty et al., 1980). Therefore, the restriction of antral follicle maturation by AMH may be due to the specific lesion of E2 production in primates. AMH may also play a role in regulating the selection of dominant follicle in primates. Similar to women (Gougeon, 1996), a single antral follicle is selected for continued development to the pre-ovulatory stage in each menstrual cycle in macaques (Bishop et al., 2009). Continuous local infusion of AMH-Ab in vivo during the follicular phase of the menstrual cycle disrupted the selection process resulting in the presence of multiple medium-to-large antral follicles at the late follicular phase. Supported by data from the current in vitro study, AMH may limit antral follicle maturation by inhibiting E2 production. Therefore, blocking endogenous AMH actions by AMH-Ab may promote the continued development of several small antral follicles, present during the early follicular phase (Bishop et al., 2009), to more advanced stages. The elevated serum E2

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AMH regulates primate follicle development

levels during the follicular phase may be due to the increased number of medium-to-large antral follicles with active function in steroidogenesis, or increased aromatase expression by antral follicles. This premise is supported by observations of a negative correlation between granulosa cell AMH mRNA/protein expression and antral follicle sizes (diameters) in women (Weenen et al., 2004; Jeppesen et al., 2013). Thus, AMH may act as a physiological gatekeeper for follicular development as suggested by clinical studies (Jeppesen et al., 2013; Dewailly et al., 2014). The reduction of AMH expression by the antral follicle may be critical to its selection for further development into a dominant follicle destined for ovulation. The data are also consistent with findings from in vivo studies in rodents and domestic animals. Treatment of AMH-knockout mice with gonadotrophin-releasing hormone antagonist followed by addition of FSH increased the number of large antral follicles compared with the wild-type controls under the same treatment (Durlinger et al., 2001). An increased number of medium and large antral follicles were observed in sheep with active AMH immunization (Campbell et al., 2012). Moreover, the elevated serum P4 levels during the luteal phase in the current study may indicate the presence of multiple corpora lutea developed from the luteinized antral follicles following AMH-Ab infusion. Although three of the five ovaries infused with neutralizing AMH-Ab displayed medium-to-large antral follicles, the other two developed a single large antral follicle. It could be that the current dose of AMH-Ab was not sufficient to block actions of endogenous AMH highly produced by those two animals. It may also due to the timing of the treatment initiation. A previous study evaluated antral follicle growth in the macaque ovary by ultrasound during the spontaneous menstrual cycle (Bishop et al., 2009). The data indicated that it is possible for the dominant follicle to be selected before Day 3 of the cycle. Therefore, AMH-Ab treatment starting from cycle day 1–4 may not effectively prevent the dominant follicle selection in those two animals with early follicle selection process. Further studies will consider AMH-Ab infusion at a higher dose beginning at the late luteal phase (Day 1–4 prior to menses) of the menstrual cycle. In summary, AMH appears to be a key local factor in regulating ovarian follicular development, whose actions vary at specific stages of folliculogenesis, and likely pertains to the selection of dominant follicle in primates. While increased AMH production may promote pre-antral follicle growth to the antral stage, a reduction in AMH production and/or function may be crucial for E2 biosynthesis and oocyte maturation during subsequent antral follicle development and dominant follicle selection. Further studies are warranted to unravel the molecular mechanisms of AMH combining with or mediating the actions of gonadotropic hormones and/or other local factors to control follicular cell proliferation, differentiation, and function during folliculogenesis in primates.

Acknowledgements We are grateful for the assistance provided by members of the Division of Comparative Medicine, the Pathology Services Unit, the Surgical Services Unit, the Endocrine Technology Support Core, the Assisted Reproductive Technologies Support Core, and the Biostatistics and Bioinformatics Unit at ONPRC.

Authors’ roles J.X. provided contributions to (i) experimental design, (ii) in vitro and in vivo experiments, (iii) data analysis and interpretation, (iv) manuscript

drafting and critical revising and (v) final approval of the version to be submitted for publication. C.V.B. provided contributions to (i) in vivo experiments, (ii) data analysis and interpretation, (iii) critical manuscript revising for important intellectual content and (iv) final approval of the version to be submitted for publication. M.S.L. provided contributions to (i) in vitro and in vivo experiments, (ii) data analysis, (iii) critical manuscript revising for important intellectual content and (iv) final approval of the version to be submitted for publication. B.S.P. provided contributions to (i) experimental design, (ii) data analysis and interpretation, (iii) critical manuscript revising for important intellectual content and (iv) final approval of the version to be submitted for publication. F.X. provided contributions to (i) in vitro experiments, (ii) data analysis and interpretation, (iii) critical manuscript revising for important intellectual content and (iv) final approval of the version to be submitted for publication.

Funding Research reported in this publication was supported by the National Institutes of Health (NIH) Eunice Kennedy Shriver National Institute of Child Health & Human Development (NI CHD) R01HD082208, NIH Office of Research on Women’s Health/NICHD K12HD043488 (Building Interdisciplinary Research Careers in Women’s Health, BIRCWH), NIH Office of the Director P51OD011092 (ONPRC), and Collins Medical Trust. 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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