Western-style diet in the presence of elevated circulating testosterone induces adipocyte hypertrophy without proinflammatory responses in rhesus macaques.

PROBLEM: Anovulatory infertility is commonly associated with hyperandrogenemia (elevated testosterone, T), insulin resistance, obesity, and white adipose tissue (WAT) dysfunction associated with adipocyte hypertrophy. However, whether hyperandrogenemia and adipocyte hypertrophy per se induce a proinflammatory response is unknown. METHOD OF STUDY: Young adult female rhesus macaques were exposed to an obesogenic Western-style diet (WSD) in the presence of elevated circulating testosterone (T+WSD) or a low-fat control diet with no exogenous T. Immune cells residing in visceral omental white adipose tissue (OM-WAT), corpus luteum and the contralateral ovary, endometrium, lymph nodes, bone marrow, and peripheral blood mononuclear cells were characterized by flow cytometry during the luteal phase of the reproductive cycle. RESULTS: Following one year of treatment, T+WSD animals became more insulin-resistant and exhibited increased body fat and adipocyte hypertrophy compared to controls. T+WSD treatment did not induce macrophage polarization toward a proinflammatory phenotype in the tissues examined. Additionally, T+WSD treatment did not affect TNFα production by bone marrow macrophages in response to toll-like receptor agonists. While the major lymphoid subsets were not significantly affected by T+WSD treatment, we observed a significant reduction in the frequency of effector memory CD8+ T-cells (Tem) in OM-WAT, but not in other tissues. Notably, OM-WAT Tem frequencies were negatively correlated with insulin resistance as assessed by the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR). CONCLUSION: This study shows that short-term T+WSD treatment induces weight gain, insulin resistance, and adipocyte hypertrophy, but does not have a significant effect on systemic and tissue-resident proinflammatory markers, suggesting that adipocyte hypertrophy and mild hyperandrogenemia alone are not sufficient to induce a proinflammatory response.

The combined impact of testosterone and Western-style diet on endometriosis severity and progression in rhesus macaques†.

Polycystic ovary syndrome (PCOS) is associated with irregular menstrual cycles, hyperandrogenemia, and obesity. It is currently accepted that women with PCOS are also at risk for endometriosis, but the effect of androgen and obesity on endometriosis has been underexplored. The goal of this study was to determine how testosterone (T) and an obesogenic diet impact the progression of endometriosis in a nonhuman primate (NHP) model. Female rhesus macaques were treated with T (serum levels approximately 1.35 ng/ml), Western-style diet (WSD; 36% of calories from fat compared to 16% in standard monkey chow) or the combination (T + WSD) at the time of menarche as part of a longitudinal study for ~7 years. Severity of endometriosis was determined based on American Society for Reproductive Medicine (ASRM) revised criteria, and staged 1-4. Stages 1 and 2 were associated with extent of abdominal adhesions, while stages 3 and 4 were associated with presence of chocolate cysts. The combined treatment of T + WSD resulted in earlier onset of endometriosis and more severe types associated with large chocolate cysts compared to all other treatments. There was a strong correlation between glucose clearance, homeostatic model assessment for insulin resistance (HOMA-IR), and total percentage of body fat with presence of cysts, indicating possible indirect contribution of hyperandrogenemia via metabolic dysfunction. An RNA-seq analysis of omental adipose tissue revealed significant impacts on a number of inflammatory signaling pathways. The interactions between obesity, hyperandrogenemia, and abdominal inflammation deserve additional investigation in NHP model species.

Increasing vitamin D levels to improve fertilization rates in cattle.

Recently, interest in supplementing vitamin D (Vit D) to improve aspects of health, mainly in human fertility, has emerged. Still, supplementation of Vit D above the minimum required levels has yet to be explored in cattle despite evidence for Vit D receptors in reproductive tissues. The objective of this study was to establish if a dose-response relationship exists between Vit D exposure and success of in vitro production (IVP) of embryos and, if acute supplementation of Vit D improves pregnancy rates during timed artificial insemination (TAI) of dairy cows. Cumulus-oocyte complexes (COCs) were obtained from ovaries acquired from a local abattoir and cultured in five different IVP treatments from three separate collections (Control, 50, 100, 150, and 200 ng/mL of 1,25(OH)2D3; n = 20-30 COCs/group). In Experiment 2, dairy breed cows (n = 100) were synchronized for TAI with the PresynchOvsynch protocol. Cows received 150,000 IU of Vit D (n = 48) or castor oil as control (n = 53) along with gonadotropin-releasing hormone (GnRH) 24 h before TAI. Serum samples were collected before and 24 h after treatment. A small cohort of cows (n = 4) received the same treatments in two separate cycles and follicular fluid (FF) was collected after 24 h for calcidiol (25OHD) analyses. Increased concentrations of Vit D resulted in decreased rates of maturation of COC (150 and 200 ng/mL vs. control and 50 ng/mL; P = 0.01). Supplementation with 50 ng/mL resulted in greater numbers of early blastocyst and blastocyst stage embryos (P < 0.009). Pregnancy at first breeding did not differ (P = 0.13) between groups, but serum 25OHD increased in treated females after 24 h (P = 0.002). The FF 25OHD levels were reflective of serum levels, however, the observed increase in the treatment cycle (P = 0.04) was parallel to an overall increase in serum 25OHD during the entire second cycle, likely due to increased environmental sunlight exposure (March, control vs. May, treatment). A similar increase in the serum 25OHD in the lactating commercial herd maintained in covered housing was not observed, although experiments were conducted during a similar timeframe. This herd had levels of 25OHD near the low end of sufficiency according to National Research Council (NRC) guidelines. We conclude mild Vitamin D supplementation with concentrations at the higher end of NRC guidelines can improve maturation rates of recovered COCs. However, longer term supplementation may be needed to appreciate any benefits on fertility.

Vitamin D is an important hormone that among other things, contributes to bone health, immunity, and reproduction. Recently, research has linked vitamin D to fertility in other species (primates), and therefore the objectives of the current research were to determine if mild supplementation with Vitamin D impacted fertility in female cattle. A dose-dependent relationship was detected between concentrations of vitamin D and embryo development. The concentration of 50 ng/mL of vitamin D appeared to be beneficial to early embryogenesis. Studies in dairy-breed females indicated serum levels of vitamin D correlated well with intrafollicular levels in the periovulatory follicle. Finally, a fertility trial investigated if a single dose of vitamin D improves fertility when administered before artificial insemination in cattle. There were no detectable benefits to this brief supplementation with vitamin D on measures of fertility in this group. It is concluded supplementation with vitamin D improves embryo development in vitro, but brief supplementation did not impact pregnancy success. Longer-term supplementation with vitamin D may be needed to appreciate any measurable benefits on fertility.

History, insights, and future perspectives on studies into luteal function in cattle.

The corpus luteum (CL) forms following ovulation from the remnant of the Graafian follicle. This transient tissue produces critical hormones to maintain pregnancy, including the steroid progesterone. In cattle and other ruminants, the presence of an embryo determines if the lifespan of the CL will be prolonged to ensure successful implantation and gestation, or if the tissue will undergo destruction in the process known as luteolysis. Infertility and subfertility in dairy and beef cattle results in substantial economic loss to producers each year. In addition, this has the potential to exacerbate climate change because more animals are needed to produce high-quality protein to feed the growing world population. Successful pregnancies require coordinated regulation of uterine and ovarian function by the developing embryo. These processes are often collectively termed "maternal recognition of pregnancy." Research into the formation, function, and destruction of the bovine CL by the Northeast Multistate Project, one of the oldest continuously funded Hatch projects by the USDA, has produced a large body of evidence increasing our knowledge of the contribution of ovarian processes to fertility in ruminants. This review presents some of the seminal research into the regulation of the ruminant CL, as well as identifying mechanisms that remain to be completely validated in the bovine CL. This review also contains a broad discussion of the roles of prostaglandins, immune cells, as well as mechanisms contributing to steroidogenesis in the ruminant CL. A triadic model of luteolysis is discussed wherein the interactions among immune cells, endothelial cells, and luteal cells dictate the ability of the ruminant CL to respond to a luteolytic stimulus, along with other novel hypotheses for future research.

The corpus luteum (CL) forms on the ovary from the cellular remnants of the follicle following ovulation. The function of the CL is to produce progesterone that is required for successful pregnancy. In the absence of an embryo or sufficient embryonic signaling, the uterus will release a prostaglandin that kills the CL in a process called luteolysis. Therefore, the CL and the embryo share a symbiotic relationship, each requiring the other to be healthy and functional for survival. The Northeast Multistate Project, one of the oldest in the nation, has produced a large body of evidence that has enhanced our understanding of how the CL functions, its regulation, and the impact of ovarian activity on fertility of cattle. This review highlights some of the important advances made in the understanding of the ruminant CL.

Physiological Action of Progesterone in the Nonhuman Primate Oviduct.

Therapies that target progesterone action hold potential as contraceptives and in managing gynecological disorders. Recent literature reviews describe the role of steroid hormones in regulating the mammalian oviduct and document that estrogen is required to stimulate epithelial differentiation into a fully functional ciliated and secretory state. However, these reviews do not specifically address progesterone action in nonhuman primates (NHPs). Primates differ from most other mammals in that estrogen levels are >50 pg/mL during the entire menstrual cycle, except for a brief decline immediately preceding menstruation. Progesterone secreted in the luteal phase suppresses oviductal ciliation and secretion; at the end of the menstrual cycle, the drop in progesterone triggers renewed estrogen-driven tubal cell proliferation ciliation secretory activity. Thus, progesterone, not estrogen, drives fallopian tube cycles. Specific receptors mediate these actions of progesterone, and synthetic progesterone receptor modulators (PRMs) disrupt the normal cyclic regulation of the tube, significantly altering steroid receptor expression, cilia abundance, cilia beat frequency, and the tubal secretory milieu. Addressing the role of progesterone in the NHP oviduct is a critical step in advancing PRMs as pharmaceutical therapies.

Matrix-free three-dimensional culture of bovine secondary follicles to antral stage: Impact of media formulation and epidermal growth factor (EGF).

Disrupted/disordered ovarian steroidogenesis is associated with several fertility disorders such as polycystic ovary syndrome in humans and cystic ovarian disease in cattle. Methods to interrogate theca cell processes as part of follicular development are necessary to further research into treatments for these types of disorders. Multilayer follicles of dairy-breed cows were placed into culture in a novel matrix-free 3D system using round bottom low-attachment plates. Follicles were first cultured in the presence of two types of media previously used for in vitro follicle maturation (basal α-MEM and basal T-199). After the optimal media was identified, impact of supplementation of epidermal growth factor (EGF) on growth and survival of bovine secondary follicles to antral stage was evaluated. No differences were observed in growth and survival of follicles cultured in basal α-MEM media or basal T-199 media, although T-199 media's high phenol red content made assessment of follicles difficult. Further studies were then performed with α-MEM media. Three cohorts of follicles were observed based on time to antrum formation: ≤ 5 days (fast), 6-19 days (slow), or survived but did not form an antrum by 21 days (no). Supplementation of EGF to the basal α-MEM media dramatically improved follicle survival rates in culture (defined as follicles that either formed an antrum or did not form an antrum but did not die during 21 day culture period) from 29% to 95.7% (Chi-square p < 0.0001). However, in follicles that survived to form an antrum there were no differences in proportion of fast, slow and no antrum follicles after addition of EGF (Chi-square p > 0.7). Fast antrum follicles treated with EGF plateaued in size earlier in culture compared to controls (p = 0.013). Slow and no antrum follicles were larger in diameter during EGF culture than controls (p's < 0.0001). Many follicles cultured in this matrix-free system that formed an antrum approached 1.5-2 mm in size, an improvement from previous single follicle culture methods used for bovine pre-antral follicles in vitro. In addition, follicles displayed functional steroidogenesis in vitro producing measureable levels of estradiol and androstenedione. This matrix-free 3D culture system provides an excellent in vitro model to explore processes associated with folliculogenesis in cattle.

Insulin-like growth factor 2 is produced by antral follicles and promotes preantral follicle development in macaques†.

Insulin-like growth factors (IGFs) are known for their involvement in endocrine and paracrine regulation of ovarian function. Although IGF2 is the predominant circulating and intraovarian form of IGFs in primate species, the stage-specific follicular expression, action, and regulation of IGF2 are not well defined. Therefore, experiments were conducted to investigate the follicular IGF production in response to steroid hormone regulation and the direct IGF actions on follicular development and function in vitro. Preantral follicles were isolated from rhesus macaque ovaries and cultured to the antral stage in media supplemented with follicle-stimulating hormone and insulin. Follicles were randomly assigned to treatment groups: (a) control, (b) trilostane (a steroid synthesis inhibitor), (c) trilostane + estradiol, (d) trilostane + progesterone, and (e) trilostane + dihydrotestosterone. Media was analyzed for IGF concentrations, which were correlated to follicle growth. Follicles produced IGF2, but not IGF1, at the antral stage. Steroid depletion decreased, whereas steroid replacement increased, IGF2 production by antral follicles. Media IGF2 levels correlated positively with antral follicle diameters. Macaque preantral follicles and granulosa cells were subsequently cultured without (control) and with recombinant human IGF2 supplementation. Follicle survival, growth, and paracrine factor production, as well as granulosa cell proliferation and gonadotropin receptor gene expression, were assessed. IGF2 addition increased follicle survival rates, diameters and inhibin B production, as well as granulosa cell proliferation. These data demonstrate that IGF2 produced by antral follicles, in response to steroid hormone regulation, could act as a paracrine factor that positively impacts preantral follicle development and function in primates.

Individual and combined effects of 5-year exposure to hyperandrogenemia and Western-style diet on metabolism and reproduction in female rhesus macaques.

STUDY QUESTION: What is the impact of prolonged exposure to hyperandrogenemia (T), Western-style diet (WSD) and the combination on metabolic and reproductive function in female rhesus macaques, particularly in the post-partum period? SUMMARY ANSWER: Combined T + WSD worsened measures of insulin sensitivity and parameters of cyclicity following prolonged (5 years) exposure, but there was no effect on post-partum metabolic function. WHAT IS KNOWN ALREADY: Women with hyperandrogenemia due to polycystic ovary syndrome are at higher risk for gestational diabetes and Type 2 diabetes post-partum, but it is unknown if this is related to hyperandrogenemia. Hyperandrogenemia in the presence of a WSD worsens metabolic function in female nonhuman primates. STUDY DESIGN, SIZE, DURATION: Female rhesus macaques began treatment near menarche (roughly 2.5 years of age) consisting of either cholesterol (control; C) or testosterone (T) implants (average serum levels 1.4 ng/ml) and exposure to standard monkey chow or a WSD (15 vs 36% of calories from fat, respectively). The four groups were maintained on treatment for 3 years, underwent a fertility trial in Year 4 and continued with treatments through Year 5. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metabolic measurements (glucose tolerance tests and double X-ray absorptiometry scans) were performed yearly, and results from 5 years of treatment are reported for all animals. Animals were bled daily for 30 days at 5 years to capture changes in ovarian cycle hormones, and ultrasound measurements were performed during the early follicular and luteal phase. MAIN RESULTS AND THE ROLE OF CHANCE: After 5 years of treatment, WSD exposure moderately increased body weight and body fat, although control animals also had a high body mass index due to ad libitum feeding. Animals in the T + WSD group had increased fasting insulin and insulin secretion during an intravenous glucose tolerance test. WSD exposure also altered ovarian cycles, delaying the time to the E2 surge, decreasing progesterone and anti-Müllerian hormone levels and increasing the number of antral follicles present by ultrasound. Longitudinal assessment of metabolic function for only those animals that became pregnant in Year 4 of treatment revealed no differences in post-partum metabolism between groups, although WSD resulted in overall elevated weights, body fat and measures of insulin resistance. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The small sample size and heterogeneity in metabolic effects observed in the T + WSD group are limitations of the current study, with only a subset of animals in this group showing impaired insulin resistance relative to controls. In addition, obesity in the C group prevented comparisons to lean animals. WIDER IMPLICATIONS OF THE FINDINGS: Hyperandrogenemia combined with WSD had a greater impact on insulin sensitivity and ovarian function than either treatment alone. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by NIH grant P50 HD071836 to C.T.R., J.H. and C.T. and P51 OD011092 for support of the Oregon National Primate Research Center. All authors declare no competing interests.

Mild hyperandrogenemia in presence/absence of a high-fat, Western-style diet alters secretory phase endometrial transcriptome in nonhuman primates.

OBJECTIVE: To identify novel transcriptomic changes to eutopic endometrium by exposure to chronic mild hypernadrogenemia (testosterone [T]) with/without exposure to an obesogenic Western-style diet (WSD). DESIGN: Two-by-two factorial arrangement of treatments. SETTING: National primate research center. ANIMALS: Rhesus macaque females were chronically exposed to T and/or consumed a WSD from menarche through adulthood. After 4.5 years of treatment, Tru-Cut endometrial biopsies were obtained at the midsecretory phase (n = 6-4/group), and paired-end sequencing of RNA was performed. Several females in the T, WSD, and T+WSD cohorts developed endometriosis within 6 months of biopsy; a separate analysis was performed contrasting diagnosis of endometriosis stage 0-2 versus stages 3 and 4 (American Society for Reproductive Medicine revised criteria). INTERVENTIONS: Chronic exposure to mild elevation of T (~five-fold elevation) and/or WSD from menarche until adulthood. MAIN OUTCOME MEASURES: Limma voom empirical Bayes pipeline was performed to detect differentially expressed RNAs (DEs) significantly impacted by treatments and endometriosis severity. Differentially expressed RNAs were then interrogated by Ingenuity Pathway Analyses and Protein Analysis through Evolutionary Relationships. RESULTS: Total DEs included C versus T, 469; C versus WSD, 525; C versus T+WSD, 549; and T versus T+WSD, 1,505. The majority of DEs mapped to the ontology pathways: heterotrimeric G-protein signaling pathways Gi alpha and Gs alpha (C vs. T), WNT signaling (C vs. WSD and T vs. T+WSD), and Huntington disease (C vs. T+WSD). A total of 2,171 DEs from eutopic endometrium were altered by the presence of stage 3 and 4 endometriosis lesions. CONCLUSIONS: The present global transcriptomic analyses demonstrate that the greatest magnitude of changes occurred in contrasts of C and T versus T+WSD, adding to the evidence that these two insults have a synergistic effect on female physiology. These data also support the concept that prior alterations to the function of eutopic endometrium increase the risk for endometriosis.

Vitamin D3 Regulates Follicular Development and Intrafollicular Vitamin D Biosynthesis and Signaling in the Primate Ovary.

There is an increasing recognition that vitamin D plays important roles in female reproduction. Recent studies demonstrated that 1α,25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, improved ovarian follicle survival and growth in vitro. Therefore, we investigated the direct effects of VD3 at the specific preantral and antral stages of follicular development, and tested the hypothesis that vitamin D receptor (VDR) and enzymes critical for vitamin D biosynthesis are expressed in the primate ovary. Fourteen adult rhesus macaques provided ovarian tissue. Secondary and antral follicles were isolated for PCR analysis on VDR, vitamin D3 25-hydroxylase, and 25-hydroxyvitamin D3-1α-hydroxylase. VDR protein localization was determined by immunohistochemistry on ovarian sections. Isolated secondary follicles were cultured under conditions of control and VD3 supplementation during the preantral or antral stage. Follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, as well as oocyte maturation were evaluated. In vivo- and in vitro-developed follicles were also assessed for genes that are critical for vitamin D biosynthesis and signaling, gonadotropin signaling, steroid and paracrine factor production, and oocyte quality. The mRNA encoding VDR, 25-hydroxylase, and 1α-hydroxylase was detectable in in vivo- and in vitro-developed preantral and antral follicles. The 25-hydroxylase was elevated in cultured follicles relative to in vivo-developed follicles, which further increased following VD3 exposure. VD3 treatment increased 1α-hydroxylase in in vitro-developed antral follicles. The absence of VD3 during culture decreased VDR expression in in vitro-developed antral follicles, which was restored to levels comparable to those of in vivo-developed antral follicles by VD3 supplementation. Positive immunostaining for VDR was detected in the nucleus and cytoplasm of granulosa cells and oocytes. While only survival was improved in preantral follicles treated with VD3, VD3 supplementation promoted both survival and growth of antral follicles with increased estradiol and AMH production, as well as oocyte maturation. Thus, Vitamin D biosynthesis and signaling systems are expressed in primate ovarian follicles. Our findings support a role for VD3 in regulating follicular development in a stage-dependent manner, as well as the intrafollicular vitamin D biosynthesis and signaling, directly in the ovary.

Anti-Müllerian hormone is a survival factor and promotes the growth of rhesus macaque preantral follicles during matrix-free culture.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector + recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.

Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys.

STUDY QUESTION: Does chronic hyperandrogenemia beginning at menarche, in the absence and presence of a western-style diet (WSD), alter ovarian and uterine structure-function in young adult rhesus monkeys? SUMMARY ANSWER: Phenotypic alterations in ovarian and uterine structure/function were induced by exogenous testosterone (T), and compounded in the presence of a WSD (T+WSD). WHAT IS KNOWN ALREADY: Hyperandrogenemia is a well-established component of PCOS and is observed in adolescent girls, indicating a potential pubertal onset of disease symptoms. Obesity is often associated with hyperandrogenemia and it is hypothesized that metabolic dysfunction exacerbates PCOS symptoms. STUDY DESIGN, SIZE, DURATION: Macaque females (n = 40) near the onset of menarche (~2.5 years of age) were assigned to a 2 by 2 factorial cohort design. Effects on reproductive characteristics were evaluated after 3 years of treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Rhesus macaques (Macaca mulatta) were fed either a normal balanced diet (n = 20) or a WSD (n = 20). Additionally, implants containing cholesterol (n = 20) or T (n = 20) were implanted subcutaneously to elevate serum T approximately 5-fold. This resulted in treatment groups of controls (C), T, WSD and T+WSD (n = 10/group). Vaginal swabbing was performed daily to detect menses. After 3 years of treatment, daily serum samples from one menstrual cycle were assayed for hormone levels. Ovarian structure was evaluated in the early follicular phase by 3D/4D ultrasound. Uterine endometrial size and ovarian/luteal vascular function was also evaluated in subgroups (n = 6/group) in the late follicular and mid-luteal phases by 3D/4D ultrasound and contrast-enhanced ultrasound, respectively. Expression of steroid hormone receptors and markers of decidualization and endometrial receptivity were assessed in endometrial biopsies at mid-luteal phase. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately 90% of menstrual cycles appeared ovulatory with no differences in frequency or duration between groups. Serum estradiol (E2) levels during the early follicular phase were greatest in the T alone group, but reduced in T+WSD (P < 0.02). Serum LH was elevated in the T group (P < 0.04); however, there were no differences among groups in FSH levels (P > 0.13). Ovarian size at menses tended to be greater in the WSD groups (P < 0.07) and antral follicles ≥1 mm were more numerous in the T+WSD group (P < 0.05). Also, females in T and T+WSD groups displayed polycystic ovarian morphology (PCOM) at greater frequency than C or WSD groups (P < 0.01). Progesterone (P4) levels during the luteal phase were reduced in the T+WSD group compared to C and T groups (P < 0.05). Blood volume (BV) and vascular flow (VF) within the corpus luteum was reduced in all treatment groups compared to C (P < 0.01, P = 0.03), with the WSD alone group displaying the slowest BV and VF (P < 0.05). C and WSD groups displayed endometrial glands at mid-luteal phase with low estrogen receptor 1 (ESR1) and progesterone receptor (PGR) mRNA and immunohistochemical staining in the functionalis zone, but appreciable PGR in the stroma. In contrast, T and T+WSD treatment resulted in glands with less secretory morphology, high ESR1 expression in the glandular epithelium and low PGR in the stroma. Endometrial levels of TIMP3 and MMP26 mRNA and immunostaining were also decreased in the T and T+WSD groups, whereas AR expression was unchanged. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Females are young adults, so effects could change as they reach prime reproductive age. The T level generated for hyperandrogenemia may be somewhat greater than the 3-4-fold increase observed in adolescent girls, but markedly less than those observed in male monkeys or adolescent boys. WIDER IMPLICATIONS OF THE FINDINGS: Alterations to ovarian and uterine structure-function observed in T and, in particular, T+WSD-treated female macaques are consistent with some of the features observed in women diagnosed with polycystic ovary syndrome (PCOS), and suggest impaired fertility. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under Award Number P50HD071836 (to RLS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Additional funding was provided by Office of the Director, NIH under Award Number P51OD011092 (Support for National Primate Research Center). Authors declare no competing interests.

Knockdown of Progesterone Receptor (PGR) in Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production.

Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGR mRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4-6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGR mRNA and P levels. Addition of hCG increased (P < 0.05) PGR mRNA and medium P levels in controls. However, a time-dependent decline (P < 0.05) in PGR mRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P < 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization.

Dynamics of Immune Cell Types Within the Macaque Corpus Luteum During the Menstrual Cycle: Role of Progesterone.

The goal of the current study was to characterize the immune cell types within the primate corpus luteum (CL). Luteal tissue was collected from rhesus females at discrete intervals during the luteal phase of the natural menstrual cycle. Dispersed cells were incubated with fluorescently labeled antibodies specific for the immune cell surface proteins CD11b (neutrophils and monocytes/macrophages), CD14 (monocytes/macrophages), CD16 (natural killer [NK] cells), CD20 (B-lymphocytes), and CD3epsilon (T-lymphocytes) for analysis by flow cytometry. Numbers of CD11b-positive (CD11b(+)) and CD14(+) cells increased significantly 3 to 4 days after serum progesterone (P4) concentrations declined below 0.3 ng/ml. CD16(+) cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases and were 3-fold increased 3 to 4 days after serum P4 decreased to baseline levels. CD3epsilon(+) cells tended to increase 3 to 4 days after P4 decline. To determine whether immune cells were upregulated by the loss of luteotropic (LH) support or through loss of LH-dependent steroid milieu, monkeys were assigned to 4 groups: control (no treatment), the GnRH antagonist Antide, Antide plus synthetic progestin (R5020), or Antide plus the estrogen receptor agonists diarylpropionitrile (DPN)/propyl-pyrazole-triol (PPT) during the mid-late luteal phase. Antide treatment increased the numbers of CD11b(+) and CD14(+) cells, whereas progestin, but not estrogen, replacement suppressed the numbers of CD11b(+), CD14(+), and CD16(+) cells. Neither Antide nor steroid replacement altered numbers of CD3epsilon(+) cells. These data suggest that increased numbers of innate immune cells in primate CL after P4 synthesis declines play a role in onset of structural regression of primate CL.

Exposure of female macaques to Western-style diet with or without chronic T in vivo alters secondary follicle function during encapsulated 3-dimensional culture.

Increased adiposity and hyperandrogenemia alter reproductive parameters in both animal models and women, but their effects on preantral follicles in the ovary remain unknown. We recently reported that Western-style diet (WSD) consumption over 1 year, with or without chronic exposure to elevated circulating T, increased the body fat percentage, elicited insulin resistance, suppressed estradiol and progesterone production, as well as altered the numbers, size, and dynamics of antral follicles in the ovary during the menstrual cycle in female macaques. Therefore, experiments were designed to compare the WSD and WSD+T effects to age-matched controls on the survival, growth, and function of isolated secondary follicles during 5 weeks of encapsulated 3-dimensional culture. Follicle survival significantly declined in the WSD and WSD+T groups compared with the control (CTRL) group. Although media progesterone levels were comparable among groups, androstenedione and estradiol levels were markedly reduced in the WSD and WSD+T groups compared with the CTRL group at week 5. Anti-Müllerian hormone levels peaked at week 3 and were lower in the WSD+T group compared with the WSD or CTRL group. Vascular endothelial growth factor levels also decreased at week 5 in the WSD+T group compared with the WSD or CTRL group. After human chorionic gonadotropin exposure, only antral follicles developed from the CTRL group yielded metaphase II oocytes. Thus, WSD with or without T exposure affects the cohort of secondary follicles in vivo, suppressing their subsequent survival, production of steroid hormones and local factors, as well as oocyte maturation in vitro.

Quantification of dynamic changes to blood volume and vascular flow in the primate corpus luteum during the menstrual cycle.

BACKGROUND: The objective of the current study was to determine changes to vascular parameters of nonhuman primate dominant ovarian structures by dynamic contrast-enhanced ultrasound (DCE-US). MATERIALS AND METHODS: Dynamic contrast-enhanced ultrasound with intravenous microbubble infusion was performed on the rhesus macaque ovary bearing the pre-ovulatory follicle and corpus luteum (CL) sequentially during the natural luteal phase (n = 8) and GnRH antagonist (antide)-induced luteal regression (n = 6). RESULTS: Changes in luteal blood volume (BV) and vascular flow (VF) were observed between stages of the luteal phase Luteal BV was highest in early stage CL, before decreasing 2.5-fold in late stage CL (P < 0.06); in contrast, luteal VF peaked at mid luteal stage (P < 0.01). Two females identified with luteal insufficiency trended toward lower peak BV, compared to typical CLs. Another female was identified with a luteal cyst on the contralateral ovary, and a CL that regressed before P levels declined. After 72 hours of antide exposure, BV was reduced 2.3-fold (P = 0.03). CONCLUSIONS: DCE-US provides a sensitive, non-invasive measurement of the dynamics of blood volume and flow in dominant ovarian structures.

Endocrine and local control of the primate corpus luteum.

The primate corpus luteum is a transient endocrine gland that differentiates from the ovulatory follicle midway through the ovarian (menstrual) cycle. Its formation and limited lifespan is critical for fertility, as luteal-derived progesterone is the essential steroid hormone required for embryo implantation and maintenance of intra-uterine pregnancy until the placenta develops. It is well-established that LH and the LH-like hormone, CG, are the vital luteotropic hormones during the menstrual cycle and early pregnancy, respectively. Recent advances, particularly through genome analyses and cellular studies, increased our understanding of various local factors and cellular processes associated with the development, maintenance and repression of the corpus luteum. These include paracrine or autocrine factors associated with angiogenesis (e.g., VEGF), and that mediate LH/CG actions (e.g., progesterone), or counteract luteotropic effects (i.e., local luteolysis; e.g., PGF2α). However, areas of mystery and controversy remain, particularly regarding the signals and events that initiate luteal regression in the non-fecund cycle. Novel approaches capable of gene "knockdown" or amplification", in vivo as well as in vitro, should identify novel or underappreciated gene products that are regulated by or modulate LH/CG actions to control the functional lifespan of the primate corpus luteum. Further advances in our understanding of luteal physiology will help to improve or control fertility for purposes ranging from preservation of endangered primate species to designing novel ovary-based contraceptives and treating ovarian disorders in women.

Progesterone inhibition of oxytocin signaling in endometrium.

Expression of the oxytocin receptor (OXTR) in the endometrium of ruminant species is regulated by the ovarian steroids progesterone (P) and estradiol (E). Near the end of the estrous cycle, long-term exposure of endometrial epithelial cells to P results in loss of genomic P receptors (PGRs), leading to an increase in E receptors (ERs). Genomic regulation of the OXTR is mediated via suppression of ER signaling by P. Upon OT binding at the plasma membrane of endometrial cells, a signaling cascade is generated stimulating release of prostaglandin F2α (PGF2α). Transport of PGF2α to the ovary results in release of OT by luteal cells in a positive feedback loop leading to luteal regression. This signaling cascade can be rapidly blocked by exposing endometrial cells to physiologic levels of P. This mini review will focus on the mechanisms by which P may act to block OXTR signaling and the luteolytic cascade in the ruminant endometrium, with special focus on both non-genomic signaling pathways and non-receptor actions of P at the level of the plasma membrane. While this review focuses on ruminant species, non-classical blockage of OXTR signaling may be important for fertility in women.