Human embryonic stem cells derived by somatic cell nuclear transfer.

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

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.

Chronically elevated androgen and/or consumption of a Western-style diet impairs oocyte quality and granulosa cell function in the nonhuman primate periovulatory follicle.

PURPOSE: To investigate the impact of chronically elevated androgens in the presence and absence of an obesogenic diet on oocyte quality in the naturally selected primate periovulatory follicle. METHODS: Rhesus macaques were treated using a 2-by-2 factorial design (n = 10/treatment) near the onset of menarche with implants containing either cholesterol (C) or testosterone (T, 4-5-fold increase above C) and a standard or "Western-style" diet alone (WSD) or in combination (T+WSD). Following ~ 3.5 years of treatment, females underwent controlled ovulation (COv, n = 7-10/treatment) cycles, and contents of the naturally selected periovulatory follicle were aspirated. Follicular fluid (FF) was analyzed for cytokines, chemokines, growth factors, and steroids. RNA was extracted from luteinizing granulosa cells (LGCs) and assessed by RNA-seq. RESULTS: Only healthy, metaphase (M) I/II-stage oocytes (100%) were retrieved in the C group, whereas several degenerated oocytes were recovered in other groups (33-43% of T, WSD, and T+WSD samples). Levels of two chemokines and one growth factor were reduced (p < 0.04) in FF of follicles with a MI/MII oocyte in WSD+T (CCL11) or T and WSD+T groups (CCL2 and FGF2) compared to C and/or WSD. Intrafollicular cortisol was elevated in T compared to C follicles (p < 0.02). Changes in the expression pattern of 640+ gene products were detected in LGC samples from follicles with degenerated versus MI/MII-stage oocytes. Pathway analysis on RNAs altered by T and/or WSD found enrichment of genes mapping to steroidogenic and immune cell pathways. CONCLUSIONS: Female primates experiencing hyperandrogenemia and/or consuming a WSD exhibit an altered intrafollicular microenvironment and reduced oocyte quality/competency, despite displaying menstrual cyclicity.

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.

Changes in immune cell distribution and their cytokine/chemokine production during regression of the rhesus macaque corpus luteum.

Our previous flow cytometry results demonstrated a significant increase in neutrophils, macrophages/monocytes, and natural killer (NK) cells in dispersed rhesus monkey corpora lutea (CL) after progesterone (P4) levels had fallen below 0.3 ng/ml for ≥3 days during the natural menstrual cycle. In this study, immunohistochemistry revealed the CD11b+ cells (neutrophils, macrophages/monocytes) present in the CL after luteal P4 synthesis ceased were distributed throughout the tissue. CD16+ cells (presumptive NK cells) were observed mainly near the vasculature in functional CL, until their numbers increased and they became widely distributed in regressing CL. To determine if the immune cells that enter luteal tissue during structural regression are functionally different from those that are present during peak function, CD11b+ or CD16+ populations were enriched from mid-late stage (functional) and regressing (days 1.8 ± 0.3 postmenses) CL using antibody-conjugated magnetic microbeads. Flow cytometry analyses revealed the majority of CD11b+ cells expressed CD14, a protein mainly produced by macrophages/monocytes. The antibody-enriched and depleted fractions were cultured for 24 h, and the media then analyzed for the production of 29 cytokines/chemokines. From the mid-late CL, the CD11b+-enriched fraction produced three cytokines/chemokines, whereas CD16+-enriched cells only produced the chemokine CCL2. However, CD11b +-enriched cells isolated from regressed CL produced eight cytokines/chemokines. The CD16+-enriched cells isolated from regressing CL produced significant levels of only three cytokines. Thus, the CD11b+ cells that appear in the rhesus macaque CL after functional regression produce several cytokines/chemokines that likely play a role in orchestrating structural regression.

Subcutaneous ovarian tissue transplantation in nonhuman primates: duration of endocrine function and normalcy of subsequent offspring as demonstrated by reproductive competence, oocyte production, and telomere length.

PURPOSE: The main purposes of the study were to investigate the endocrine function of ovarian tissue transplanted to heterotopic subcutaneous sites and the reproductive competence and telomere length of a nonhuman primate originating from transplanted tissue. METHODS: Ovarian cortex pieces were transplanted into the original rhesus macaques in the arm subcutaneously, in the abdomen next to muscles, or in the kidney. Serum estradiol (E2) and progesterone (P4) concentrations were measured weekly for up to 8 years following tissue transplantation. A monkey derived from an oocyte in transplanted ovarian tissue entered time-mated breeding and underwent controlled ovarian stimulation. Pregnancy and offspring were evaluated. Telomere lengths and oocytes obtained following controlled ovarian stimulation were assessed. RESULTS: Monkeys with transplants in the arm and abdomen had cyclic E2 of 100 pg/ml, while an animal with arm transplants had E2 of 50 pg/ml. One monkey with transplants in the abdomen and kidney had ovulatory cycles for 3 years. A monkey derived from an oocyte in transplanted tissue conceived and had a normal gestation until intrapartum fetal demise. She conceived again and delivered a healthy offspring at term. Controlled ovarian stimulations of this monkey yielded mature oocytes comparable to controls. Her telomere length was long relative to controls. CONCLUSIONS: Heterotopic ovarian tissue transplants yielded long-term endocrine function in macaques. A monkey derived from an oocyte in transplanted tissue was reproductively competent. Her telomere length did not show epigenetically induced premature cellular aging. Ovarian tissue transplantation to heterotopic sites for fertility preservation should move forward cautiously, yet optimistically.

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.

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.

Primate follicular development and oocyte maturation in vitro.

The factors and processes involved in primate follicular development are complex and not fully understood. An encapsulated three-dimensional (3D) follicle culture system could be a valuable in vitro model to study the dynamics and regulation of folliculogenesis in intact individual follicles in primates. Besides the research relevance, in vitro follicle maturation (IFM) is emerging as a promising approach to offer options for fertility preservation in female patients with cancer. This review summarizes the current published data on in vitro follicular development from the preantral to small antral stage in nonhuman primates, including follicle survival and growth, endocrine (ovarian steroid hormone) and paracrine/autocrine (local factor) function, as well as oocyte maturation and fertilization. Future directions include major challenges and strategies to further improve follicular growth and differentiation with oocytes competent for in vitro fertilization and subsequent embryonic development, as well as opportunities to investigate primate folliculogenesis by utilizing this 3D culture system. The information may be valuable in identifying optimal conditions for human follicle culture, with the ultimate goal of translating the experimental results and products to patients, thereby facilitating diagnostic and therapeutic approaches for female fertility.

Oocyte maturation and in vitro hormone production in small antral follicles (SAFs) isolated from rhesus monkeys.

PURPOSE: The small antral follicles (SAFs) from the ovarian medulla can be a potential source of oocytes for infertility patients, but little is known about their ability to yield mature oocytes. This study evaluated the response of these SAFs to a stimulatory bolus of human corionic gonadotropin (hCG) in vitro. METHODS: Oocyte nuclear maturation and hormone production (estradiol [E2], progesterone [P4]), antimullerian hormone [AMH]) by individual intact SAFs (n = 91; >0.5 mm; n = 5 monkeys) was evaluated after 34 h of culture in the absence (control) or presence of hCG. RESULTS: Of the total cohort (n = 91), 49 % of SAFs contained degenerating oocytes. The percentage of healthy oocytes able to reinitiate meiosis to the metaphase I (MI) and MII was greater (p < 0.05) after hCG compared to controls. E2, P4 and AMH levels were higher (p < 0.05) in SAF cultures containing germinal vesicle (GV) oocytes compared to those with MII oocytes regardless of hCG exposure. SAF with MI oocytes produced more E2, but less (p < 0.05) P4 and AMH compared to SAFs containing GV oocytes (p < 0.05). Follicles ≥1 mm produced more (p < 0.05) E2, whereas follicle diameter did not correlate with P4 or AMH levels. Only P4 increased (p < 0.05) in response to hCG, regardless of follicle size or oocyte maturity. SAFs containing degenerating oocytes produced similar levels of E2, P4 and AMH compared to SAFs containing healthy oocytes. CONCLUSIONS: These data indicate, for the first time, that oocytes within primate SAFs can reinitiate meiosis in vitro in the absence of hCG, but nuclear maturation is enhanced in SAFs cultured with hCG. Oocyte nuclear maturation within SAFs in is associated with decreased E2, P4 and AMH levels. Furthermore, hormone content within the culture media does not necessarily reflect oocyte quality.

Amphiregulin promotes the maturation of oocytes isolated from the small antral follicles of the rhesus macaque.

BACKGROUND: In non-primates, the epidermal growth factor (EGF) and EGF-related ligands such as amphiregulin (AREG) serve as critical intermediates between the theca/mural cells and the cumulus-oocyte-complex (COC) following the mid-cycle LH surge. Studies were designed in primates (1) to analyze AREG levels in follicular fluid (follicular fluid) obtained from pre-ovulatory follicles, as well as (2) to assess dose-dependent effects of AREG on oocytes from small antral follicles (SAFs) during culture, including meiotic and cytoplasmic maturation. METHODS: Controlled ovulation protocols were performed on rhesus monkeys (n=12) to determine AREG content within the single, naturally selected dominant follicle after an ovulatory stimulus. Using healthy COCs (n=271) obtained from SAFs during spontaneous cycles (n=27), in vitro maturation (IVM) was performed in the absence or presence of physiological concentrations of AREG (10 or 100 ng/ml) with or without gonadotrophins (FSH, 75 mIU/ml; LH, 75 mIU/ml). At the end of the culture period, oocyte meiotic maturation was evaluated and ICSI was performed (n=111), from which fertilization and early embryo development was followed in vitro. RESULTS: AREG levels in follicular fluid from pre-ovulatory follicles increased (P<0.05) following an ovulatory bolus of hCG at 12, 24 and 36 h post-treatment. At 12 h post-hCG, AREG levels in follicular fluid ranged from 4.8 to 121.4 ng/ml. Rhesus macaque COCs incubated with 10 ng/ml AREG in the presence of gonadotrophins displayed an increased percentage of oocytes that progressed to the metaphase II (MII) stage of meiosis (82 versus 56%, P<0.05) and a decreased percentage of metaphase I (MI) oocytes (2 versus 23%, P<0.05) relative to controls, respectively. The percentage of either MI or MII oocytes at the end of the culture period was not different between oocytes cultured with 100 ng/ml AREG or in media alone. Fertilization and first cleavage rates obtained by ICSI of all IVM MII oocytes were 93 and 98%, respectively, and did not vary among treatment groups. Of the MII oocytes that fertilized (n=103), 37 were randomly selected and maintained in culture to assess developmental potential. A total of 13 early blastocysts were obtained, with four embryos developing to expanded blastocysts. CONCLUSIONS: These data indicate that AREG levels increase in rhesus macaque pre-ovulatory follicles after an ovulatory stimulus, and a specific concentration of AREG (10 ng/ml) enhances rhesus macaque oocyte nuclear maturation but not cytoplasmic maturation from SAFs obtained during the natural menstrual cycle. However, owing to the small number of samples in some treatment groups, further studies are now required.

Role of the DLL4-NOTCH system in PGF2alpha-induced luteolysis in the pregnant rat.

We investigated the expression and cell localization of NOTCH1, NOTCH4, and the delta-like ligand DLL4 in corpus luteum (CL) from pregnant rats during prostaglandin F2alpha (PGF2alpha)-induced luteolysis. We also examined serum progesterone (P(4)) and CL proteins related to apoptosis after local administration of the notch inhibitor N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT). Specific staining for NOTCH1 and NOTCH4 receptors was detected predominantly in large and small luteal cells. Furthermore, in line with the fact that the notch intracellular domain is translocated to the nucleus, where it regulates gene expression, staining was evident in the nuclei of luteal cells. In addition, we detected diffuse cytoplasmic immunostaining for DLL4 in small and large luteal cells, in accordance with the fact that DLL4 undergoes proteolytic degradation after receptor binding. The mRNA expression of Notch1, Notch4, and Dll4 in CL isolated on Day 19 of pregnancy decreased significantly after administration of PGF2alpha. Consistent with the mRNA results, administration of PGF2alpha to pregnant rats on Day 19 of pregnancy decreased the protein fragment corresponding to the cleaved forms of NOTCH1/4 CL receptors. In contrast, no significant changes were detected in protein levels for the ligand DLL4. The local intrabursal administration of DAPT decreased serum P(4) levels and increased luteal levels of active caspase 3 and the BAX:BCL2 ratio 24 h after the treatment. These results support a luteotropic role for notch signaling to promote luteal cell viability and steroidogenesis, and they suggest that the luteolytic hormone PGF2alpha may act in part by reducing the expression of some notch system members.

Dynamics of the transcriptome in the primate ovulatory follicle.

Experiments were designed to evaluate changes in the transcriptome (mRNA levels) in the ovulatory, luteinizing follicle of rhesus monkeys, using a controlled ovulation model that permits analysis of the naturally selected, dominant follicle at specific intervals (0, 12, 24 and 36 h) after exposure to an ovulatory (exogenous hCG) stimulus during the menstrual cycle. Total RNA was prepared from individual follicles (n= 4-8/timepoint), with an aliquot used for microarray analysis (Affymetrix Rhesus Macaque Genome Array) and the remainder applied to quantitative real-time PCR (q-PCR) assays. The microarray data from individual samples distinctly clustered according to timepoints, and ovulated follicles displayed markedly different expression patterns from unruptured follicles at 36 h. Between timepoint comparisons revealed profound changes in mRNA expression profiles. The dynamic pattern of mRNA expression for steroidogenic enzymes (CYP17A, CYP19A, HSD3B2, HSD11B1 and HSD11B2), steroidogenic acute regulatory protein (StAR) and gonadotrophin receptors [LH/choriogonadotrophin receptor (LHCGR), FSH receptor (FSHR)] as determined by microarray analysis correlated precisely with those from blinded q-PCR assays. Patterns of mRNA expression for epidermal-growth-factor-like factors (amphiregulin, epiregulin) and processes [hyaluronan synthase 2 (HAS2), tumor necrosis factor alpha-induced protein 6 (TNFAIP6)] implicated in cumulus-oocyte maturation/expansion were also comparable between assays. Thus, several mRNAs displayed the expected expression pattern for purported theca (e.g. CYP17A), granulosa (CYP19A, FSHR), cumulus (HAS2, TNFAIP6) cell and surface epithelium (HSD11B)-related genes in the rodent/primate pre-ovulatory follicle. This database will be of great value in analyzing molecular and cellular pathways associated with periovulatory events in the primate follicle (e.g. follicle rupture, luteinization, inflammatory response and angiogenesis), and for identifying novel gene products controlling mammalian fertility.

Dynamics of the primate ovarian surface epithelium during the ovulatory menstrual cycle.

BACKGROUND: Epithelial ovarian cancer (EOC) risk correlates strongly with the number of ovulations that a woman experiences. The primary source of EOC in women is the ovarian surface epithelium (OSE). Mechanistic studies on the etiology of OSE transformation to EOC cannot be realistically performed in women. Selecting a suitable animal model to investigate the normal OSE in the context of ovulation should be guided by the model's reproductive similarities to women in natural features that are thought to contribute to EOC risk. METHODS: We selected the non-human primate, rhesus macaque, as a surrogate to study the normal OSE during the natural menstrual cycle. We investigated OSE morphology and marker expression, plus cell proliferation and death in relation to menstrual cycle stage and ovulation. RESULTS: OSE cells displayed a morphological range from squamous to columnar. Cycle-independent parameters and cycle-dependent changes were observed for OSE histology, steroid receptor expression, cell death, DNA repair and cell adhesion. Contrary to findings in non-primates, primate OSE cells were not manifestly cleared from the site of ovulation, nor were proliferation rates affected by ovulation or stage of the menstrual cycle. DNA repair proteins were more highly expressed in OSE than in other ovarian cells. CONCLUSIONS: This study identifies significant differences between primate and non-primate OSE. In contrast to established views, ovulation-induced death and proliferation are not indicated as prominent contributors to EOC risk, but disruption of OSE cadherin-mediated adhesion may be, as could the loss of ovary-mediated chronic suppression of proliferation and elevation of DNA repair potential.

Ovarian surface epitheliectomy in the non-human primate: continued cyclic ovarian function and limited epithelial replacement.

BACKGROUND: The fifth leading cause of cancer deaths among women is ovarian cancer (OC), which originates primarily in the ovarian surface epithelium (OSE) that surrounds the ovary. Permanent removal of the OSE could provide a novel strategy to substantially reduce OC risk, while retaining the benefits of ovarian function, including gameto- and steroidogenesis. It must be determined whether ovarian surface epitheliectomy (OSEx) carries deleterious side effects, including loss of menstrual cyclicity, infertility or scarring (e.g. adhesions), prior to any clinical application of this strategy. To achieve this, we selected the non-human primate, rhesus macaque, for long-term (12 month) studies on the effects of OSEx. METHODS: Rhesus macaque females underwent OSEx by detergent treatment and were then monitored for menstrual cyclicity (menstruation, steroidogenesis and follicle development) and adverse side effects (tissue scarring or adhesions). Ovaries were collected at 6 or 12 months and examined for evidence of tissue damage, follicle rupture and regression of the corpus luteum. The ovarian surface was examined immunohistologically for signs of epithelial replacement, using markers for OSE and fimbrial epithelium (FE), a possible alternative source of pelvic tumors diagnosed as OC. RESULTS: After OSEx, menstrual cycle length, estrogen and progesterone production, follicle rupture and luteal regression appeared normal. No evidence of adhesions was seen. At 6 and 12 months post-OSEx, the ovarian surface was sparsely populated by cells expressing OSE and FE markers. Proliferative activity in this population was notably low. CONCLUSIONS: OSEx may provide a novel method to reduce the risk of OC, without sacrificing ovarian function, although the effects on fertility remain to be tested. The absence of epithelial replacement via enhanced proliferation suggests OSEx does not increase malignant potential. Complete and permanent OSEx may be feasible.

The science behind 25 years of ovarian stimulation for in vitro fertilization.

To allow selection of embryos for transfer after in vitro fertilization, ovarian stimulation is usually carried out with exogenous gonadotropins. To compensate for changes induced by stimulation, GnRH analog cotreatment, oral contraceptive pretreatment, late follicular phase human chorionic gonadotropin, and luteal phase progesterone supplementation are usually added. These approaches render ovarian stimulation complex and costly. The stimulation of multiple follicular development disrupts the physiology of follicular development, with consequences for the oocyte, embryo, and endometrium. In recent years, recombinant gonadotropin preparations have become available, and novel stimulation protocols with less detrimental effects have been developed. In this article, the scientific background to current approaches to ovarian stimulation for in vitro fertilization is reviewed. After a brief discussion of the relevant aspect of ovarian physiology, the development, application, and consequences of ovarian stimulation strategies are reviewed in detail.

Ovulation in the absence of the ovarian surface epithelium in the primate.

The ovarian surface epithelium (OSE) has a prominent role in ovarian cancer in women, but no studies have been conducted to evaluate its role in normal ovarian function. Data from other species suggest the OSE is needed for ovulation. We have tested whether the OSE is needed for follicle rupture, a necessary step in ovulation, using the nonhuman primate, rhesus macaque. The OSE was removed in two different short-term protocols spanning a single periovulatory interval--one protocol used a cytology brush to remove the OSE only from the follicle apex, and one used mild detergent to remove the entire OSE--and in one long-term protocol spanning 6 wk (two periovulatory intervals) that removed the entire OSE with detergent. Serum levels of estrogen and progesterone (E and P) were monitored, and sectioned ovaries were examined for evidence of successful OSE removal and follicle rupture. In the short-term protocols, removal of the OSE over the follicle apex did not prevent follicle rupture (n = 4 ovaries), but removal of the entire OSE using detergent did in four of six cases. In the long-term protocol, when ovaries were collected after the second periovulatory interval, all the ovaries (n = 5) showed evidence of follicle rupture. In all the protocols, E and P production appeared unaffected. Detergent penetrated up to 40 microm into the ovary. This may have transiently disrupted the stroma and caused follicle rupture failure. We conclude that the primate OSE is not essential for ovulation and perhaps can be removed without lasting consequence.

Cumulus-oocyte complexes from small antral follicles during the early follicular phase of menstrual cycles in rhesus monkeys yield oocytes that reinitiate meiosis and fertilize in vitro.

The stage at which follicle-enclosed cumulus-oocyte complexes achieve developmental competence in primates is unknown. Therefore, studies were designed to characterize the ability of oocytes in small antral follicles present during the menstrual cycle to spontaneously resume meiosis, fertilize, and support early embryo development. Ovaries were removed from adult rhesus monkeys (n = 12) during the early follicular phase (Days 3-4) of spontaneous cycles. Small antral follicles were divided into five groups according to their diameter; group I: <0.5 mm; group II: 0.5-0.99 mm; group III: 1.0-1.49 mm; group IV: 1.5-1.99 mm; and group V: 2.0-2.5 mm. The cumulus-oocyte complex from healthy small antral follicles (devoid of dark oocytes or granulosa cells) were extracted (n = 199) and cultured for 48 h under different conditions: in TALP (tyrode, albumin, lactate, pyruvate) medium alone, SAGE medium alone, or plus gonadotropins. At 48 h, oocyte meiotic status and diameter were measured after treatment of cumulus-oocyte complexes with hyaluronidase. Cumulus-oocyte complexes derived from follicles of 0.5- to 2-mm diameter contain oocytes that typically reinitiate meiosis in the absence or presence of gonadotropins and fertilize via in vitro fertilization or intracytoplasmic sperm injection. Moreover, the inseminated oocytes can reach the morula stage but arrest. Thus, the ability of these oocytes to complete maturation, as monitored from subsequent embryonic development after fertilization, is suboptimal. Further studies on primate IVM of oocytes from SAFs are warranted in order for them to be considered as an additional, novel source of gametes for fertility preservation in cancer patients.

Survival, growth, and maturation of secondary follicles from prepubertal, young, and older adult rhesus monkeys during encapsulated three-dimensional culture: effects of gonadotropins and insulin.

A three-dimensional culture system supports the development of primate preantral follicles to the antral stage with appreciable steroid production. This study assessed i) whether in vitro developmental competence of follicles is age dependent, ii) the role of gonadotropins and insulin in supporting folliculogenesis, and iii) anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) production by growing follicles. Ovaries were obtained from prepubertal, young, and older adult rhesus macaques. Secondary follicles were encapsulated into alginate beads and cultured individually for 40 days in media containing 0.05 or 5  µg/ml insulin, with or without recombinant human (rh) FSH (500  mIU/ml). No follicles survived in the culture without rhFSH. In the presence of rhFSH, survival was lower for follicles from older animals, whereas growth, i.e. follicle diameter, was less by day 40 for follicles from prepubertal animals. The surviving follicles were categorized as no-grow (NG; ≤ 250 µm), slow-grow (SG; 250-500 µm), and fast-grow (FG; ≥ 500  µm) according to their diameters. SG follicles cultured with 5 µg/ml insulin produced more ovarian steroids than those cultured with 0.05  µg/ml insulin by week 5. SG and FG follicles produced more AMH and VEGF than the NG, and levels peaked at weeks 2 and 5 respectively. After 100  ng/ml rh chorionic gonadotropin treatment for 34 h, more healthy oocytes were retrieved from young adults whose follicles were cultured with 5  µg/ml insulin. This culture system offers an opportunity to characterize the endocrine and paracrine function of primate follicles that influence follicle growth and oocyte maturation.