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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 Gn RH 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To date, ultrasonography of monkey ovaries is rare and typically of low resolution. The objectives of this study were to use state-of-the-art, high-resolution, transabdominal ultrasonography with real-time Doppler capabilities to: (1) determine whether one can reliably detect in real time the large dominant follicle, the corpus luteum (CL), and small (<2 mm) antral follicles on the ovaries of rhesus monkeys during the natural menstrual cycle; and (2) predict the follicular response of rhesus ovaries to controlled ovarian stimulation (COS) protocols. Rhesus monkeys were selected for transabdominal ultrasonography using a GE Voluson 730 Expert Doppler System at discrete stages of the menstrual cycle. Subsequently, serial ultrasound scanning was employed to observe growth of antral follicles and the CL. Finally, females were scanned to assess follicular growth during COS. The dominant structure and small antral follicles (<2 mm) were reliably visualized in real time. The follicle destined to ovulate could be identified by size differential by day 3 of the follicular phase. The number of small antral follicles present before onset of COS protocol correlated positively with the number of metaphase II-stage oocytes collected after treatment. The results of this study demonstrate that the population dynamics of antral follicle pools can be noninvasively evaluated in monkeys during natural and pharmacologic ovarian cycles. Am. J. Primatol. 71:384–392, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

In women, the corpus luteum is the source of circulating relaxin. No previous studies have addressed whether the corpus luteum is also a relaxin target organ. We determined relaxin receptor LGR7 mRNA expression in human term pregnancy corpora lutea and nonhuman primate corpora lutea obtained during the menstrual cycle. Real-time reverse transcription-PCR demonstrated the expression of LGR7 mRNA in both human and rhesus monkey corpora lutea. Rhesus monkey corpora lutea were obtained from naturally cycling animals following documented luteinizing hormone (LH) surges at early, mid-, mid-late, and late luteal phases. Luteal expression of LGR7 mRNA did not show temporal variation. Since the primate corpus luteum is LH dependent, we assessed LGR7 mRNA expression in corpora lutea from rhesus monkeys treated with a gonadotropin-releasing hormone (GnRH) antagonist, which significantly suppressed pituitary LH levels. GnRH antagonist treatment, which also inhibits both progesterone and relaxin production, resulted in a fivefold increase in luteal LGR7 mRNA expression. These data suggest that luteal LGR7 mRNA expression may be regulated by relaxin and/or LH and that the primate corpus luteum is a target organ for relaxin. [ABSTRACT FROM AUTHOR]

Molecular targets of rapid non-genomic steroid actions are not well known compared to those of the classical transcription pathway, but ion channels have recently been identified to be steroid-sensitive. Especially, in the ovary, the very organ producing high amounts of sex steroids, their rapid actions are not well examined. We now identified a yet unknown target for sex steroids, a voltage-dependent K+ channel (Kv4.2) that contributes to a transient outward K+ current (IA) in human granulosa cells (GCs). Sex steroid hormones at concentrations typical for the ovary (1 µM) blocked Kv4.2 thereby attenuating IA by about 25% within seconds. We also found both Kv4.2 (KCND2) mRNA and protein in endocrine cells of the human and rhesus macaque ovary, emphasizing the physiological relevance of this channel. Therefore, we propose a role as fast-responding steroid sensor for the Kv4.2 channel. The direct regulation of K+ channel activity by sex steroids might represent a yet unknown mechanism of rapid steroid action in close proximity to the site of steroid production in the primate ovary. Our data might also be important for Kv4 channels in the brain and the cardiovascular system where rapid steroid effects are discussed in the context of prevention of cell death. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Pituitary gonadotrophins, notably LH, and ovarian steroids, such as progesterone, play essential roles in the events leading to ovulation of the mature follicle and development of the subsequent corpus luteum. Rhesus monkeys (Macaca mulatta) undergoing controlled ovarian stimulation cycles comparable to those in clinical IVF/assisted reproduction treatment protocols provide a non-human primate model for studying peri-ovulatory events. The ability to manipulate the intrafollicular steroid milieu, via oral administration of a steroid synthesis inhibitor, with or without steroid replacement, allows one to distinguish between gonadotrophin-initiated, steroid-dependent versus steroid-independent processes. The length of the periovulatory interval (onset of the LH surge to follicle rupture) is long (36-40 h) in primates and can be considered in terms of early (<12 h) versus later (>24 h) events. Granulosa cells lose their proliferative activity and differentiate into progesteron e-secreting cells during the early peri-ovulatory interval. The rapid increase in progesterone synthetic capacity and expression of progesterone receptors suggests that this steroid has early actions, e.g. in controlling cell cycle machinery or differentiation. However, it is not until later that morphological luteinization is evident. By this stage, progesterone may serve as a potent anti-apoptotic factor and regulator of tissue remodelling through control of protease expression and activity, angiogenesis, or other events. Application of modern techniques to study LH- and progesterone-responsive gene expression will further unravel ovulatory and luteinization processes in specific compartments of the primate follicle. [ABSTRACT FROM AUTHOR]

Continual administration of low doses of the antiprogestin ZK 137 316 permits ovarian/menstrual cyclicity, but prevents pregnancy in female rhesus monkeys. The sites of contraceptive action remain unknown. This study determined whether chronic, low-dose antiprogestin exposure during follicular development impairs oocyte maturation in vivo, as well as fertilization and preimplantation embryogenesis in vitro. Adult, female rhesus monkeys exhibiting normal menstrual cycles received vehicle (n=9) or 0.03 mg ZK 137 316 (n=8)/kg body weight i.m. daily for 3 months. Controlled ovarian stimulation with recombinant gonadotropins was initiated in the 3rd month. Oocytes collected from preovulatory follicles were evaluated for nuclear maturity and inseminated in vitro. Preimplantation embryonic development was monitored in vitro. The total number of oocytes and percentage collected at each nuclear stage were similar in both groups. More (P<0.05) atretic oocytes were recovered following antiprogestin relative to vehicle treatment. Fertilization rates and percentages of embryos that progressed to the morula stage were similar between groups, but antiprogestin-treated females exhibited less (P<0.05) normal cleavage. Embryonic development was accelerated by 1 day (P<0.05) from the 16-cell to the morula stage in the antiprogestin group relative to vehicle. Despite this, the majority of embryos became blastocysts within 6 days in vitro in the antiprogestin group, but fewer expanded (P=0.09) and hatched (P<0.05) compared to vehicle. During in vivo treatment with chronic, low-dose antiprogestin, oocytes retained their ability to resume and complete meiosis as well as fertilize following insemination in vitro. However, preimplantation embryogenesis in vitro was impaired, particularly during the later stages of blastocyst development. Thus, antiprogestin exposure during follicular development altered oocyte functions that are critical for normal preimplantation embryogenesis; this may contribute to pregnancy prevention. [Copyright &y& Elsevier]

Objective: To investigate the effect of slow cryopreservation on the morphology and function of primate primordial follicles within ovarian tissue slices.Design: Fresh monkey ovarian tissue was frozen by slow cryopreservation and thawed for analysis of morphologic and functional parameters.Setting: University-affiliated laboratory.Animals: Rhesus monkey ovarian tissue.Intervention(s): None.Main Outcome Measure(s): Histologic analysis, follicle counting, assessment of protein abundance and localization.Result(s): After freezing and thawing, 89% of the primordial follicles maintained their laminar-based architecture, with sizes close to those of fresh fixed follicles. Molecular markers of early follicle health (activin subunits and the phosphorylated form of the signaling protein Smad2 [pSmad2]) were present in fresh and frozen-thawed primordial follicles. Stroma cells, but not follicles, had a higher level of TUNEL staining. Granulosa cells within the follicles of frozen-thawed ovarian tissue cultured for 48 hours had the capacity to proliferate and sustained expression of the activin subunits and nuclear pSmad2.Conclusion(s): This study provides evidence that markers of early follicle growth and development are preserved after slow cryopreservation and thaw, with little effect on follicle morphology and function. [ABSTRACT FROM AUTHOR]

Abstract: Background: The study was conducted to determine whether the phosphodiesterase (PDE) 3 inhibitor ORG 9935 prevents the resumption of meiosis in primate oocytes during natural menstrual cycles. Study Design: Regularly cycling adult female macaques (n=8) were followed during the follicular phase and then started on a 2-day treatment regimen of human recombinant gonadotropins to control the timing of ovulation. Monkeys received no further treatment (controls) or ORG 9935. Oocytes were recovered by laparoscopic follicle aspiration 27 h after an ovulatory stimulus, cultured in vitro in the absence of inhibitor and inseminated. The primary outcome was the meiotic stage of the oocyte. Results: In six ORG 9935 cycles, five of the recovered oocytes were germinal vesicle (GV)-intact, and one exhibited GV breakdown (GVBD). In contrast, all three oocytes that recovered during control cycles were GVBD (p<.05). None of the ORG 9935-treated oocytes underwent fertilization compared with 2/3 (67%) from controls. Conclusions: These results demonstrate that ORG 9935 blocks resumption of meiosis in the naturally selected dominant follicle in primates and suggest that PDE3 inhibitors have potential clinical use as contraceptives in women. [Copyright &y& Elsevier]

Abstract: To determine if anti-angiogenic agents disrupt primate ovarian function, vehicle or a general angiostatic compound (TNP-470), specific antagonists of vascular endothelial growth factor (soluble VEGF receptor-1, sVEGFR-1; anti-VEGF monoclonal antibody, VEGF Ab) and/or an angiopoietin antagonist (Ang-2) were administered to rhesus monkeys: (1) locally via injection into the preovulatory follicle at midcycle or the developing corpus luteum at the midluteal phase; or (2) systemically via subcutaneous injection in the early follicular phase or at midcycle during the natural menstrual cycle. Compared to controls, intrafollicular injection of TNP-470 or sVEGFR-1 decreased circulating progesterone (P) levels in the subsequent luteal phase. Treatment with sVEGFR-1, but not TNP-470, also decreased the incidence of ovulation. Intrafollicular injection of Ang-2 also prevented ovulation, as well as any functional luteal phase. In the absence of elevated P, serum estradiol levels rose to peak levels 11–12 days post-Ang-2 treatment, at which time another large antral follicle was observed on the contralateral (noninjected) ovary. Intraluteal and systemic injection of VEGF antagonists alone or with Ang-2 had minimal effects. Thus, anti-angiogenic factors can act locally in the primate follicle to disrupt the gametogenic (oocyte release) and endocrine (steroid) functions of the ovary. However, further studies are needed to optimize delivery of angiogenic agents before they can be meaningfully evaluated as possible contraceptive agents. [Copyright &y& Elsevier]

Abstract: To determine whether phosphodiesterase (PDE) 3 inhibitors prevent the resumption of meiosis by primate oocytes in vivo, rhesus macaques were stimulated to develop multiple preovulatory follicles by administering human recombinant gonadotropins, and follicles were aspirated 34 h after an ovulatory stimulus (human chorionic gonadotropin [hCG]). Monkeys received no further treatment (controls) or the PDE3 inhibitor ORG 9935 (a) exclusively in the periovulatory interval beginning 6–12 h prior to receiving hCG at 200 mg/kg every 12 h orally (PER200) or a 200 mg/kg oral loading dose followed by 50 mg/kg sc every 6 h (PER50) or (b) throughout the ovarian stimulation protocol with daily increases until a dose of 200 mg/kg bid was administered onward from the eighth day of ovarian stimulation (EXT200). The primary outcome was the number of oocytes that had resumed meiosis (germinal vesicle breakdown [GVBD]) at collection. At initial aspiration, 85% of oocytes recovered from control animals (n=4) had progressed to GVBD compared with 53% (p&#60;.01), 23% (p&#60;.01), and 13% (p&#60;.01) recovered from animals in the PER200 (n=2), PER50 (n=1) and EXT200 (n=3) groups, respectively. Although spontaneous maturation of oocytes was observed during follow-up culture in the absence of ORG 9935, none of the oocytes in the PER50 or EXT200 underwent normal fertilization in vitro. These results demonstrate that the PDE3 inhibitor ORG 9935 blocks oocyte maturation during gonadotropin-stimulated ovarian cycles in rhesus macaques and suggest that PDE3 inhibitors have potential clinical use as contraceptives in women. [Copyright &y& Elsevier]

To determine the practicality of controlled ovulation of the dominant follicle as a technique to study meiotic maturation of oocytes during contraceptive research, we developed a technique for aspiration of the single dominant follicle using a dual-needle continuous irrigation technique 27 hours after an ovulatory stimulus. All of the oocytes (3/3) recovered from control animals, but only 1/6 (17%) of oocytes from animals treated with the meiotic inhibitor ORG 9935 exhibited germinal vesicle breakdown, indicating resumption of meiosis. [ABSTRACT FROM AUTHOR]