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Abstract: Background: The study was conducted to determine whether a phosphodiesterase (PDE) 3 inhibitor has potential as a novel contraceptive in primates. Methods: Regularly cycling adult female cynomolgus macaques of proven fertility (n=16) were treated for 7 months with placebo (controls) or the PDE3 inhibitor ORG 9935 as a daily food treat (150 mg/kg) or as a weekly depot injection (150 mg/kg, sc). After 1 month, a male of proven fertility was introduced into each group. Females underwent weekly monitoring of progesterone (P) and ultrasound evaluation for pregnancy if P remained elevated (1.0 ng/mL) >3 weeks. ORG 9935 values were evaluated using high-performance liquid chromatography. Results: Overall, the pregnancy rate in ORG 9935-treated monkeys (4/8, 50%) did not differ from controls (7/8, 88%; p=.5). However, no animal became pregnant in a cycle when the serum level of ORG 9935 exceeded 300 nmol/L. Moreover, two treated monkeys who mated throughout the treatment phase and did not conceive became pregnant within four cycles after stopping ORG 9935. The other two animals were discontinued prematurely from the protocol. Conclusions: These results demonstrate that ORG 9935 may prevent pregnancy in primates at serum concentrations above 300 nmol/L and that the effect is reversible. [Copyright &y& Elsevier]

Studies were designed to examine the expression and activity of four caspases that contribute to the initial (caspases-2, -8, and -9) and final (caspase-3) events in apoptosis in the rat corpus luteum (CL) during pregnancy (days 7, 17, 19, and 21 of gestation), postpartum (days / and 4), and after injection (0, 8, 16, 24, and 36 h) of the physiological luteolysin PGF2α. In addition, the temporal relationship of caspase expression/activity relative to steroid production and luteal regression was evaluated. During pregnancy, the activity of all four caspases was significantly greater on day 19, before a decline in CL progesterone (P) and CYP1 1A1 levels at day 21 of gestation. The levels of the caspase-3 active fragment (p17, measured by Western blot) also increased at days 19 and 21 of pregnancy. lrnmunohistochemical analyses detected specific staining for the caspases in luteal cells (large and small) as well as in endothelial cells. However, the percentage of apoptotic cells did not increase in the CL until postpartum. Following PGF2α injection, there was a significant decrease in CL P by 24 h, although the activity of all four caspases did not increase until 36 h posttreatment. The active p17 fragment of caspase-3 also significantly increased at 36 h post- PGF2α. These results suggest that an increase in the activity of caspases-2, -8, -9, and -3 is associated with the early events of natural luteolysis at the end of pregnancy. Also, the exogenous administration of the luteolysin PGF2α may regulate members of the caspase family. [ABSTRACT FROM AUTHOR]

Objective: To compare LH, FSH, P, and E(2) levels obtained from blood spot vs. plasma (single-visit study) and to determine whether blood spots can document circulating hormone levels during ovulatory cycles (menstrual-cycle study).Design: Cross-sectional study.Setting: Academic center.Patient(s): Women 18-35 years of age with regular menstrual cycles and no recent use of hormonal contraception.Intervention(s): Women contributed both a blood spot sample from a finger-stick and a plasma sample through venipuncture on a random day within their menstrual cycle (n = 100, single study visit). Five additional women were followed for an entire menstrual cycle with biweekly venipuncture and daily self-collected blood spot sampling. Samples were analyzed for FSH, LH, P, and E(2).Main Outcome Measure(s): Correlation between blood spot and plasma levels.Result(s): Significant positive correlations were found between the blood spot and plasma samples in the single-visit study (r(2): FSH, 0.91; LH, 0.93; P, 0.83; and E(2,) 0.70). Two of the 5 menstrual-cycle study women had ovulatory cycles based on P levels (>3 ng/mL) and an LH surge. Daily blood spot sampling was better able to document hormonal changes than biweekly venipuncture.Conclusion(s): Blood spot monitoring of FSH, LH, P, and, to a lesser extent, E(2), appears to be as valid as traditional plasma assays for clinical research and care. [ABSTRACT FROM AUTHOR]

Selection of the species-specific number of follicles that will develop and ovulate during the ovarian cycle can be overridden by increasing the levels of pituitary gonadotropin hormones, FSH and LH. During controlled ovarian stimulation (COS) in nonhuman primates for assisted reproductive technology (ART) protocols, the method of choice (but not the only method) has been the administration of exogenous gonadotropins, either of nonprimate or primate origin. Due to species-specificity of the primate LH (but not FSH) receptor, COS with nonprimate (e.g., PMSG) hormones can be attributed to their FSH activity. Elevated levels of FSH alone will produce large antral follicles containing oocytes capable of fertilization in vitro (IVF). However, there is evidence that LH, probably in lesser amounts, increases the rate of follicular development, reduces heterogeneity of the antral follicle pool, and improves the viability and rate of pre-implantation development of IVF-produced embryos. Since an endogenous LH surge typically does not occur during COS cycles (especially when a GnRH antagonist is added), a large dose of an LH-like hormone (i.e., hCG) may be given to reinitiate meiosis and produce fertilizable oocytes. Alternate approaches using exogenous LH (or FSH), or GnRH agonist to induce an endogenous LH surge, have received lesser attention. Current protocols will routinely yield dozens of large follicles with fertilizable eggs. However, limitations include non/poor-responding animals, heterogeneity of follicles (and presumably oocytes) and subsequent short luteal phases (limiting embryo transfer in COS cycles). However, the most serious limitation to further improvements and expanded use of COS protocols for ART is the lack of availability of nonhuman primate gonadotropins. Human, and even more so, nonprimate gonadotropins are antigenic in monkeys, which limits the number of COS cycles to as few as 1 (PMSG) or 3 (recombinant hCG) protocols in macaques. Production and access to sufficient supplies of nonhuman primate SH, LH and CG would overcome this major hurdle. [ABSTRACT FROM AUTHOR]

Estrogen promotes the growth of some ovarian cancer cells at nanomolar concentrations, but has been shown to inhibit growth of normal ovarian surface epithelial (OSE) cells at micromolar concentrations (1 μg/ml). OSE cells express the estrogen receptor (ER)-α, and are the source of 90% of ovarian cancers. The potential sensitivity of OSE cells to estrogen stresses the importance of understanding the estrogen-dependent mechanisms at play in OSE proliferation and transformation, as well as in anticancer treatment. We investigated the effects of estradiol on cell proliferation in vitro, and demonstrate an intracellular locus of action of estradiol in cultured rhesus ovarian surface epithelial (RhOSE) cells. We show that ovarian and breast cells are growth-inhibited by micromolar concentrations of estradiol, and that this inhibition correlates with estrogen receptor expression. We further show that normal rhesus OSE cells do not activate ERK or Akt in response to estradiol, nor does estradiol block the ability of serum to stimulate ERK or induce cyclin D expression. Contrarily, estradiol inhibits serum-dependent retinoblastoma protein (Rb) phosphorylation and blocks DNA synthesis. This inhibition does not formally arrest cells, and is reversible within hours of estrogen withdrawal. Our data are consistent with growth inhibition by activation of Rb and indicate that sensitivity to hormone therapy in anticancer treatment can be modulated by cell cycle regulators downstream of the estrogen receptor. [Copyright &y& Elsevier]

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]

Circulating levels of Ang-2 and sTie-2 receptor were detectable but invariant in women during COS cycles. During the postimplantation period, the rise in Ang-2 (but not sTie-2) levels probably reflects placental rather than luteal production. [Copyright &y& Elsevier]

A preface for Basic and applied biology of the primate reproductive tract: in honor of the career of Dr Robert M Brenner is presented.

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: 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. [ABSTRACT FROM AUTHOR]

Objective: To determine whether angiopoietin (ANGPT)-1 and -2 are detectable in the circulation of nonhuman primates and women and whether these levels fluctuate in association with ovarian activity.Design: Prospective.Setting: National Primate Research Center, medical center, and infertility clinic.Patient(s): Adult female rhesus monkeys; 15 women donating oocytes for infertility treatment.Intervention(s): Controlled ovarian stimulation with gonadotropins, removal of the corpus luteum and ovaries, oocyte retrieval, and ET.Main Outcome Measure(s): Circulating levels of ANGPT-1 and ANGPT-2.Result(s): Serum ANGPT-1 and ANGPT-2 levels were detectable and invariant in maintaining an ANGPT-1 to -2 ratio >1 in [1] macaques over the course of the natural menstrual cycle, during a controlled ovulation protocol, and after removal of the corpus luteum or ovaries and [2] women undergoing controlled ovarian simulation. In contrast, the ANGPT-1 to -2 ratio was markedly decreased (<<1) at mid-to-late gestation in macaques and in the follicular fluid of women undergoing controlled ovarian simulation because of increased levels of ANGPT-2.Conclusion(s): The ovary and its dominant structures are not major contributors to circulating levels of ANGPT-1 or ANGPT-2. The physiologic importance of the rising levels of ANGPT-2 after the luteal-placental shift in pregnancy is unknown. [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]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To examine the small antral follicle (SAF) cohort in ovaries of adult rhesus monkeys after consumption of a Western-style diet (WSD), with or without chronically elevated androgen levels since before puberty.Design: Cholesterol or T (n = 6 per group) implants were placed SC in female rhesus macaques beginning at 1 year of age (prepubertal), with addition of a WSD (high fat/fructose) at 5.5 years (menarche approximately 2.6 years). Ovaries were collected at 7 years of age. One ovary per female was embedded in paraffin for morphologic and immunohistochemical analyses. The SAFs (<2.5 mm) were dissected from the other ovary obtained at or near menses in a subgroup of females (n = 3 per group) and processed for microarray analyses of the SAF transcriptome. Ovaries of adult monkeys consuming a standard macaque diet (low in fats and sugars) were obtained at similar stages of the menstrual cycle and used as controls for all analyses.Setting: Primate research center.Animal(s): Adult, female rhesus monkeys (Macaca mulatta).Intervention(s): None.Main Outcome Measures: Histologic analyses, SAF counts and morphology, protein localization and abundance in SAFs, transcriptome in SAFs (messenger RNAs [mRNAs]).Result(s): Compared with controls, consumption of a WSD, with and without T treatment, increased the numbers of SAFs per ovary, owing to the presence of more atretic follicles. Numbers of granulosa cells expressing cellular proliferation markers (pRb and pH3) was greater in healthy SAFs, whereas numbers of cells expressing the cell cycle inhibitor (p21) was higher in atretic SAFs. Intense CYP17A1 staining was observed in the theca cells of SAFs from WSD with or without T groups, compared with controls. Microarray analyses of the transcriptome in SAFs isolated from WSD and WSD plus T-treated females and controls consuming a standard diet identified 1,944 genes whose mRNA levels changed twofold or more among the three groups. Further analyses identified several gene pathways altered by WSD and/or WSD plus T associated with steroid, carbohydrate, and lipid metabolism, plus ovarian processes. Alterations in levels of several SAF mRNAs are similar to those observed in follicular cells from women with polycystic ovary syndrome.Conclusion(s): These data indicate that consumption of a WSD high in fats and sugars in the presence and absence of chronically elevated T alters the structure and function of SAFs within primate ovaries. [ABSTRACT FROM AUTHOR]

Background: ADRB-2 was implicated in rodent ovarian functions, including initial follicular growth. In contrast, ADRB-2 expression and function in nonhuman primate and human ovary were not fully known but innervation and significant levels of norepinephrine (NE), which is a ligand at the ADRB-2, were reported in the ovary. Methods: We studied expression of ADRB-2 in human and rhesus monkey ovary (RT-PCR, immunohistochemistry; laser micro dissection) and measured levels of norepinephrine (NE; ELISA) in monkey follicular fluid (FF). 3D cultures of monkey follicles (4 animals) were exposed to NE or the ADRB-2 agonist isoproterenol (ISO), and follicular development (size) was monitored. Upon termination expression of ADRB-2, FSH receptor and aromatase genes were examined. Results: Immunohistochemistry and RT-PCR of either human follicular granulosa cells (GCs) obtained by laser micro dissection or isolated monkey follicles revealed ADRB-2 in GCs of primordial, primary, secondary and tertiary follicles. Staining of GCs in primordial and primary follicles was intense. In large preantral and antral follicles the staining was heterogeneous, with positive and negative GCs present but GCs lining the antrum of large follicles were generally strongly immunopositive. Theca, interstitial, and ovarian surface epithelial cells were also positive. NE was detected in FF of preovulatory antral monkey follicles (0.37 + 0.05 ng/ml; n = 7; ELISA) but not in serum. We examined preantral follicles ranging from 152 to 366 μm in diameter in a 3D culture in media supplemented with follicle stimulating hormone (FSH). Under these conditions, neither NE, nor ISO, influenced growth rate in a period lasting up to one month. Upon termination of the cultures, all surviving follicles expressed aromatase and FSH receptors, but only about half of them also co-expressed ADRB-2. The ADRB-2 expression was not correlated with the treatment but was positively correlated with the follicular size at the beginning and at the end of the culture period. Hence, expression of ADRB-2 was found in the largest and fastest-in vitro growing follicles. Conclusions: The results imply ADRB-2-mediated actions in the development of primate follicles. Drugs interfering with ADRB-2 are used to treat medical conditions and may have unexplored effects in the human ovary. [ABSTRACT FROM AUTHOR]

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]

Fertility impairment and loss due to cancer or its treatment is a significant survivorship consideration for many pediatric, adolescent, and young adult cancer survivors. Chemotherapeutics, radiation, and surgery can impact the future fertility of men, women, and children with cancer. The field of oncofertility, founded to ensure the reproductive future of cancer survivors, gained momentum with 5 years of funding through a 2007 National Institutes of Health Roadmap Grant for Biomedical Research. This report from working group meetings at the fifth annual Oncofertility Consortium Conference speaks to the present state of oncofertility research and clinical care, existing gaps, and future directions for the field. This summary from conference participants and leaders in the field addresses the science, clinical specialties, and academic scholarship that can guide the field as the Roadmap Grant funding comes to a close. [ABSTRACT FROM AUTHOR]

Mutations in mitochondrial DNA (mtDNA) are associated with severe human diseases and are maternally inherited through the egg's cytoplasm. Here we investigated the feasibility of mtDNA replacement in human oocytes by spindle transfer (ST; also called spindle-chromosomal complex transfer). Of 106 human oocytes donated for research, 65 were subjected to reciprocal ST and 33 served as controls. Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA. The mtDNA can be efficiently replaced in human oocytes. Although some ST oocytes displayed abnormal fertilization, remaining embryos were capable of developing to blastocysts and producing embryonic stem cells similar to controls. [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]

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. [ABSTRACT FROM AUTHOR]

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]

Background: This study was conducted to determine whether increased body mass index (BMI) affects oral contraceptive (OC) pharmacokinetics and suppression of hypothalamic-pituitary-ovarian (HPO) axis activity.Study Design: Ovulatory reproductive-age women with normal weight (BMI <25 kg/m(2); n=10) and with obesity (BMI >30 kg/m(2); n=10) received OCs for two cycles (prospective cohort). Subjects were admitted for two 48-h inpatient stays at the beginning and end of the hormone-free interval. Ethinyl estradiol and levonorgestrel (LNG) levels were evaluated during both inpatient stays. Gonadotropin pulsatility (follicle-stimulating hormone and luteinizing hormone) was measured during the second inpatient stay. Estradiol (E(2)) and progesterone (P) were measured daily during inpatient stays and twice per week in Cycle 2.Results: BMI was greater in the obese compared to the normal-BMI group [37.3 kg/m(2) (SD, 6.0) vs. 21.9 kg/m(2) (SD, 1.6); p<.05]. The LNG half-life was significantly longer in the obese group (52.1+/-29.4 vs. 25.6+/-9.3 h, p<.05), which correlated with a lower maximum LNG concentration on Cycle 2, Day 1 [1.9 ng/mL (SD, 0.5) vs. 2.5 ng/mL (SD, 0.7)] and a longer time to reach steady state (10 vs. 5 days) in obese women. There were no significant differences in volume of distribution between groups. LH pulse parameters did not differ statistically between groups but trended toward greater HPO activity in the obese group. Additionally, more obese (6/10 vs. 3/10 normal BMI, p>.05) women exhibited E(2) levels consistent with development of a dominant follicle and P levels consistent with ovulation (2/10 vs. 1/10) during Cycle 2.Conclusions: Compared to women with normal BMI, obese women exhibit differences in OC pharmacokinetics that are associated with greater HPO activity. [ABSTRACT FROM AUTHOR]

Abstract: Background: This study was conducted to determine whether increased body mass index (BMI) affects oral contraceptive (OC) pharmacokinetics and suppression of hypothalamic–pituitary–ovarian (HPO) axis activity. Study Design: Ovulatory reproductive-age women with normal weight (BMI <25 kg/m2; n=10) and with obesity (BMI >30 kg/m2; n=10) received OCs for two cycles (prospective cohort). Subjects were admitted for two 48-h inpatient stays at the beginning and end of the hormone-free interval. Ethinyl estradiol and levonorgestrel (LNG) levels were evaluated during both inpatient stays. Gonadotropin pulsatility (follicle-stimulating hormone and luteinizing hormone) was measured during the second inpatient stay. Estradiol (E2) and progesterone (P) were measured daily during inpatient stays and twice per week in Cycle 2. Results: BMI was greater in the obese compared to the normal-BMI group [37.3 kg/m2 (SD, 6.0) vs. 21.9 kg/m2 (SD, 1.6); p<.05]. The LNG half-life was significantly longer in the obese group (52.1±29.4 vs. 25.6±9.3 h, p<.05), which correlated with a lower maximum LNG concentration on Cycle 2, Day 1 [1.9 ng/mL (SD, 0.5) vs. 2.5 ng/mL (SD, 0.7)] and a longer time to reach steady state (10 vs. 5 days) in obese women. There were no significant differences in volume of distribution between groups. LH pulse parameters did not differ statistically between groups but trended toward greater HPO activity in the obese group. Additionally, more obese (6/10 vs. 3/10 normal BMI, p>.05) women exhibited E2 levels consistent with development of a dominant follicle and P levels consistent with ovulation (2/10 vs. 1/10) during Cycle 2. Conclusions: Compared to women with normal BMI, obese women exhibit differences in OC pharmacokinetics that are associated with greater HPO activity. [Copyright &y& Elsevier]

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]

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]

This study was designed to evaluate the timecourse of ovarian and pituitary endocrine events throughout the menstrual cycle in the vervet monkey, and whether circulating luteinizing hormone (LH) or the uterus regulates the functional lifespan of the vervet corpus luteum. Daily saphenous blood samples were collected from adult females (1) during spontaneous menstrual cycles (n = 7), and (2) during cycles in which a gonadotropin-releasing hormone antagonist (acyline) was administered for 3 days at midluteal phase (n = 3), and (3) for 30 days following recovery from hysterectomy (n = 4). Estradiol (E) and progesterone (P) levels were assayed using electrochemoluminescent assays. Gonadotropin levels were measured by radioimmunoassay using reagents developed for the assay of follicle-stimulating hormone and LH in macaques. Spontaneous cycles exhibited a midcycle E rise (476±49 pg/ml), engendering an LH surge, 12±1 days after onset of menses, followed by a luteal phase with peak P levels of 4.7±0.9 ng/ml. Histologic evaluation of the ovaries at late follicular phase or early luteal phase revealed the presence of a single, large Graafian follicle or developing corpus luteum, respectively. Acyline treatment caused a significant (P<0.05) decline in P levels (2.9±0.5 vs 0.5±0.3 ng/ml, 0 vs 48 h post-treatment) and premature menstruation compared with untreated controls (P<0.05). Hysterectomy had no apparent effect on the monthly pattern or levels of circulating E or P. Thus, the characteristics and regulation of the ovarian cycle in vervets appear similar to those in women and macaques, with cyclicity dependent on pituitary gonadotropin hormones and independent of a uterine luteolytic factor. Am. J. Primatol. 69:890–900, 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The vervet monkey was evaluated as a primate model for use in assisted reproductive technologies (ARTs). Eight adult female vervets were hormonally monitored for their potential use as egg donors and those six females displaying regular menstrual cycles were subjected to controlled ovarian stimulation with recombinant human gonadotropins. Three animals failed to respond while laparoscopic follicular aspiration was performed on the other three females at 27–30 h post-human chorionic gonadotropin administration. A total of 62, 40, and 18 oocytes was recovered from these three animals of which 30, 20, and 4, respectively, matured to the metaphase II stage and were subsequently inseminated using intracytoplasmic sperm injection. An average of 40±15% (SEM) of the inseminated oocytes were fertilized based on pronucleus formation and timely cleavage. One embryo from each of the two stimulated females developed into expanded blastocysts. Two adult male vervets were assessed as sperm donors. Neither adjusted well to the restraint and collection procedure required for penile electroejaculation. Samples collected via rectal electroejaculation were very low in sperm motility and concentration; however, cauda epididymal aspirations from one male yielded an adequate concentration of motile sperm. These results emphasize the need to establish species-specific ovarian stimulation protocols and semen collection techniques if vervets are to be considered for basic and applied (ARTs) research on primate gametes or embryos. Am. J. Primatol. 69:917–929, 2007. © 2007 Wiley-Liss, Inc. [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]

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]

Presents a paper which reviewed progress in the use of nuclear technology as it relates to mammalian cloning. What Hans Spemann, a German developmental biologist suggested in a 1938 experiment; Indepth look at reasons for cloning; How the cloning of rhesus monkeys was done; Information on nuclear transfer in the treatment of human infertility; Detailed information on the ethics of human cloning.

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]