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Background: Administration of low doses of an antiprogestin to rhesus monkeys permits ovarian/menstrual cyclicity, suppresses endometrial proliferation and prevents pregnancy without adverse or toxic side-effects after 5-6 months of daily treatment. The purpose of this study was to test the reversibility with respect to restoration of fertility after 1 year of low-dose antiprogestin treatment., Methods: This experiment included a daily 1 year vehicle- or antiprogestin-treatment interval followed by a 9 month post-treatment interval for adult, female rhesus monkeys (n = 5/group) of proven fertility and exhibiting regular menstrual cycles. Co-habitation occurred with a male of proven fertility and vaginal swabs were taken to identify the presence of sperm during the treatment (antiprogestin females) and post-treatment intervals (vehicle and antiprogestin females)., Results: Mating and vaginal sperm were evident in all antiprogestin females during, and, in both groups, after treatment. Based on ultrasonography, none of the antiprogestin-treated females became pregnant during the treatment interval. However, upon cessation of treatment, pregnancy rates were similar between antiprogestin-treated (3/5) relative to vehicle-treated (4/5) females with live, healthy infants born in both groups. There were no differences between groups in fetal measurements, gestation lengths, live birth rates and infant weights., Conclusions: The reversal of the anti-fertility effects of chronic, low-dose antiprogestin treatment supports the clinical feasibility of potent and selective antiprogestins as potential contraceptives for women.

Background: The purpose of this study was to determine whether phosphodiesterase (PDE) 3 inhibitors selectively prevent the resumption of meiosis in primates., Methods: Immature oocytes (intact germinal vesicles) obtained from large pre-ovulatory follicles following ovarian stimulation in rhesus macaques were incubated with or without various doses of the PDE3 inhibitors, Cilostamide, Milrinone or ORG 9935, or a selective PDE4 inhibitor, Rolipram. Oocytes were observed for germinal vesicle breakdown (GVBD) as an indicator of resumption of meiosis., Results: At 24 h, 72 of 121 (60%) control oocytes progressed to GVBD compared with 9/34 (27%, P < 0.01), 4/36 (11.1%, P < 0.01) and 0/28 (0%, P < 0.01) oocytes incubated with ORG 9935 at 0.1, 0.5 and 1.0 micromol/l respectively. Similar results were achieved at 24 h with 1.0 micromol/l Cilostamide (2/24 oocytes, 8%, P < 0.01) and 100 micromol/l Milrinone (2/32, 6%, P < 0.01). In contrast, no significant difference in GVBD was noted between control oocytes and those incubated with up to 100 micromol/l Rolipram for 24 h (43/58, 74%) or 48 h (44/58, 76%)., Conclusions: These experiments establish the specificity and dose-dependent ability of PDE3, but not PDE4, inhibitors to block resumption of meiosis in macaque oocytes in vitro. Thus, PDE3 inhibitors have potential use as contraceptives in primates.

Continual administration of low doses of the antiprogestin ZK 137 316 was previously reported to permit ovarian/menstrual cyclicity, but disrupt endometrial growth in macaques. The contraceptive efficacy of this regimen was tested in female rhesus monkeys (10 per group) treated daily with vehicle (controls), 0.01 or 0.03 mg ZK 137 316 per kg body weight for 30 days before and during continual co-habitation with males of proven fertility. Treatment continued until confirmation of pregnancy or for 5 months after pair-housing with males. Mating and vaginal sperm were evident in all females. A cumulative pregnancy rate of 90% (9/10) was observed in the controls. Of the 10 animals receiving 0.01 mg/kg, four conceived during the first 2 months of pairing (P = 0.06) with no further conceptions. No pregnancies were observed in the 0.03 mg/kg group (P < 0.01). Timely, overt menses occurred at a higher frequency in the 0.01 mg/kg group than the 0.03 mg/kg group. However, corpora lutea were present in ovaries from both groups during the last treatment cycle, indicating that ovarian cycles occurred. Thus, chronic administration of low-dose ZK 137 316 that permits continued ovarian cyclicity and a high incidence of timely menses, prevents pregnancy in non-human primates. This regimen may provide a novel method of contraception for women.

The efficacy of follicle stimulating hormone (FSH) as an alternative to luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) for the initiation of periovulatory events in primate follicles is unknown. A single bolus of 2500 IU recombinant (r)-hFSH was compared to 1000 IU r-HCG for its ability to promote oocyte nuclear maturation and fertilization, granulosa cell luteinization and corpus luteum function following r-hFSH (60 IU/day) induction of multiple follicular development in rhesus monkeys. Following the r-hFSH bolus, bioactive luteinizing hormone concentrations were <3 ng/ml. Peak concentrations of serum FSH (1455+/-314 mIU/ml; mean+/-SEM) were attained 2-8 h after r-hFSH, and declined by 96 h. Bioactive HCG concentrations peaked between 2-8 h after r-HCG and remained > or = 100 ng/ml for >48 h, while immunoreactive FSH concentrations were at baseline. The proportion of oocytes resuming meiosis and undergoing in-vitro fertilization (IVF) were comparable for r-hFSH (89%; 47+/-19%) and r-HCG (88%; 50+/-17%). In-vitro progesterone production and expression of progesterone receptors in granulosa cells did not differ between groups. Peak concentrations of serum progesterone in the luteal phase were similar, but were lower 6-9 days post-FSH relative to HCG. Thus, a bolus of r-hFSH was equivalent to r-HCG for the reinitiation of oocyte meiosis, fertilization and granulosa cell luteinization, but a midcycle FSH surge did not sustain normal luteal function in primates.

The long-term effects of the antiprogestin ZK 137 316 on reproductive tract morphology in rhesus macaques were investigated. The monkeys were injected daily (i.m.) for five menstrual cycles with vehicle or 0.01, 0.03 or 0.1 mg ZK 137 316/kg body weight. Reproductive tracts (n = 3/ group) were collected during the mid-luteal phase (day 8) of the fifth cycle in the control, 0.01 and 0.03 mg/kg groups, or 6-7 days after the oestradiol peak in the 0.1 mg/kg group. ZK 137 316 treatment resulted in a dose-dependent atrophy of the endometrium, marked by reduced mitotic activity in the glands, compaction of the stroma, degradation of spiral arteries and dilation of veins. There was no effect of ZK 137 316 on myometrial or oviductal weight. Treatment with 0.1 and 0.03 mg/kg, but not 0.01 mg/kg resulted in fully ciliated and secretory oviducts, indicating a dose-dependent blockade of progesterone antagonism of oestrogen-dependent oviductal differentiation. In the endometrium, the suppressive action of progesterone on oestrogen and progestin receptors was also blocked by ZK 137 316 in a dose-dependent manner. However, endometrial atrophy appeared due to inhibition of progesterone action together with a blockade of oestrogen-dependent proliferation. The profoundly suppressed endometrium produced by chronic low-dose ZK 137 316 treatment is unlikely to support implantation. Such treatment may therefore provide a novel contraceptive modality.

Large doses of antiprogestin typically disrupt menstrual cyclicity. A chronic low-dose regimen of the potent new antiprogestin ZK 137 316, which permits continued menstrual cyclicity but alters gonadal-reproductive tract activity, was established. Rhesus monkeys received vehicle (n = 6) or 0.01 (n = 8), 0.03 (n = 8) or 0.1 (n = 5) mg ZK 137 316/kg body weight daily for five menstrual cycles (C-1 to C-5). Oestradiol, progesterone and gonadotrophin profiles were normal during cycles involving vehicle and 0.01 and 0.03 mg ZK 137 316/kg body weight. In the 0.1 mg/kg group, mid-cycle oestradiol and gonadotrophin surges, and subsequent progesterone production, were absent in C-3 and C-5. Ovarian cyclicity was accompanied by timely menstruation in the vehicle and 0.01 mg/kg groups. By C-3, half the animals in the 0.03 mg/kg group and all animals in the 0.1 mg/kg group were amenorrhoeic. A corpus luteum was noted during the mid-luteal phase of C-5 in the vehicle, 0.01 mg/kg and 0.03 mg/kg groups. Large antral and cystic follicles were evident in the 0.1 mg/kg group. Thus, a daily treatment with 0.01 mg/kg ZK 136317 permitted normal menstrual cyclicity in macaques. While the daily administration of 0.03 mg/kg ZK 136 317 allowed ovarian cyclicity, menstruation was disrupted in some animals. Increasing the dose to 0.1 mg/kg antagonized pituitary function and resulted in anovulation and amenorrhoea. A chronic low-dose regimen of the antiprogestin ZK 137 316, which permits normal ovarian/menstrual cyclicity, has potential as a contraceptive in women.

During in-vitro fertilization (IVF) cycles, a large bolus of human chorionic gonadotrophin (HCG) is used to induce periovulatory events, but the efficacy of lower doses is undefined. Following follicular stimulation in rhesus monkeys, oocyte nuclear maturation, IVF, granulosa cell luteinization and corpus luteum function were compared after injection of 100, 300 or 1000 IU recombinant HCG or 1000 IU urinary HCG. Bioactive HCG rose to peak concentrations within 2 h that were proportional to the dose administered (100 < 300 < 1000 IU, recombinant HCG = urinary HCG). The duration of surge values (>100 ng/ml) was also dose-dependent (0 h, 100 IU; 24 h, 300 IU; >48 h, 1000 IU, recombinant and urinary HCG). While the proportions of oocytes resuming meiosis and undergoing IVF were similar among groups, fewer animals yielded fertilizable oocytes following 100 and 300 IU (five of nine) compared to 1000 IU recombinant and urinary HCG (nine of 10). Peak values of serum progesterone in the luteal phase were similar, but declined 2 days earlier after 100 and 300 IU relative to 1000 IU recombinant and urinary HCG. Thus, 3-10 fold lower doses of HCG elicit low amplitude surges of short duration that induce periovulatory events such as re-initiation of oocyte meiosis and granulosa cell luteinization. However, oocyte fertilization and luteal function may optimally require surges of higher amplitude and longer duration similar to those produced by standard doses of 1000 IU recombinant or urinary HCG.

We previously demonstrated, in luteinizing hormone (LH)-deficient macaques, that follicular growth and maturation occurred with administration of exogenous (recombinant human) follicle stimulating hormone (r-hFSH) alone, and that the oocytes recovered fertilized at a notably higher rate than their counterparts from animals receiving both r-hFSH and r-hLH (Zelinski-Wooten et al., 1995). Here, the developmental potential of embryos produced from animals treated with r-hFSH alone or in combination with r-hLH was evaluated. Embryos (n = 127) were cryopreserved, thawed and either co-cultured on buffalo rat liver cells until the hatched blastocyst stage or transferred to synchronized recipients. Although embryos from each treatment group demonstrated a similar ability to develop to hatched blastocysts with a definitive inner cell mass, a significant difference was seen in cryosurvival (56 versus 78%) and in developmental rate to the hatched blastocyst (12 versus 10 days) between embryos from the r-hFSH alone and the combination group respectively. Pregnancies resulted following oviductal embryo transfers in both groups, with corpus luteum rescue occurring on days 12-16 of the luteal phase. In summary, r-hFSH alone during the pre-ovulatory interval is adequate for the gametogenic events required to produce embryos that develop either in vitro or in vivo; however, exposure to r-hLH may improve embryo viability and the rate of development.

Both follicle stimulating hormone (FSH) and luteinizing hormone (LH) are proposed requirements for follicular growth and steroidogenesis; however, the role of LH in primate folliculogenesis is unclear. Follicular stimulation by recombinant human FSH (n = 5) with and without recombinant LH (1:1; n = 6) following 90 days of gonadotrophin-releasing hormone (GnRH) antagonist (Antide) treatment in macaques was evaluated. Human chorionic gonadotrophin (HCG) was administered when six follicles > or = 4 mm were observed. Oocytes were aspirated 27 h later and inseminated in vitro. Chronic Antide reduced serum oestradiol and bioactive LH to concentrations observed in hypophysectomized rhesus monkeys. Multiple follicular growth required a longer interval following recombinant FSH (12 +/- 1 days) than recombinant FSH+recombinant LH (9 +/- 0.2 days), but the total number of follicles/animal did not differ between groups. The day prior to HCG, oestradiol concentrations were 4-fold less following recombinant FSH compared to recombinant FSH+recombinant LH. With recombinant FSH, more oocytes completed meiosis to metaphase II (51%) and fertilized (89 +/- 5%) relative to recombinant FSH+recombinant LH (12 and 52 +/- 11% respectively). Follicular growth and maturation in LH-deficient macaques occurred with FSH alone. Thus, LH is not required for folliculogenesis in primates. Higher fertilization rates following follicular stimulation with FSH alone suggest that the presence of LH with FSH (1:1) during the pre-ovulatory interval impairs gametogenic events in the periovulatory period.

We and others have detected progesterone receptors (PR) in the primate (macaque and human) corpus luteum by immunocytochemistry. However, we have been unable to measure PR in the corpus luteum by conventional ligand binding assay, presumably because high endogenous concentrations of progesterone (P) in luteal tissue prevented specific binding of radiolabeled hormone to PR during the assays. To test this hypothesis, we treated monkeys with the 3 beta-hydroxysteroid dehydrogenase inhibitor, Trilostane, to reduce levels of endogenous P before conducting binding assays for PR in luteal tissue. To obtain adequate tissue for saturation analysis, rhesus monkeys (n = 6) were superovulated by treating them with hFSH beginning at menses (day 1) for 6 days, then with hFSH and hLH (days 6-9), followed by hCG (day 10). Trilostane (600 mg) was given 5 days after hCG treatment (day 15), and binding assays were conducted 18 h later. Trilostane significantly reduced mean (+/- SE) serum levels of P from 97.8 +/- 16 to 2.7 +/- 1.3 nmol/L within 18 h (P < 0.001). Strong nuclear staining of PR was detected by immunocytochemistry in both Trilostane-treated and control (not Trilostane-treated) tissue, but ligand binding was measurable only in Trilostane-treated monkeys. Scatchard transformations of saturation curves revealed high affinity binding of [3H] R5020 in luteal cytosol and nuclear extracts with an approximate dissociation constant (Kd) of 4.8 and 1.37 nmol/L, respectively. Also, the PR-specific monoclonal antibody, JZB-39, shifted a [3H]R5020-bound luteal macromolecule on sucrose gradients. Mean cytosolic and nuclear binding of [3H]R5020 were 0.31 +/- 0.09 and 0.06 +/- 0.02 fmol/micrograms DNA, respectively. Similar binding of [3H]R5020 was demonstrated in corpora lutea obtained during spontaneous menstrual cycles after Trilostane treatment (n = 3). These results show unequivocally that the PR in macaque luteal tissue can bind P with high affinity and suggest a receptor-mediated action of P in the primate corpus luteum.

The amplitude and duration of the midcycle LH surge required for periovulatory changes in the primate follicle are incompletely defined. We reported that short (4- to 14-h) LH surges were insufficient to induce periovulatory events after multiple follicular development in macaques. In contrast, an 18- to 24-h LH surge induced oocyte maturation plus granulosa cell luteinization, but did not support corpus luteum function. In this study, the periovulatory changes following LH surges of 48 h elicited using pituitary (pit) or recombinant (r) human (h) LH were compared to those after 24-h LH surge durations or after urinary hCG (u-hCG) treatment. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. On day 10, animals (n = 3-5/group) received 1) a single injection of u-hCG [79 +/- 3 micrograms RP-1 equivalents (equiv), im], 2) two injections of pit-hLH (91 +/- 4 micrograms RP-1 equiv, im), 3) one injection of r-hLH (21 +/- 1 micrograms RP-1 equiv, im), or 4) two injections of r-hLH (21 +/- 1 micrograms RP-1 equiv). Oocytes and granulosa cells were obtained via follicle aspiration 27 h after the initial LH or hCG injection. In all groups, serum estradiol rose to similar peak levels by day 10. Circulating LH-like bioactivity was elevated for more than 48 h after u-hCG. Peak serum LH bioactivities were proportional to the administered LH doses, as determined in the in vitro bioassay. Two injections of either r-hLH or pit-hLH elicited surge levels (> 100 ng/mL) of bioactive LH for 36-48 h, whereas one injection sustained surge levels for only 18-24 h. The proportions of oocytes resuming meiosis (68-76%) were similar in all groups. Immunocytochemical staining for progesterone receptor and in vitro progesterone production by granulosa cells in all LH-treated groups were comparable to those of cells form the hCG-treated group. Peak levels of progesterone in the luteal phase were comparable in monkeys treated with two doses of pit-hLH and r-hLH (18.5 +/- 10.4 vs. 8.1 +/- 1.5 ng/mL) and approached that in u-hCG treated monkeys (39.5 +/- 18.0 ng/mL). However, progesterone levels in animals treated once with r-hLH (3.4 +/- 1.5 ng/mL) were less (P < 0.05) than those in u-hCG-treated monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)

The actions of the mid-cycle gonadotrophin surge to convert the pre-ovulatory follicle into the corpus luteum are not well understood in primate species. In experiments titrating the surge duration required in macaque monkeys during artificial in-vitro fertilization (IVF)-related cycles, short (< or = 14 h) LH surges similar to those in rodents and domestic animals failed to initiate peri-ovulatory events. Attenuated (24 h) surges, one-half the duration of spontaneous surges in primates, reinitiated oocyte meiosis and initial luteinization of granulosa cells. However, only surges of > or = 48 h sustained luteal development and function to produce luteal phases of approximately 13 days. Three approaches-immunocytochemistry of steroid receptors, binding of radiolabelled steroid, and reverse transcription-polymerase chain reaction of mRNA-indicate that progesterone receptor (PR), but not oestrogen receptor, is expressed in the macaque corpus luteum. Studies on cells collected before and after the gonadotrophin surge identified a novel action of LH to induce PR expression in luteinizing granulosa cells. Thus, the LH surge requirements vary between non-primate and primate species, as well as between peri-ovulatory events, and the LH surge may promote cellular recognition of paracrine and autocrine factors (e.g. progesterone) that become predominant in the developing corpus luteum.

Local modulation of follicular and gametogenic functions by ovarian androgens and estrogens in mammalian species has been proposed. This study examined the effects of elevated androgen/estrogen ratios during follicular maturation in vivo by inhibiting aromatase activity in rhesus monkeys. To obviate steroid feedback effects, gonadotropin-treated animals were used. Beginning at menses (day 1), animals received human (h) FSH (60 IU/day, im) on days 1-6, followed by hFSH plus hLH (60 IU/day, im) on days 7-9 to promote the growth of multiple follicles. Ovulatory maturation was induced by hCG (1000 IU, im) on day 10. On days 8-10, four animals received an aromatase inhibitor, 1,4,6-androstatrien-3,17-dione (ATD; 1-1.25 g, orally, twice/day), while five served as controls and received no further treatment. Within 8 h of ATD treatment, a 63% reduction in serum estradiol levels relative to control values was evident, which reached maximal suppression (84%) by day 10. A marked elevation (17-fold) in serum androstenedione and a lesser increase (2.6-fold) in serum testosterone occurred with aromatase inhibition, yielding androstenedione/estradiol (18.0) and testosterone/estradiol (1.9) ratios greater than those in controls (0.6 and 0.3, respectively). ATD treatment did not alter follicular diameters or the total number of follicles per animal (20 +/- 3) relative to control values (16 +/- 3). Of the total cohort classified, the proportion of oocytes collected at prophase I was greater (P < 0.05) after ATD treatment (31%) than in controls (11%). Completion of oocyte meiosis to metaphase II was retarded (P < 0.05) in ATD-treated (4%) compared to control (26%) animals. Furthermore, the in vitro fertilization rate of metaphase II oocytes from ATD-treated animals (9%) was reduced (P < 0.05) relative to that in controls (25%). While basal progesterone production by luteinizing granulosa cells in vitro was similar between groups, the addition of hCG in vitro enhanced progesterone secretion by cells from ATD-treated animals (3.1 +/- 0.3-fold over basal) to a greater extent (P = 0.05) than in controls (1.5 +/- 0.3-fold). Progesterone receptor was detected by immunocytochemistry in nuclei of luteinizing granulosa cells from ATD-treated animals as well as controls. Serum progesterone profiles and the length of the luteal phase were similar between groups. Thus, acute elevation of serum androgen/estrogen ratios in vivo during follicular maturation was detrimental to the gametogenic functions of the primate follicle, but did not alter follicular growth, events of early luteinization, or subsequent luteal function.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factors (IGFs) are mitogenic peptides that are important for fetal and maternal tissue growth during pregnancy. They circulate complexed primarily with a serum binding protein, IGFBP-3, which regulates the availability of the IGFs to their target tissues. We previously reported that in pregnant women, serum IGFBP-3 levels, assessed by Western ligand blotting, decline markedly beginning at 6 weeks gestation due to a circulating protease that cleaves IGFBP-3 into a 29-kilodalton (kDa) protein and lower mol wt (M(r)) fragments. In the current study, we compared IGFBP profiles, IGFBP-3 and IGFBP-1 levels, and IGFBP protease activities in sera from pregnant and nonpregnant women, baboons, and rhesus monkeys, using Western ligand blotting, IGFBP-specific immunoassays, IGFBP-3 protease assay, and zymographic gel electrophoresis. Serum IGFBP profiles in nonpregnant human and nonhuman primates were similar and were not cycle-dependent. IGFBP-3 (37-43 kDa), IGFBP-2 (31 kDa), and IGFBP-1 (28 kDa) were identified in all three species using IGFBP-specific human antisera. A 24-kDa IGFBP was also present and is believed to be IGFBP-4. Serum IGFBP-1 levels increased throughout gestation in human and nonhuman primates. Serum IGFBP-2 and putative IGFBP-4 were barely detectable in all three species from midgestation to term, but increased several days postpartum. In contrast, serum IGFBP-3 profiles differed markedly between species during gestation. Rather than the decrease seen in human pregnancy serum, there was an increase in circulating IGFBP-3 levels in nonhuman primates. Furthermore, for both baboon and rhesus monkey, the M(r) of serum IGFBP-3 was about 2 kDa greater in pregnant than in nonpregnant animals, and deglycosylation studies suggested that the higher M(r) forms may be alternatively glycosylated or may have a unique primary structure. As in nonpregnant women, serum IGFBP-3 protease activity was absent in nonpregnant and pregnant baboons. However, rhesus monkey serum contained a calcium-dependent protease that cleaved recombinant human IGFBP-3 into unique fragments, compared to the human pregnancy enzyme. Unlike human pregnancy serum, which proteolyzes IGFBP-3, in human nonpregnancy serum, rhesus serum incubated under similar conditions did not result in proteolysis of rhesus IGFBP-3, suggesting that the IGFBP-3 protease in human pregnancy serum is not present in the circulation of the rhesus monkey. To assess proteolytic activity in these sera, zymographic polyacrylamide gel analysis, using gelatin as a substrate, was performed. A minor band of proteolytic activity (72 kDa) was observed in all three species throughout gestation.(ABSTRACT TRUNCATED AT 400 WORDS)

After stimulation of multiple follicular development, endogenous LH surges elicited by GnRH or GnRH agonist were of insufficient duration (4-14 h) to evoke oocyte maturation and luteinization in this species. In this study, periovulatory LH surge requirements were further titrated using hLH as the ovulatory stimulus. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. To induce ovulatory maturation on day 10, animals received: 1) hCG (1000 IU, im; n = 8); 2) highly-purified, urinary hLH (2542 IU, im; n = 4); or 3) hLH (2542 IU, im) followed by three injections of hLH (200 IU, im) at 8-h intervals (0800, 1600, 2400 h) daily during the luteal phase until menses (n = 3). Oocytes and luteinizing granulosa cells were obtained via follicle aspiration 27 h after the initial hLH or hCG injection. Estradiol and progesterone levels were measured in daily serum samples by RIA. Bioactive LH levels were determined at selected intervals within 36 h of the hLH ovulatory stimulus. Nuclear maturity of oocytes was evaluated as an indicator for reinitiation of meiosis. Luteinizing granulosa cells were processed for indirect immunocytochemistry using a monoclonal antibody to human progesterone receptor. In vitro progesterone production by luteinizing granulosa cells over 24 h was also assessed in the absence and presence of hCG. In all groups, serum estradiol rose to similar peak levels on day 10. After hLH, bioactive LH levels peaked (1262 +/- 79 ng/mL; mean +/- SEM) by 2-6 h, declined thereafter but remained above surge levels (100 ng/mL) for 18-24 h. Within 24 h of hLH injection, serum progesterone increased to 13 +/- 3 nmol/L, but returned to baseline in 1-6 days. In contrast, higher levels of progesterone were observed after hCG (114 +/- 51 nmol/L) and during luteal phase treatment with hLH (137 +/- 25 nmol/L) and the luteal phase was longer (11.5 +/- 0.4 and 14.3 +/- 0.7 days, respectively). Of the total cohort of oocytes aspirated, the proportion of oocytes resuming meiotic maturation (metaphase I plus metaphase II) was similar after hCG (76%) and hLH (74%). However, the proportion of oocytes maturing to metaphase II tended to be less (P = 0.08) after hLH (13%) than hCG (22%). Fertilization rates were similar between the two groups. Progesterone receptor was detected in nuclei of luteinizing granulosa cells from all animals receiving hCG, but only in some given hLH.(ABSTRACT TRUNCATED AT 400 WORDS)

The amplitude and duration of the midcycle LH surge required for ovulatory maturation of the follicle and its enclosed oocyte in primates are unknown. To titrate periovulatory LH requirements, female rhesus monkeys received human gonadotropins (FSH with/without LH) for 9 days beginning at menses to promote the development of multiple preovulatory follicles. The next day, animals (n = 4-6/group) received: 1) no ovulatory stimulus; 2) 1000 IU hCG, im; 3) one injection of 100 micrograms GnRH, sc (GnRH-1); 4) three injections of GnRH (GnRH-3) at 3-h intervals (0800, 1100, and 1400 h); or 5) two injections of 50 micrograms GnRH agonist (GnRHa), sc, 8 h apart (0800 and 1700 h) to induce ovulatory maturation. Follicles were aspirated 27 h after the hCG or initial GnRH/GnRHa injection or on days 8 and 10 in animals receiving no ovulatory stimulus. Nuclear maturity of oocytes was evaluated as a marker for reinitiation of meiosis. Estradiol and progesterone levels were determined in daily serum samples by RIA. Levels of LH(-like) bioactivity were measured at selected intervals after hCG injection and within 24 h of GnRH/GnRHa treatment. In all groups, estradiol continuously rose to similar peak levels on day 10. The hCG treatment markedly elevated circulating LH-like bioactivity for up to 3 days. In GnRH-1, bioactive LH increased to 433.1 +/- 170.2 ng/mL (mean +/- SEM; n = 3) within 1-2 h, but then decreased to baseline (4.9 +/- 1.5 ng/mL) within 6 h. GnRH-3 and GnRHa treatment extended the interval of elevated bioactive LH to 8 and 14 h, respectively. There was no difference in the peak levels of LH(-like) bioactivity reached after hCG, GnRH, or GnRHa injection. Functional luteal phases were absent in monkeys receiving no ovulatory stimulus, whereas hCG treatment increased progesterone levels to 101 +/- 9 nmol/L (n = 6) and elicited functional luteal phases of 11.8 +/- 0.4 days. In contrast, only one animal in the GnRH/GnRHa groups (i.e. one GnRH-3 monkey) displayed elevated progesterone levels in the luteal phase. Of the total cohort of oocytes aspirated from follicles, a greater (P less than 0.05) proportion were classified as being in metaphase I or II of meiosis after hCG treatment (86%) compared to no ovulatory stimulus (13%), GnRH-1 (0%), GnRH-3 (43%), and GnRHa (12%). Thus, GnRH elicits a transient LH surge that can be extended by GnRH-3 or GnRHa in stimulated cycles of monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)

The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

A luteotropic role for prostaglandins (PGs) during the luteal phase of the menstrual cycle of rhesus monkeys was suggested by the observation that intraluteal infusion of a PG synthesis inhibitor caused premature luteolysis. This study was designed to identify PGs that promote luteal function in primates. First, the effects of various PGs on progesterone (P) production by macaque luteal cells were examined in vitro. Collagenase-dispersed luteal cells from midluteal phase of the menstrual cycle (Day 6-7 after the estimated surge of LH, n = 3) were incubated with 0-5,000 ng/ml PGE2, PGD, 6 beta PGI1 (a stable analogue of PGI2), PGA2, or PGF2 alpha alone or with hCG (100 ng/ml). PGE2, PGD2, and 6 beta PGI1 alone stimulated (p less than 0.05) P production to a similar extent (2- to 3-fold over basal) as hCG alone, whereas PGA2 and PGF2 alpha alone had no effect on P production. Stimulation (p less than 0.05) of P synthesis by PGE2, PGD2, and 6 beta PGI1 in combination with hCG was similar to that of hCG alone. Whereas PGA2 inhibited gonadotropin-induced P production (p less than 0.05), that in the presence of PGF2 alpha plus hCG tended (p = 0.05) to remain elevated. Second, the effects of various PGs on P production during chronic infusion into the CL were studied in vivo. Saline with or without 0.1% BSA (n = 12), PGE2 (300 ng/h; n = 4), PGD2 (300 ng/h; n = 4), 6 beta PGI1 (500 ng/h; n = 3), PGA2 (300 ng/h; n = 4), or PGF2 alpha (10 ng/h; n = 8) was infused via osmotic minipump beginning at midluteal phase (Days 5-8 after the estimated LH surge) until menses. In addition, the same dose of PGE, PGD, PGI, or PGA was infused in combination with PGF2 alpha (n = 3-4/group) for 7 days. P levels over 5 days preceding treatment were not different among groups. In 5 of 8 monkeys receiving PGF2 alpha alone, P declined to less than 0.5 ng/ml within 72 h after initiation of infusion and was lower (p less than 0.05) than controls. The length of the luteal phase in PGF2 alpha-infused monkeys was shortened (12.3 +/- 0.9 days; mean +/- SEM, n = 8; p less than 0.05) compared to controls (15.8 +/- 0.5). Intraluteal infusion of PGE, PGD, PGI, or PGA alone did not affect patterns of circulating P or luteal phase length.(ABSTRACT TRUNCATED AT 400 WORDS)

The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Removal of the carbohydrates from hCG results in an antagonist (degly-hCG) that competitively inhibits hCG/LH-stimulated adenylate cyclase in macaque luteal tissue in vitro, but its effect in vivo is controversial. To examine the effect of degly-hCG on the lifespan and steroidogenic activity of the primate corpus luteum, the antagonist was administered to female rhesus monkeys (n = 7) beginning at the midluteal phase of the menstrual cycle. In a control cycle the saline vehicle was infused via an osmotic minipump directly into the corpus luteum. In a subsequent cycle, one of three dose rates of degly-hCG (0.001, 0.009, and 0.09 nmol/h) was infused into the corpus luteum. Pump implantation and infusion began 5-9 days after the midcycle LH surge and continued for 7 days. Peripheral venous blood was collected daily from day 8 of the cycle until menses, and serum progesterone levels were determined by RIA. Progesterone levels and patterns were similar in animals that received either the saline vehicle or degly-hCG, and the length of the luteal phase in monkeys receiving any dose of degly-hCG (16.4 +/- 0.5 days) was not different from that in animals receiving a control infusion (16.1 +/- 0.9 days). In a corollary study, an intraluteal infusion of degly-hCG (0.009 nmol/h) in the midluteal phase did not prevent stimulation of progesterone levels after im injection of hCG (15 IU/day for 5 days). We conclude that whereas degly-hCG is a useful tool to examine gonadotropin action in vitro, it is not a potent gonadotropin antagonist in vivo.

Intraluteal infusion of the prostaglandin (PG) synthesis inhibitor, sodium meclofenamate (Mec) causes premature luteolysis in rhesus monkeys. To evaluate further the actions of PG synthesis inhibitors in primate luteal function, we examined the in vitro effects of Mec and another inhibitor, flurbiprofen (Flur), on PG, cAMP, and progesterone (P) production by macaque luteal tissue obtained at midluteal phase of the menstrual cycle. First, collagenase dispersed luteal cells were incubated with 0-100 microM Mec or Flur, either alone or in the presence of 10 microM arachidonic acid (AA) to assess PGF2 alpha and PGE2 synthesis. Levels of both PGF2 alpha and PGE2 were stimulated (P less than 0.05) by AA (3.3- and 5.8-fold, respectively). Maximal suppression (P less than 0.01) of basal and AA-stimulated PGF2 alpha and PGE2 synthesis was elicited by 1 microM Mec and Flur. Second, adenylate cyclase activity, measured by the conversion of alpha 32P-ATP to alpha 32P-cAMP, was monitored in luteal homogenates exposed to increasing doses of Mec and Flur either alone or with maximal stimulatory doses of hCG, PGE2, or PGI2. Mec elicited a dose-dependent reduction (P less than 0.01) in control activity (incubated with 50 microM GTP), as well as inhibiting hCG- and PG-stimulated activity. The presence of 100 microM Mec suppressed (P less than 0.01) hCG-, PGE2- and PGI2-stimulated activity to control levels, but had no effect on activity stimulated by GMP-P(NH)P or forskolin. In contrast, Flur at any dose did not alter control activity or that stimulated by hormonal or nonhormonal activators. Third, P production by dispersed luteal cells was quantified during exposure to 0, 1, and 100 microM Mec or Flur alone or with maximal stimulatory doses of hCG, PGE2, PGD2, 6 beta PGI1, PGA2, or dibutyryl cAMP (dbcAMP). All hormones and dbcAMP stimulated (P less than 0.01) P synthesis 2-3 fold over basal levels, except PGA2, which had no effect. The presence of 100 microM Mec reduced (P less than 0.01) basal P production by 62% and abolished (P less than 0.05) hCG-, PG-, and dbcAMP-induced stimulation. Conversely, neither 1 microM Mec nor either dose of Flur affected P synthesis in the absence or presence of hormones or dbcAMP. These data indicate that: 1) Mec and Flur are potent inhibitors of PG synthesis in primate luteal cells in vitro and 2) higher doses of Mec suppress PG- and gonadotropin-sensitive adenylate cyclase activity and P production.(ABSTRACT TRUNCATED AT 250 WORDS)

Twenty-three rhesus monkeys were subjected to 9 days of ovarian hyperstimulation with sequential exposure to human follicle-stimulating hormone (hFSH) and then human luteinizing hormone (hLH) + hFSH. Six animals (26%) did not exhibit sustained, elevated levels of circulating estradiol, primarily due to the occurrence of a premature surge of endogenous LH (n = 4). Seventeen animals (74%) responded with supraphysiologic levels of circulating estradiol (peak value: means = 4480 pg/ml) and received human chorionic gonadotropin (hCG) on Day 10. Oocytes were collected 26 h later by aspiration of large antral follicles. Oocyte quantity (means = 18/animal) and quality (63% mature) were evaluated by in vitro fertilization (IVF), embryonic development, and embryo transfer to foster mothers. Modified conditions for the successful fertilization of oocytes used a Tyrode's augmented (TALP) medium supplemented with 0.3% bovine serum albumin (BSA). Oocytes were inseminated at the metaphase II stage with ejaculated, washed sperm (50 100 x 10(3)/ml) preexposed at ambient temperature to caffeine and dibutyryl cyclic adenosine 3'5'-monophosphate. Successful fertilization ranged from 26% to 75%. In one experiment, 5 of 11 embryos produced by IVF developed in vitro to hatched blastocysts. Embryo freezing employed a propanediol-based protocol and was applied to early cleavage-stage embryos with 100% (5 of 5) post-thaw survival. Two frozen-thawed embryos were transferred transtubally on 3 occasions into rhesus monkeys during the early luteal phase of spontaneous menstrual cycles. One pregnancy resulted, which proceeded normally to the unassisted delivery of a male offspring 170 days after the LH surge. We conclude that this sequential regimen of human gonadotropins provides a cohort of oocytes from rhesus monkeys that will complete meiotic maturation and fertilize in vitro, with embryonic development proceeding in vitro and in vivo. The production of putative antibodies to human gonadotropins, assessed by the presence of Protein A-precipitated hCG binding components in sera, limits the repeated use of monkeys in the hyperstimulation protocol. Nevertheless, this model system should facilitate further studies on oocyte maturation, fertilization, and early embryogenesis in primates.