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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.

Follicular stimulation was compared in macaques receiving sequential gonadotropin treatment which was terminated after seven, eight, or nine days depending on the time required to attain preselected criteria of follicular maturation. Although estradiol levels and follicle sizes varied, the number of follicles and oocytes/animal, oocyte nuclear maturity, IVF rates and progesterone levels during the luteal phase were similar among groups. Reducing the duration of gonadotropin treatment to individualize follicular stimulation regimens does not compromise follicle or gamete quality.

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)

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)