Desensitization of testicular ornithine decarboxylase to gonadotropin releasing hormone and gonadotropic hormones

Prior exposure of the testis to gonadotropin releasing hormone, luteinizing hormone or follicle stimulating hormone caused the testis refractory to these hormones in terms of ornithine decarboxylase activity at 24 h. Luteinizing hormone caused desensitization in the Leydig cells while the levels of ornithine decarboxylase in the seminiferous tubules were unaltered. In gonadotropin releasing hormone desensitized testis all the other treated compounds namely, luteinizing hormone, follicle stimulating hormone, prostaglandin F2 alpha, norepinephrine and cyclic AMP caused stimulation of ornithine decarboxylase activity. The testis desensitized with LH responded to cyclic AMP and norepinephrine whereas prostaglandin E2 or gonadotropin releasing hormone caused less stimulation of ornithine decarboxylase activity. These results indicate that testicular desensitization to gonadotropin releasing hormone and luteinizing hormone is not due to a post cyclic AMP block.     

Tissue specific compartmental analysis of gonadotropin stimulation of ovarian ornithine decarboxylase

Luteinizing hormone is known to stimulate the enzyme ornithine decarboxylase in the ovary. Highly purified human follicle stimulating hormone that is devoid of significant biologically active luteinizing hormone can also induce ornithine decarboxylase activity in intact immature rats with a time course of induction similar to that reported for luteinizing hormone. A maximum of 8–10-fold stimulation above controls was observed 4 h following intravenous administration of human follicle stimulating hormone. This stimulation followed a strict dose response relationship. Ovine luteinizing hormone and human chorionic gonadotropin always induced more ovarian ornithine decarboxylase activity than that achieved by maximally effective doses of follicle stimulating hormone. This could not be attributed solely to the ability of specific cell population to respond to the respective gonadotropins. Although granulosa cells contained little receptor for luteinizing hormone/human chorionic gonadotropin and the residual tissue contained little receptor for follicle stimulating hormone, each tissue responded to these gonadotropins in a manner suggestive of the mediation by one or more diffusable factors. A relationship between gonadotropin induced 3’5’-cyclic adenosine monophosphate (cyclic adenosine monophosphate) concentration and ornithine decarboxylase activity suggests that the mediation of gonadotropin stimulated ovarian ornithine decarboxylase is not solely through cyclic adenosine monophosphate, indicating the presence of other factors in the induction of gonadotropin increased ornithine decarboxylase activity.     

Vascular endothelial growth factor production in growing pig antral follicles

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4-5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.     

Gap junction amplification in rat ovarian granulosa cells: I. A direct response to follicle-stimulating hormone

The ability of follicle-stimulating hormone (FSH), the estrogens, estradiol-17β and diethylstilbestrol, and the estrogen antagonists, clomiphene and enclomiphene citrate to affect the growth and internalization of hypophysectomized rat granulosa cell gap junction membranes was compared in ovarian follicles assigned to one of four follicle size classes (60–149, 150–249, 250–319, and 320–450 μm diameter). In the absence of exogenous hormone stimulation, atresia prevents follicle growth beyond 320 μm in diameter but surface gap junction membrane increases throughout this early follicle growth. Internalization of gap junction membrane is first detected at the 150- to 249-μm follicle stage and also increases with follicle size. Therefore, growth and turnover of gap junction membrane occur at a basal rate in the absence of gonadotropin or steroid hormone stimulation. Estrogen and estrogen antagonist injections result in no significant differences in the amount of surface or internalized junction membrane in the three smallest follicle size classes when compared to the untreated hypophysectomized animals. However, estrogen but not estrogen antagonists rescues growing follicles from atresia and permits their further growth into the 320- to 450-μm follicle size class. As a result of the additional follicle growth, both surface and internalized junction membrane increase beyond that seen in the largest follicles from hypophysectomized animals. In contrast to other treatments, FSH stimulation promotes amplification of gap junction membrane in all size classes and, like estrogen, rescues follicles from atresia and promotes their entry into the 320- to 450-μm follicle size class. Surface gap junction membrane is amplified two- to fourfold over other treatments in the first three follicle size classes, but reaches maximal levels in the 250- to 319-μm follicles. The internalized junction membrane which first appears in the 150- to 249-μm size class is dramatically increased over other treatments in the 250- to 319- and 320- to 450-μm size classes. These studies indicate that exogenous estrogen stimulation promotes gap junction growth indirectly by sustaining the basal rate of junction synthesis in follicles rescued from atresia. In contrast, exogenous FSH stimulation directly amplifies the developmental sequence of gap junction growth and turnover. During early follicle growth, FSH stimulation preferentially promotes increases in surface gap junctions while internalization of surface junctions is increased during later follicle growth.     

Production of prostaglandins by porcine preovulatory follicular tissues and their roles in intrafollicular function

Prostaglandin production in vitro by theca and granulosa cells isolated from prepubertal pig ovaries was quantified in order to investigate the role of prostaglandins in intrafollicular function. Prepubertal gilts were slaughtered without treatment (O h, control) or treated with 1000 IU pregnant mare's serum gonadotropin (PMSG) and slaughtered at 36 or 72 h, or at 75 h following treatment with 500 IU of hCG at 72 h. Theca and granulosa cells were isolated from preovulatory follicles and cultured for 24 h alone or with follicle-stimulating hormone (FSH) or luteinizing hormone (LH). In vitro accumulation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) was measured by radioimmunoassay. On a per follicle basis theca produced more of each prostaglandin (approx. 10-fold) than granulosa at each stage of follicular development; production by each tissue type increased with development of the follicle, responding to administration of gonadotropin (PMSG) in vivo. Neither tissue type was generally responsive to further gonadotropin stimulation in vitro. However, production of PGE2 by granulosa cells was increased by addition of gonadotropin, particularly LH, in vitro, with the greatest response observed in tissue obtained at 36 and 72 h after PMSG. There were no functional correlates between prostaglandin production and steroidogenesis by either tissue type and we conclude that prostaglandins do not have an obligatory role in follicular steroidogenesis. However, these data provide additional circumstantial evidence for a role of PGE2 in granulosa cell luteinization, and possibly in ovulation. The data also indicate that prostaglandins derived from thecal tissue in relatively large quantities may play an important role in ovulation.     

Stimulation of folliculogenesis in domestic cats with human FSH and LH

Folliculogenesis in response to exogenous stimulation by human urinary follicle stimulating hormone (huFSH) and human menopausal gonadotropin (hMG) was evaluated in the domestic queen (Felis catus). The role of LH and/or FSH in folliculogenesis was examined by measuring concentrations of estradiol 17beta (E(2)) and progesterone (P) in the serum. Additionally, changes in the number and size of follicles from before the administration of exogenous hormones to surgical oocyte collection were monitored. Findings indicated that in queens receiving huFSH or hMG followed by human chorionic gonadotropin (hCG) to induce ovulation, the numbers of follicles from 1 to 3 mm increase with statistical significance (P<0.005) from before the initiation of treatment to surgical collection of oocytes. Although E(2) concentrations in cats receiving hMG increased above baseline by the third exogenous hormone injection, mean E(2) concentrations did not increase in the groups that received both huFSH and hCG, or hCG only, until after the administration of hCG. This suggests that the exogenous administration of LH contained in both hMG and hCG was necessary for E(2) to rise to levels associated with estrus.     

Gonadotropin and steroid control of granulosa cell proliferation during the periovulatory interval in rhesus monkeys

Progesterone produced in response to the midcycle gonadotropin surge is essential for ovulation and luteinization of the primate follicle. Because cell-cycle arrest is associated with the initiation of luteinization, this study was designed to determine the dynamics and regulation of granulosa cell proliferation by gonadotropin and progesterone during the periovulatory interval in the primate follicle. Granulosa cells or ovaries were obtained from macaques undergoing controlled ovarian stimulation either before (0 h) or as long as 36 h following the administration of an ovulatory hCG bolus with or without a 3beta-hydroxysteroid dehydrogenase inhibitor with or without a nonmetabolizable progestin. The percentage of cells staining positive for Ki-67, a nuclear marker for cell proliferation, decreased (P < 0.05) within 12 h of hCG administration in a steroid-independent manner. Levels of cyclin D2 and E mRNA did not decline during the periovulatory interval; however, cyclin B1 mRNA was reduced significantly by 12 h. Steroid depletion increased (P < 0.05) cyclin B1 mRNA at both 12 and 36 h post-hCG and was reversible by progestin replacement at 36 h. The cyclin-dependent kinase inhibitor p21(Cip1) was transiently increased 12 h post-hCG, whereas p27(Kip1) mRNA levels increased at 36 h in a steroid-independent fashion. These data suggest that a gonadotropin bolus inhibits mitosis in granulosa cells early (12 h) in the periovulatory interval, whereas progesterone may play a later, antiproliferative role in luteinized cells of primates.     

Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat

Summary The observation that tissue-type plasminogen activator (tPA) activity increased dramatically in preovulatory follicles has led to the hypothesis that plasminogen activation is causally related to follicle rupture. With immunohistochemistry, we have studied the appearance of tPA in ovaries of immature rats induced to ovulate and in adult cycling rats. Treatment of immature female rats with a single dose of pregnant mare serum gonadotropin (PMSG) induced follicular maturation. A subsequent human chorionic gonadotropin (hCG) injection resulted in follicle rupture 12–14 h later. PMSG treatment alone did not induce appearance of tPA-immunoreactive cells in any ovarian compartment. After hCG stimulation, however, theca cells, granulosa cells, and oocytes of pre- and postovulatory follicles displayed distinct tPA immunoreactivity. Fibroblastlike cells in the theca layers and tunica albuginea of the follicle apex also demonstrated localized cytoplasmic tPA reactivity. In addition to tPA synthesis in preovulatory follicles, hCG also induced tPA staining in the theca (but not granulosa) layers of non-ovulatory follicles. At 24 h after hCG treatment, there was a marked tPA staining in developing corpora lutea, ovulated ova, and oviductal epithelium. Ovaries from regularly cycling adult rats displayed a similar ovulation-related pattern of tPA immunostaining. The appearance of tPA in different cell types of the preovulatory follicle and in the fibroblast-like cells at the follicle apex, strengthens the hypothesis of a direct involvement of tPA in follicle rupture. Presence of tPA in postovulatory oocytes, cumulus cells, and surrounding oviductal epithelium may also indicate a role for tPA in the transfer of eggs in the oviduct.This work was supported by NIH Research Grants HD-14084; 12303     

Steroid-induced final maturation in brook trout (Salvelinus fontinalis) oocytes in vitro: the effects of forskolin and phosphodiesterase inhibitors

The effects of phosphodiesterase inhibitors and forskolin on steroid-induced germinal vesicle breakdown (GVBD) were investigated in brook trout (Salvelinus fontinalis) oocytes using an in vitro incubation technique. Follicles were first treated with a collagenase solution to remove the follicle wall. Denuded oocytes were examined, using scanning electron microscopy. In all experiments GVBD was induced by the use of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one. Cyclic adenosine 3',5'-monophosphate (cAMP) levels were measured (by protein-binding assay) in control and forskolin-treated oocytes. Collagenase treatment removed a majority of the follicle wall, as shown by scanning electron microscopy. Partially denuded (PD) oocytes were slightly more sensitive to steroid treatment than intact follicles (IF), as shown by ED50 values; but PD oocytes did not respond to gonadotropin (GTH) stimulation. Both 3-isobutyl-1-methyl-xanthine (IBMX) and SQ20,006 (Squibb) blocked GVBD, but IBMX was more inhibitory. Forskolin also blocked steroid-induced GVBD. Kinetics of inhibition studies were performed using IBMX, forskolin, and cycloheximide. IBMX and cycloheximide inhibited GVBD if added during the first 18 h following steroid stimulation, whereas forskolin blocked GVBD if added within 12 h after steroid treatment. Forskolin, at levels that block GVBD in vitro, significantly increased cAMP in both IF and PD oocytes, but the response of IF was greater than that of PD oocytes.     

Hormonal stimulation, in vivo, of the ovary of European eel in the yellow stage

The immature ovary of yellow eel was stimulated by a long-term treatment with a gonadotropin II rich extract of carp pituitary. Ovarian follicles of treated yellow eels showed an accumulation of lipidic vacuoles in the oocyte cytoplasm and a thickened follicular envelope (in this regard, they resembled the ovarian follicles of previously studied silver eels). The treatment also resulted in a significant but limited increase in the gonadosomatic ratio and mean follicle size (these values were much lower than those measured in silver eels). No change in pigmentation or general morphology was observed. Thus, under our experimental conditions, a gonadotropic stimulation of the yellow eel leads to some but not the totality of the ovarian modifications which occur physiologically during silvering.     

Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of³H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication. A single injection of pregnant mare serum gonadotropin could thus maximally stimulate by 18 hr³H-thymidine incorporation into DNA of the ovary of immature hamsters. Neutralization of pregnant mare serum gonadotropin by an antiserum to ovine follicle stimulating hormone only during the initial 8–10 hr and not later could inhibit the increase in³H-thymidine incorporationin vitro observed at 18 hr, suggesting that the continued presence of gonadotropin stimulus was not necessary for this response. The other indices of follicular growth monitored such as ovarian weight, serum estradiol and uterine weight showed discernible increase at periods only after the above initial event. A single injection of estrogen (diethyl stilbesterol or estradiol-l7β) could similarly cause 18 hr later, a stimulation in the rate of incorporation of³H-thymidine into DNAin vitro in ovaries of immature rats. The presence of endogenous gonadotropins, however, was obligatory for observing this response to estrogen. Evidence in support of the above was two-fold: (i) administration of antiserum to follicle stimulating hormone or luteinizing hormone along with estrogen completely inhibited the increase in³H-thymidine incorporation into ovarian DNAin vitro; (ii) a radioimmunological measurement revealed following estrogen treatment, the presence of a higher concentration of endogenous follicle stimulating hormone in the ovary. Finally, administration of varying doses of ovine follicle stimulating hormone along with a constant dose of estrogen to immature rats produced a dose-dependent increment in the incorporation of³H-thymidine into ovarian DNAin vitro. These observations suggested the potentiality of this system for developing a sensitive bioassay for follicle stimulating hormone.     

Plasma concentration changes in LH and FSH following electrochemical stimulation of the medial preoptic are or dorsal anterior hypothalamic area of estrogen- or androgen-sterilized rats.

Plasma concentraption changes in luteinizing hormone (LH) and follicle stimulation hormone (FSH) following elctrochemical stimulation (ECS) of the medial preoptic area (MPOA) or dorsal anterior hypothalamic area (DAHA) of estrogen-or androgen-sterilized rats were compared with normal proestrous rats in which spontaneous gonadotropin surges had been blocked with Nembutal (control). ECS of control rats, AST, and ESR provoked marked increases in FSH by 120 minutes and peak levels by 180 minutes poststimulation. No differenses were seen when ESR of ASR plasma levels were compared with control values or to each other (ESR vs. ASR). DAHA-ECS did not provoke a marked LH rise in ASR and ESR. MPOA-ECS of control, ASR, and ESR resulted in the release of LH and FSH. FSH peaked in all groups at 240 minutes but at 120 and 180 minutes poststimulation plasma FSH levels were greater in ASR and ESR than controls. FSH in ESR was significantly higher (p. 05) at 120 and 240 minutes poststimulation. LH was elevated in all groups following MPOA-EDS but in ESR markedly greater LH levels were obtained at 60 and 180 minutes compared with controls or ASR. Preoptic stimulation of progesterone-treated ASR (P-ASR) did not induce greater release of FSH than in control, ASR, ESR, or P-ESR. Stimulation of P-ASR resulted in greater plasma LH rise by 60 minutes which was greater than ASR, P-ASR, ESR, or controls, followed by decreased levels to baseline in P-ESR. MPOA-ECS controls ovulated (8-13 eggs) whereas neither ASR nor ESR Fallopian tubes contained eggs the following morning. No DAHA-stimulated groups ovulated. All P-ASR but no P-ESR ovulated after MPOA-ECS. It is concluded that while exposure of neonatal female rats to estrogen renders them more sensitive than ASR to preoptic stimulation, the ovaries of these amimals are much less responsive to the gonadotropin released into plasma than are those of ASR or normal rats.     

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.     

LH-stimulation of the adenylyl cyclase system in follicles and oocytes of Xenopus laevis

The presence of an adenylyl cyclase sensitive to LH in Xenopus laevis is studied. The assay for adenylyl cyclase in membranes and homogenates from Xenopus laevis follicles and oocytes is characterized and the aim is centered on the appearance of LH-response through oogenesis. The results show a stimulation by LH in whole follicle and oocytes surrounded by the follicle cells. The oocytes become responsive to LH from stage III onwards, suggesting an action of the gonadotropin on the monolayer of follicular cells in early stages of follicle development.     

Ovarian stimulation of squirrel monkeys (Saimiri boliviensis boliviensis) using pregnant mare serum gonadotropin

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (< or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.     

Early folliculogenesis in primate ovaries: testing the role of estrogen.

The purpose of this study was to examine the effect of an exogenous estrogen, diethylstilbestrol (DES), on follicle development in the ovary of a juvenile primate. The immature cynomolgus monkey (12-22 mo) was used as a model since ovaries at this age lack endogenous gonadotropin support but are capable of responding to exogenous hormonal stimulation. In addition, the pituitary gland receives virtually no GnRH stimulation and under these conditions lacks responsiveness to estrogen feedback. Two groups of three monkeys each received DES for 14 days. Members of the second group also were given GnRH antagonist to assure no GnRH action upon the gonadotropes. The left ovary of each monkey was removed just prior to Day 1 of DES treatment and served as the control. The right ovary was removed on Day 14 of treatment. Both ovaries from each monkey were prepared for evaluation by light microscopy. Results indicated that both the number of preantral follicles and the mean number of medium-sized (0.5-1 mm in diameter) developing antral follicles decreased significantly (p less than 0.05) in the DES-treated ovaries with no increase in early-atretic antral follicles. These data suggest that DES, at the amount administered, inhibits the growth of both preantral and medium-sized antral follicles in the primate. Whether these effects are manifest directly at the follicle level or are mediated by other mechanisms remains to be determined.     

Use of ultrasound biomicroscopy to evaluate induced ovarian follicular growth and ovulation in mice

Recent advances in image technology, including significant gains in spatial resolution, have made realtime sequential ovarian evaluations possible in small rodents, allowing longitudinal (continued) studies of the ovarian cycle and reducing the required number of experimental animals. The aim of this study was to evaluate exogenous stimulated follicular growth in mice using high-resolution ultrasound technology. Female mice (n = 15) received a 5 IU intraperitoneal injection of equine chorionic gonadotropin (eCG) and 48 h later a 5 IU injection of human chorionic gonadotropin (hCG), and were allowed to mate thereafter. In experiment 1, animals (n = 7) were evaluated every 6 h, from 3 to 51 h after eCG injection, with an ultrasound biomicroscopy (UBM) equipped with a realtime 45 MHz microvisualization probe (RMV 707b). The ovaries were identified and follicular population quantified, and follicles were classified according to the diameter as small (≤449 μm) or large (≥450 μm). A significant change in the distribution of follicle population according to category was observed only 45 h after eCG injection (P < 0.05). In experiment 2, animals (n = 8) were evaluated every 2 h, from 2 h to 10 h after hCG treatment. The largest follicles reached a maximum size (596.7 ± 106.0 μm) 5.8 ± 2.3 h after hCG injection. As expected, the population of large follicles decreased thereafter, indicating the progress of ovulations, but large follicles were still detected late after treatment (10.1 ± 1.1 h). In conclusion, UBM can be used to evaluate follicle dynamics in superstimulated mice (C57BL/6 and BALB/c); significant changes in follicle distribution only occur at later stages after eCG stimulation; and hCG-induced ovulations may not occur synchronously in mice.     

Beta-endorphin/beta-lipotropin release and gonadotropin secretion after acute exercise in physically conditioned males

beta-endorphin (beta-EP) and beta-lipotropin (beta-LPH) concentrations were measured in the basal state and after acute exercise for 15 min or until exhaustion in 6 physically conditioned male volunteers. Serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone and prolactin were also measured in the basal state. In addition, the concentrations of the gonadotropins (LH and FSH) were determined after exercise and the gonadotropin response to gonadotropin releasing hormone was assessed before and after exercise. The data show that acute exercise stimulates the release of both beta-EP and beta-LPH which return to base-line levels within 60 min after exercise. This is in contrast to our previously described results in physically unconditioned male volunteers in whom only beta-LPH release was noted after exercise. Serum LH concentrations declined after exercise reaching nadir values between 60 to 150 min after exercise. As we previously reported in physically unconditioned male volunteers, serum FSH concentrations did not change with exercise and the gonadotropin response to LRH stimulation was uninfluenced by exercise. Serum testosterone and prolactin concentration were within the normal range for healthy adult males. We speculate that the difference in beta-EP release with exercise in physically conditioned and unconditioned males represents a difference in processing of the opioid precursor molecule (pro-opiomelanocortin, POMC) in the two groups.     

Application of noninvasive techniques to monitor reproductive function in a brown hyena(Hyena brunnea)

Estrus in a 10-yr-old brown hyena was described by monitoring urinary steroid metabolites. These studies were repeated the succeeding year and combined with ultrasonography, vaginal cytology, and laparoscopy to further aid in identifying natural and induced estrus. Collection of semen from a 14-yr-old male brown hyena was accomplished by electroejaculation. Use of frozen semen once during natural estrus and again during estrus induced with follicle stimulating hormone (FSH) did not result in pregnancy. Further studies with this animal indicate that stimulation with gonadotropin releasing hormone (GNRH), rather than FSH, may be the more appropriate treatment for stimulating ovulation in canids or related species. While this study did not result in conception, a significant step toward that end was realized by utilizing urinary estrogen to monitor the estrus cycle.     

Role of gonadotropins and insulin in controlling steroidogenesis and growth of antral bovine follicles in perifusion culture

Bovine follicles (2 to 4 mm in diameter) were isolated from the ovaries of 4-to 6-mo-old Holstein calves and placed in perifusion culture. Groups of 6 to 8 follicles/flask were cultured for 4 or 21 h with 1) no hormones; 2) tonic follicle-stimulating hormone (FSH) (10 ng/ml) and luteinizing hormone (LH) pulses (4 ng/ml) once every 4 h; 3) insulin (200 I.U./l); or 4) tonic FSH, LH pulses and insulin. After 0, 4 and 21 h of perifusion culture, each follicle was incubated in 1 ml of medium containing 3(H)-thymidine for 1 h. The 3(H)-thymidine incorporated into DNA of the follicle as well as the amount of estradiol-17 (E2) and testosterone (T) secreted into the medium were determined. Follicles treated with or without gonadotropins secreted higher levels of E2 and T after 4 h of perifusion compared to the 0 h controls. This elevated secretion rate was not maintained and 3(H)-thymidine incorporation was not increased over 0 h control values after 21 h of culture. Insulin suppressed the T secretion after 4 h in culture and increased 3(H)-thymidine incorporation at both 4 and 21 h of culture. After 21 h of culture, the gonadotropin and insulin treatment also enhanced 3(H)-thymidine incorporation. These results demonstrate that insulin is more mitogenic than the gonadotropin treatment tested, suggesting that insulin or insulin-like factors may play a physiological role in the growth of bovine follicles in vivo.