Gonadotropin Challenge Research Articles

In vitro exposure of vitellogenic rainbow trout ovarian follicles to endocrine disrupting chemicals can alter basal estradiol-17β production and responsiveness to a gonadotropin challenge

Endogenous estrogens play major roles in many aspects of female reproductive development in fish. In order to develop a relatively high-throughput assay to determine the potential impact on reproductive development, vitellogenic rainbow trout ovarian follicles were exposed to a suite of contaminants in vitro and then assessed for the ability to produce estradiol-17β (E2) after a 500 ng/ml salmon gonadotropin (sGTH) challenge. There was a positive correlation between ovarian follicle size and E2 production, but an inverse correlation between size and responsiveness to sGTH. Significant impacts on E2 levels were observed following treatment with different endocrine disrupting chemicals, such as 17α-ethinylestradiol (EE2), prochloraz, or trenbolone. EE2 was remarkably potent and significantly reduced ovarian follicle responsiveness to sGTH at concentrations as low as 0.1 nM. Of the other contaminants tested, only tamoxifen impacted E2 levels, and only at concentrations near the limits of solubility. Flutamide, fluoxetine, 4-hydroxy tamoxifen, hydroxyflutamide, and norfluoxetine had little or no impact. Quantitative PCR analyses of steroidogenesis-related genes were carried out on EE2 treated ovarian follicles, but significant transcriptional responses to EE2 were not observed. Overall, this study suggests that xenoestrogens and anti-estrogens are more likely to interfere with ovarian E2 synthesis than other classes of EDCs. This also provides a template for further testing of the effects of EDCs on ovarian function.

Dehydroepiandrosterone sulfate levels reflect endogenous luteinizing hormone production and response to human chorionic gonadotropin challenge in older female macaque (Macaca fascicularis)

We propose that the adrenal gland of an older higher primate female animal model will respond to human chorionic gonadotropin (hCG) hormone challenge by secreting additional dehydroepiandrosterone sulfate (DHEAS). Such a response in surgically and chemically castrated animals will provide proof of concept and a validated animal model for future studies to explore the rise in DHEAS during the menopausal transition of women. Twenty-four 18- to 26-year-old female cynomolgus monkeys were screened for ovarian function and then either ovariectomized (n = 4) or treated with a gonadotropin-releasing hormone agonist (GnRHa; n = 20) to block ovarian steroid production. After a recovery period from surgical procedure or down-regulation, a single-dose challenge (1,000 IU/animal, IM) of hCG was then administered to determine if luteinizing hormone (LH)/chorionic gonadotropin could accelerate circulating DHEAS production. Serum DHEAS, bioactive LH, and urinary metabolites of ovarian sex steroids were monitored before, during, and after these treatments. Circulating LH bioactivity and immunoreactive DHEAS concentrations were suppressed in all animals 14 days postadministration of GnRHa. Urinary metabolites of estradiol and progesterone remained low after the surgical procedure or a flare reaction to GnRHa. Circulating DHEAS levels were increased after hCG administration, and the increase in individual animals was proportional to the pretreatment DHEAS at baseline. Circulating DHEAS concentrations were positively correlated to endogenous LH bioactive concentrations prior to hCG challenge and were subsequently further elevated by the hCG challenge while no concomitant change in ovarian steroid hormone excretion was observed. These data demonstrate a positive adrenal androgen response to LH/chorionic gonadotropin in older female higher primates and suggest a mechanism for the rise in adrenal androgen production during the menopausal transition in women. These results also illustrate that the nonhuman primate animal model can be effectively used to investigate this phenomenon.

Altered sex hormone concentrations and gonadal mRNA expression levels of activin signaling factors in hatchling alligators from a contaminated Florida lake

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin betaA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly.

Gonadotropin stimulation as a challenge to calibrate cisplatin induced ovarian damage in the female rat

Cisplatin administration for treatment of cancer causes damage to the ovaries, potentially leading to ovarian failure. To extend our previous work, we tested the hypothesis that serum anti-Mullerian hormone (AMH) levels after ovarian stimulation can be used as a biomarker to calibrate the degree of ovarian damage following cisplatin. Female rats were injected with two weekly doses of cisplatin followed by an injection of pregnant mare serum gonadotropin to stimulate the ovaries. Sera and ovaries were collected 54 h after gonadotropin challenge. Analysis of serum and ovarian AMH by Western blot and ELISA revealed that increasing doses of cisplatin caused dose-dependent decrease in AMH after gonadotropin stimulation. Immunohistochemical analysis demonstrated that the AMH positive follicles declined in a dose-dependent manner after cisplatin, resulting in AMH levels that are lower after cisplatin exposure. Cisplatin damage to the ovaries can be identified by the serum AMH levels following exogenous ovarian stimulation.

Excessive Ovarian Production of Nerve Growth Factor Facilitates Development of Cystic Ovarian Morphology in Mice and Is a Feature of Polycystic Ovarian Syndrome in Humans

Although ovarian nerve growth factor (NGF) facilitates follicular development and ovulation, an excess of the neurotrophin in the rodent ovary reduces ovulatory capacity and causes development of precystic follicles. Here we show that ovarian NGF production is enhanced in patients with polycystic ovarian syndrome (PCOS) and that transgenically driven overproduction of NGF targeted to the ovary results in cystic morphology, when accompanied by elevated LH levels. NGF levels are increased in the follicular fluid from PCOS ovaries and in the culture medium of granulosa cells from PCOS patients, as compared with non-PCOS patients. Ovaries from transgenic mice carrying the NGF gene targeted to thecal-interstitial cells by the 17alpha-hydroxylase gene promoter produce more NGF than wild-type (WT) ovaries and are hyperinnervated by sympathetic nerves. Antral follicle growth is arrested resulting in accumulation of intermediate size follicles, many of which are apoptotic. Peripubertal transgenic mice respond to a gonadotropin challenge with a greater increase in plasma 17-hydroxyprogesterone, estradiol, and testosterone levels than WT controls. Transgenic mice also exhibit a reduced ovulatory response, delayed puberty, and reduced fertility, as assessed by a prolonged interval between litters, and a reduced number of pups per litter. Sustained, but mild, elevation of plasma LH levels results in a heightened incidence of ovarian follicular cysts in transgenic mice as compared with WT controls. These results suggest that overproduction of ovarian NGF is a component of polycystic ovarian morphology in both humans and rodents and that a persistent elevation in plasma LH levels is required for the morphological abnormalities to appear.

Inhibin B response to EFORT is associated with the outcome of oocyte retrieval in the subsequent in vitro fertilization cycle

Objective: To examine whether the magnitude of the rise in inhibin B levels after gonadotropin challenge is associated with subsequent response to ovarian stimulation during IVF. Design: Inhibin B serum levels after EFORT (exogenous follicle-stimulating hormone ovarian reserve test). Setting: Academic clinical practice. Patient(s): Serum samples from women who had undergone ovarian reserve screening with FSH in preparation for IVF. Thirteen of these women had a poor response in IVF (canceled cycle for low estradiol and/or no oocytes retrieved), and 19 had a good response (≥10 oocytes retrieved). Intervention(s): EFORT test. Main Outcome Measure(s): Baseline (day 3) serum E 2 (bE 2), FSH (bFSH), and inhibin B (bInhB) levels and inhibin B and E 2 levels 24 hours after EFORT (ΔInhB and ΔE 2). Result(s): The mean bInhB and ΔInhB levels were significantly higher in good vs. poor responders. The odds ratio of having a good response for women with a ΔInhB of 202 pg/mL was 51.8 times (95% CI = 6.1–1,244) the corresponding odds for women with a ΔInhB of 49 pg/mL. As expected, ΔE 2 was also significantly higher in good vs. poor responders; however, combination of ΔE 2 plus ΔInhB did not improve the odds for predicting IVF response. Conclusion(s): Our data suggest that ΔInhB after EFORT may provide a method for predicting ovarian response to hyperstimulation in a subsequent IVF cycle.

Progesterone receptors: expression and regulation in the mammalian ovary.

The authors have briefly discussed the molecular structure, regulation, and function of progesterone receptors in the mammalian ovary. Particularly important is the contrast in the regulatory mechanisms of PR induction in the ovary (gonadotropins/membrane receptor mediated) and other well-known progesterone target tissues, such as the uterus and mammary gland (estrogen/nuclear receptor mediated). Future research will focus on how the PR gene responds to these hormonal regulatory signals in this cell-specific manner. Equally important in this discussion has been the mounting evidence indicating that PRs are an essential component of the ovulatory process. The observation that PR-/- knockout mice are incapable of undergoing ovulation, even in response to gonadotropin challenge, further supports the previous physiological evidence indicating that PRs in preovulatory follicles are induced before, and are necessary for, ovulation. Further studies are required to determine the identity of PR-regulated target genes during the periovulatory period. Although our knowledge of PR structure, regulation, and function has increased dramatically during the past decade, many exciting questions remain related to the regulation and function(s) of PRs in the ovary and other tissues.

Sex-reversed and normal turtles display similar sex steroid profiles

In many oviparous reptiles, incubation temperature determines hatchling sex. Manipulation of the embryonic hormonal environment can result in sex reversal. We measured circulating estradiol 17-β (E2) and testosterone (T) levels in temperature-determined and sex-reversed snapping turtles (Chelydra serpentina). Eight-month-old turtles have sex steroid profiles that are sex specific and are consistent with gonadal sex. Males had higher levels of T than females, both before and after treatment with follicle stimulating hormone (FSH). In males, T levels increased approximately 11-fold after FSH challenge, whereas in females, T levels remained low. In contrast, levels of E2 after FSH challenge were higher in females than in males, but showed no difference before treatment. Sex-reversed individuals appear to have normal sex steroid profiles of T and E2, both at basal levels and in response to gonadotropin challenge. Our study indicates that the steroidogenic capability of these juvenile turtles was consistent with gonadal morphology and suggests that these sex-reversed individuals have normal gonadal function. © 1996 Wiley-Liss, Inc.

Poor semen quality in Irish wolfhounds: a clinical, hormonal and spermatological study.

On the basis of clinical observations, Irish wolfhounds were suspected as being subject to a relatively high incidence of soft testicular consistency and low semen quality. Thirty-seven Irish wolfhounds and 67 dogs of other breeds (control group) were therefore examined. Conventional andrological studies were performed in both groups, these included libido testing, palpation and measurement of testes and semen evaluation. Semen evaluation was performed both manually and by videomicrographic analysis. Testosterone concentrations were measured both before and after human chorionic gonadotropin challenge in the Irish wolfhounds. The Irish wolfhounds had lower libidos than the control group. More Irish wolfhounds (21.2 per cent) exhibited low semen quality than the control group (6.1 per cent). Soft testicular consistency occurred more frequently in Irish wolfhounds (25.7 per cent) than in the control group (10.5 per cent). In the control group, bodyweight correlated significantly with total sperm count. Serum testosterone concentration did not correlate with semen quality parameters.

A dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat

In the present study rats were dosed from weaning, through puberty and gestation, to Day 15 of lactation with methoxychlor at 25, 50, 100, or 200 mg/kg/day. Morphological landmarks of puberty were measured, including the ages at vaginal opening, first estrus, and first estrous cycle in females and at preputial separation in males. In the female, estrous cyclicity, fertility, litter size, number of implantation sites, organ weights, and ovarian and uterine histology were also measured. The viability of the offspring (F1) and their fertility were evaluated using a continuous breeding protocol. Males were necropsied after breeding, the reproductive organs were weighed, and the cauda epididymal sperm counts were determined. One testis was used for histopathology, while the other was used to quantify interstitial fluid (IF) content, IF testosterone concentration, and testicular sperm production. Testosterone and androgen-binding protein were measured in the caput epididymis, and sperm motility and morphology were evaluated from a caudal sample. The serum and pituitary were saved for hormonal determinations. Methoxychlor accelerated the age at vaginal opening and first estrus, and the vaginal smears were cornified. Growth was retarded at 100 and 200 mg/kg/day and fertility was reduced when the females were bred with untreated or similarly treated males. In the highest-dose group, the mated females went from constant estrus into pseudopregnancy following mating, but they had no implants. In males, methoxychlor treatment markedly reduced growth, seminal vesicle weight, cauda epididymal weight, caudal sperm content, and pituitary weight. Puberty was delayed in the two highest-dosage groups. Testicular sperm measures were much less affected than caudal measures. Testis weight and histology were slightly affected, and testicular sperm production, sperm morphology, and motility were unaffected. Endocrine function of the testes and pituitary was altered by methoxychlor administration. Leydig cell testosterone production, in response to human chorionic gonadotropin challenge, was reduced and pituitary levels of prolactin, thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH) were altered. In contrast, serum levels of prolactin, FSH, and luteinizing hormone were unaffected. Serum TSH was reduced by 50% of control at 100 and 200 mg/kg/day, while pituitary levels were increased. Gonadotropin-releasing hormone concentration in the mediobasal hypothalamus was also elevated. In spite of the many reproductive alterations, the fertility of treated males was not reduced when they were mated with untreated females. Growth and viability of the offspring (F1) from the 50 mg/kg/day treatment group were normal, but in the females, vaginal opening was accelerated, estrous cyclicity was abnormal in the rats during middle age, and fecundity was reduced.

Steroidogenic responsiveness of the monkey corpus luteum to exogenous chorionic gonadotropin.

Groups of female rhesus monkeys were given a 5-day regimen of im injections of hCG (30, 60, 90, 180, and 360 IU, respectively) beginning 2, 6, 10, or 14 days after the midcycle LH surge (day 0). Serum progesterone concentrations in the day 2 treatment group did not differ markedly from values observed in control monkeys throughout normal menstrual cycles. In contrast, monkeys receiving hCG beginning on days 6, 10 or 14 had immediate significant increases in serum progesterone in response to the first injection of hCG. Dramatic responses were seen in the day 6 and 10 groups (2.5- and 5-fold elevations in serum progesterone, respectively); progesterone values plateaued at about 13 ng/ml during the hCG treatment interval. Monkeys receiving hCG on day 14 had 4-fold elevations in serum progesterone, but concentrations did not exceed 6 ng/ml. Serum estradiol increased significantly after hCG to concentrations between 200-300 pg/ml in all treatment groups; peak values were seen at the time of or in the days immediately after the last hCG injection. Serum testosterone concentrations were not significantly altered by hCG administration at any stage of the luteal phase. hCG did not sustain serum steroid hormone concentrations in monkeys with the corpus luteum removed on day 10 of the luteal phase. A 10-day regimen of increasing hCG doses beginning on day 10 of the luteal phase mimicked the steroid hormone secretion patterns observed in control monkeys during early pregnancy. The data show that the qualitative and quantitative steroidogenic capacities of monkey corpora lutea after a gonadotropin challenge are profoundly affected by luteal age.