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Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are products of incomplete combustion. In female mouse embryos primordial germ cells proliferate before and after arriving at the gonadal ridge around embryonic (E) 10 and begin entering meiosis at E13.5. Now oocytes, they arrest in the first meiotic prophase beginning at E17.5. We previously reported dose-dependent depletion of ovarian follicles in female mice exposed to 2 or 10 mg/kg-day BaP E6.5-15.5. We hypothesized that embryonic ovaries are more sensitive to gestational BaP exposure during the mitotic developmental window, and that this exposure results in persistent oxidative stress in ovaries and oocytes of exposed F1 female offspring. We orally dosed timed-pregnant female mice with 0 or 2 mg/kg-day BaP in oil from E6.5-11.5 (mitotic window) or E12.5-17.5 (meiotic window). Cultured E13.5 ovaries were utilized to investigate the mechanism of BaP-induced germ cell death. We observed statistically significant follicle depletion and increased ovarian lipid peroxidation in F1 pubertal ovaries following BaP exposure during either prenatal window. Culture of E13.5 ovaries with BaP induced germ cell DNA damage and release of cytochrome c from the mitochondria in oocytes, confirming that BaP exposure induced apoptosis via the mitochondrial pathway. Mitochondrial membrane potential, oocyte lipid droplet (LD) volume, and mitochondrial-LD colocalization were decreased and mitochondrial superoxide levels were increased in the MII oocytes of F1 females exposed gestationally to BaP. Results demonstrate similar sensitivity to germ cell depletion and persistent oxidative stress in F1 ovaries and oocytes following gestational BaP exposure during mitotic or meiotic windows., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in glutathione (GSH) synthesis, have decreased tissue GSH. We previously showed that Gclm-/- embryos have increased sensitivity to the prenatal in vivo ovarian toxicity of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) compared with Gclm+/+ littermates. We also showed that BaP-induced germ cell death in cultured wild type embryonic ovaries is caspase-dependent. Here, we hypothesized that GSH deficiency increases sensitivity of cultured embryonic ovaries to BaP-induced germ cell death. 13.5 days post coitum (dpc) embryonic ovaries of all Gclm genotypes were fixed immediately or cultured for 24 h in media supplemented with DMSO vehicle or 500 ng/ml BaP. The percentage of activated caspase-3 positive germ cells varied significantly among groups. Within each genotype, DMSO and BaP-treated groups had increased germ cell caspase-3 activation compared to uncultured. Gclm+/- ovaries had significantly increased caspase-3 activation with BaP treatment compared to DMSO, and caspase-3 activation increased non-significantly in Gclm-/- ovaries treated with BaP compared to DMSO. There was no statistically significant effect of BaP treatment on germ cell numbers at 24 h, consistent with our prior observations in wild type ovaries, but Gclm-/- ovaries in both cultured groups had lower germ cell numbers than Gclm+/+ ovaries. There were no statistically significant BaP-treatment or genotype-related differences among groups in lipid peroxidation and germ cell proliferation. These data indicate that Gclm heterozygous or homozygous deletion sensitizes embryonic ovaries to BaP- and tissue culture-induced germ cell apoptosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Astronauts are exposed to charged particles during space travel, and charged particles are also used for cancer radiotherapy. Premature ovarian failure is a well-known side effect of conventional, low linear energy transfer (LET) cancer radiotherapy, but little is known about the effects of high LET charged particles on the ovary. We hypothesized that lower LET (16.5 keV/µm) oxygen particles would be less damaging to the ovary than we previously found for iron (LET = 179 keV/µm). Adult female mice were irradiated with 0, 5, 30 or 50 cGy oxygen ions or 50 cGy oxygen plus dietary supplementation with the antioxidant alpha lipoic acid (ALA). Six-hour after irradiation, percentages of ovarian follicles immunopositive for γH2AX, a marker of DNA double strand breaks, 4-HNE, a marker of oxidative lipid damage and BBC3 (PUMA), a proapoptotic BCL-2 family protein, were dose dependently increased in irradiated mice compared to controls. One week after irradiation, numbers of primordial, primary and secondary follicles per ovary were dose dependently decreased, with complete absence of follicles in the 50 cGy groups. The ED 50 for primordial follicle destruction was 4.6 cGy for oxygen compared to 27.5 cGy for iron in our previous study. Serum FSH and LH concentrations were significantly elevated in 50 cGy groups at 8 week. Supplementation with ALA mitigated the early effects, but not the ultimate depletion of ovarian follicles. In conclusion, oxygen charged particles are even more potent inducers of ovarian follicle depletion than charged iron particles, raising concern for premature ovarian failure in astronauts exposed to both particles during space travel., (© 2017 Society for Reproduction and Fertility.)

Females deficient in the glutamate cysteine ligase modifier subunit (Gclm) of the rate-limiting enzyme in glutathione synthesis are more sensitive to ovarian follicle depletion and tumorigenesisby prenatal benzo[a]pyrene (BaP) exposure than Gclm+/+ mice. We investigated effects of prenatal exposure to BaP on reproductive development and ovarian mutations in Kras, a commonly mutated gene in epithelial ovarian tumors. Pregnantmice were dosed from gestational day 6.5 through 15.5 with 2mg/kg/day BaP or vehicle. Puberty onset occurred 5 days earlier in F1 daughters of all Gclm genotypes exposed to BaP compared to controls. Gclm+/- F1 daughters of Gclm+/- mothers and wildtype F1 daughters of wildtype mothers had similar depletion of ovarian follicles following prenatal exposure to BaP, suggesting that maternal Gclm genotype does not modify ovarian effects of prenatal BaP. We observed no BaP treatment or Gclm genotype related differences in ovarian Kras codon 12 mutations in F1 offspring., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic by-products in culture media. However, culture conditions perturb the native state of the follicle, and these methods do not distinguish between metabolism occurring within oocytes or granulosa cells. We applied the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) at 740-nm two-photon excitation to examine the spatial distribution of free and protein-bound nicotinamide adenine dinucleotide hydride (NADH) during primordial through preovulatory stages of follicular development in fresh ex vivo murine neonatal and gonadotropin stimulated prepubertal ovaries. We obtained subcellular resolution phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD+) separately for oocyte nucleus and oocyte cytoplasm. We found that dynamic changes in oocyte nucleus free/bound NADH paralleled the developmental maturation of primordial to primary follicles. Immunohistochemistry of NAD+-dependent deacetylase SIRTUIN 1 (SIRT1) in neonatal ovary revealed that increasing SIRT1 expression in oocyte nuclei was inversely related to decreasing free/bound NADH during the primordial to primary follicle transition. We characterized oocyte metabolism at these early stages to be NADH producing (glycolysis/Krebs). We extended the results of prior studies to show that cumulus and mural granulosa cell metabolism in secondary through preovulatory follicles is mainly NADH producing (glycolysis/Krebs cycle), while oocyte metabolism is mainly NADH consuming (oxidative phosphorylation). Taken together, our data characterize dynamic changes in free/bound NADH and SIRT1 expression during early follicular development and confirm results from previous studies defining antral and preovulatory follicle metabolism in culture., (© 2016 by the Society for the Study of Reproduction, Inc.)

The polycyclic aromatic hydrocarbon pollutant benzo[a]pyrene (BaP) is a known developmental gonadotoxicant. However, the mechanism of BaP-induced germ cell death is unclear. We investigated whether exposure to BaP induces apoptotic germ cell death in the mouse fetal ovary or testis. Mouse fetal gonads were dissected at embryonic day 13.5 days postcoitum (dpc) and fixed immediately or cultured for 6, 24, 48, or 72 h with various concentrations of BaP (1-1000 ng/ml). Germ cells numbers, apoptosis, and proliferation were evaluated by immunostaining. Treatment of fetal ovaries with BaP for 72 h concentration-dependently depleted germ cells. Treatment with BaP elevated the expression of BAX protein at 6 h and activated downstream caspases-9 and -3 at 24 h in a concentration-dependent manner in germ cells of fetal ovaries. As a consequence, ovarian germ cell numbers were significantly and concentration-dependently decreased at 48 h. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, prior to exposure to 1000 ng/ml BaP prevented BaP-mediated ovarian germ cell death; there were no effects of BaP or z-VAD-fmk on germ cell proliferation. No significant effects of BaP exposure on caspase 3 activation or germ cell numbers were observed in fetal testes after 48 h of culture. Our findings show that BaP exposure increases caspase-dependent and BAX-associated germ cell apoptosis in the mouse fetal ovary, leading to germ cell depletion. In contrast, the cultured 13.5 dpc fetal testis is relatively resistant to BaP-induced germ cell death. This study provides a novel insight into molecular mechanisms by which BaP has direct gonadotoxicity in the mouse fetal ovary., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP), are ubiquitous environmental pollutants and potent ovarian toxicants. The transcription factor NRF2 is an important regulator of the cellular response to electrophilic toxicants like BaP and to oxidative stress. NRF2 regulates transcription of genes involved in the detoxification of reactive metabolites of BaP and reactive oxygen species. We therefore hypothesized that Nrf2-/- mice have accelerated ovarian aging and increased sensitivity to the ovarian toxicity of BaP. A single injection of BaP dose-dependently depleted ovarian follicles in Nrf2+/+ and Nrf2-/- mice, but the effects of BaP were not enhanced in the absence of Nrf2. Similarly, Nrf2-/- mice did not have increased ovarian BaP DNA adduct formation compared to Nrf2+/+ mice. Ovarian follicle numbers did not differ between peripubertal Nrf2-/- and Nrf2+/+ mice, but by middle age, Nrf2-/- mice had significantly fewer primordial follicles than Nrf2+/+ mice, consistent with accelerated ovarian aging., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Glutathione (GSH) is the one of the most abundant intracellular antioxidants. Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH. Our prior work showed that GSH plays antiapoptotic roles in ovarian follicles. We hypothesized that Gclm(-/-) mice have accelerated ovarian aging due to ovarian oxidative stress. We found significantly decreased ovarian GSH concentrations and oxidized GSH/oxidized glutathione redox potential in Gclm(-/-) vs Gclm(+/+) ovaries. Prepubertal Gclm(-/-) and Gclm(+/+) mice had similar numbers of ovarian follicles, and as expected, the total number of ovarian follicles declined with age in both genotypes. However, the rate of decline in follicles was significantly more rapid in Gclm(-/-) mice, and this was driven by accelerated declines in primordial follicles, which constitute the ovarian reserve. We found significantly increased 4-hydroxynonenal immunostaining (oxidative lipid damage marker) and significantly increased nitrotyrosine immunostaining (oxidative protein damage marker) in prepubertal and adult Gclm(-/-) ovaries compared with controls. The percentage of small ovarian follicles with increased granulosa cell proliferation was significantly higher in prepubertal and 2-month-old Gclm(-/-) vs Gclm(+/+) ovaries, indicating accelerated recruitment of primordial follicles into the growing pool. The percentages of growing follicles with apoptotic granulosa cells were increased in young adult ovaries. Our results demonstrate increased ovarian oxidative stress and oxidative damage in young Gclm(-/-) mice, associated with an accelerated decline in ovarian follicles that appears to be mediated by increased recruitment of follicles into the growing pool, followed by apoptosis at later stages of follicular development.

Oxidative stress occurs when cellular mechanisms to regulate levels of reactive oxygen species (ROS) are overwhelmed due to overproduction of ROS and/or deficiency of antioxidants. This chapter describes accumulating evidence that oxidative stress is involved in ovarian toxicity caused by diverse stimuli, including environmental toxicants. There is strong evidence that ROS are involved in initiation of apoptosis in antral follicles caused by several chemical and physical agents. Although less attention has been focused on the roles of ROS in primordial and primary follicle death, several studies have shown protective effects of antioxidants and/or evidence of oxidative damage, suggesting that ROS may play a role in these smaller follicles as well. Oxidative damage to lipids in the oocyte has been implicated as a cause of persistently poor oocyte quality after early life exposure to several toxicants. Developing germ cells in the fetal ovary have also been shown to be sensitive to toxicants and ionizing radiation, which induce oxidative stress. Recent studies have begun to elucidate the mechanisms by which ROS mediate ovarian toxicity., (© 2014 Elsevier Inc. All rights reserved.)

Proper functioning of the ovary is critical to maintain fertility and overall health, and ovarian function depends on the maintenance and normal development of ovarian follicles. This review presents evidence about the potential impact of oxidative stress on the well-being of primordial, growing and preovulatory follicles, as well as oocytes and early embryos, examining cell types and molecular targets. Limited data from genetically modified mouse models suggest that several antioxidant enzymes that protect cells from reactive oxygen species (ROS) may play important roles in follicular development and/or survival. Exposures to agents known to cause oxidative stress, such as gamma irradiation, chemotherapeutic drugs, or polycyclic aromatic hydrocarbons, induce rapid primordial follicle loss; however, the mechanistic role of ROS has received limited attention. In contrast, ROS may play an important role in the initiation of apoptosis in antral follicles. Depletion of glutathione leads to atresia of antral follicles in vivo and apoptosis of granulosa cells in cultured antral follicles. Chemicals, such as cyclophosphamide, dimethylbenzanthracene, and methoxychlor, increase proapoptotic signals, preceded by increased ROS and signs of oxidative stress, and cotreatment with antioxidants is protective. In oocytes, glutathione levels change rapidly during progression of meiosis and early embryonic development, and high oocyte glutathione at the time of fertilization is required for male pronucleus formation and for embryonic development to the blastocyst stage. Because current evidence suggests that oxidative stress can have significant negative impacts on female fertility and gamete health, dietary or pharmacological intervention may prove to be effective strategies to protect female fertility.

Oxidative stress has been implicated in various aspects of aging, but the role of oxidative stress in ovarian aging remains unclear. Our previous studies have shown that the initiation of apoptotic cell death in ovarian follicles and granulosa cells by various stimuli is initiated by increased reactive oxygen species. Herein, we tested the hypothesis that ovarian antioxidant defenses decrease and oxidative damage increases with age in mice. Healthy, wild-type C57BL/6 female mice aged 2, 6, 9, or 12 mo from the National Institute on Aging Aged Rodent Colony were killed on the morning of metestrus. Quantitative real-time RT-PCR was used to measure ovarian mRNA levels of antioxidant genes. Immunostaining using antibodies directed against 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) was used to localize oxidative lipid, protein, and DNA damage, respectively, within the ovaries. TUNEL was used to localize apoptosis. Ovarian expression of glutathione peroxidase 1 (Gpx1) increased and expression of glutaredoxin 1 (Glrx1), glutathione S-transferase mu 2 (Gstm2), peroxiredoxin 3 (Prdx3), and thioredoxin 2 (Txn2) decreased in a statistically significant manner with age. Statistically significant increases in 4-HNE, NTY, and 8-OHdG immunostaining in ovarian interstitial cells and follicles were observed with increasing age. Our data suggest that the decrease in mRNA expression of mitochondrial antioxidants Prdx3 and Txn2 as well as cytosolic antioxidants Glrx1 and Gstm2 may be involved in age-related ovarian oxidative damage to lipid, protein, DNA, and other cellular components vital for maintaining ovarian function and fertility.

The antioxidant tripeptide glutathione (GSH) protects ovarian follicles against oxidative damage that may lead to apoptotic death. The rate-limiting step in synthesis of GSH is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic subunit (GCLC), and a modifier subunit (GCLM). We hypothesized that GSH depletion in vivo or in vitro with buthionine sulfoximine (BSO), a specific inhibitor of GCL activity, would increase ovarian and granulosa cell GCL subunit expression. Ovarian glutathione levels are lowest on proestrous morning and increase to their highest levels on estrus and metestrus. Therefore, we treated rats on proestrous morning or on proestrous morning and again 12h later to prevent the normal increase in ovarian glutathione between proestrus and estrus. Ovarian Gclc and Gclm mRNA levels and GCLC protein levels increased transiently by 1.4-1.5-fold at 8 h, but not at 12 or 24 h, after a single dose of BSO administered to adult rats on the morning of proestrus. GCLC protein levels were also modestly increased 1.4-fold at 12 h after a second dose of BSO. GCLM protein levels increased 1.4-fold at 24 h after a single dose of BSO, but not at other time points. BSO treatment did not significantly alter ovarian GCL enzymatic activity or the intraovarian localization of either GCL subunit mRNA. Treatment of a human granulosa cell line or primary rat granulosa cells with BSO suppressed intracellular GSH; however, there was no compensatory upregulation of GCL subunit protein or mRNA levels. These results demonstrate that ovarian follicles and granulosa cells are minimally able to respond to acute GSH depletion by upregulating expression of GCL.

Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous or estrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis.

Ovarian levels of the antioxidant tripeptide glutathione (GSH) increase following gonadotropin administration, suggesting that GSH synthesis in the ovary may be associated with follicular growth. In situ hybridization with (35)S-labeled riboprobes was used to localize ovarian mRNA expression of the catalytic and modulatory subunits of glutamate cysteine ligase (Gclc and Gclm), the rate-limiting enzyme in GSH synthesis, during each stage of the rat estrous cycle. Gclm was highly expressed in the granulosa cells and oocytes of healthy, growing follicles, not in atretic follicles. Gclc was also highly expressed in follicles; however, unlike Gclm, Gclc was also expressed in corpora lutea and interstitial cells. In a subsequent experiment, the hypothesis that GSH synthesis occurs in healthy, but not in apoptotic, follicles was tested. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to detect apoptotic cells in the ovaries, and in situ hybridization for Gclm and Gclc was performed in adjacent sections of the same ovaries. TUNEL staining was found to be significantly associated with absence of Gclm hybridization in granulosa cells and oocytes and with lack of strong Gclc hybridization in granulosa cells. These results suggest that follicular apoptosis may be associated with down-regulation of Gclm and Gclc transcription in granulosa cells and oocytes., (Copyright 2003 Wiley-Liss, Inc.)

Glutathione (GSH), an antioxidant and conjugator of electrophilic toxicants, prevents toxicant-mediated destruction of ovarian follicles and oocytes. Ovarian GSH has previously been shown to change with estrous cycle stage in rats, suggesting that the gonadotropin hormones may regulate ovarian GSH synthesis. The present studies tested the hypotheses that [1] estrous cycle-related changes in ovarian GSH result from cyclic changes in protein and mRNA expression of the rate-limiting enzyme in GSH synthesis, glutamate cysteine ligase (GCL, also called gamma-glutamylcysteine synthetase), and [2] that these changes result from gonadotropin-mediated regulation of GCL subunit expression. In the first experiment, ovaries were harvested from cycling adult female rats on each stage of the estrous cycle. In the second experiment immature female rats were injected with pregnant mare's serum gonadotropin (PMSG) to stimulate follicular development or with vehicle and killed 8, 24, or 48 h later. In both experiments the ovaries were harvested for [1] total GSH assay, [2] Western analysis for GCL catalytic (GCLc) and regulatory (GCLm) subunit protein levels, or [3] Northern analysis for Gclc and Gclm mRNA levels. Ovarian GSH concentrations and Gclc and Gclm mRNA levels, but not GCL subunit protein levels, varied significantly with estrous cycle stage. PMSG administration significantly increased ovarian GSH concentrations 24 and 48 h later. GCLm protein levels increased significantly at 24 h and 48 h following PMSG. GCLc protein levels did not increase significantly following PMSG. Gcl subunit mRNA levels were not significantly increased at any time point by the planned ANOVA; however, an increase in Gelc at 48 h was identified by t-testing. These results support the hypothesis that gonadotropins regulate ovarian GSH synthesis by modulating GCL subunit expression.

Peripheral administration of N-methyl-D,L-aspartate (NMA), an analogue of the excitatory amino acid aspartate, elicits LH and prolactin (PRL) release in rats, most likely by increasing endogenous releasing-hormone secretion. These experiments were carried out to assess the degree to which NMA stimulates FSH and to analyze the relationship between endocrine status and responsiveness to NMA in female rats, in contrast to male rats, as described in the companion paper [Biol Reprod 48:000-000]. In experiment 1, estrous rats (n = 10) and diestrous rats (n = 10) and in experiment 2, estrous rats (n = 11) and rats ovariectomized (OVX) 8 days previously (n = 10) were fitted with atrial catheters and injected s.c. with 100 micrograms of an LHRH antagonist or vehicle at 2100 h. Starting at 0900 h the next day (metestrus, proestrus, or Day 9 post-OVX), blood was withdrawn every 10 min for 3 h. Each animal received i.v. 5 mg NMA after the first hour and i.v. 500 ng LHRH after the second hour. NMA significantly increased LH in metestrous and proestrous females, and LHRH antagonist blunted the increases. In OVX females, LH decreased after NMA. FSH was not affected by NMA in any group. PRL increased after NMA in proestrous and metestrous animals. LHRH caused surge-like LH and small FSH increases in vehicle groups; these increases did not differ in amplitude between intact and OVX animals and were blunted by pretreatment with LHRH antagonist. In experiment 3, 10 diestrous rats were fitted with atrial catheters and were serially bled at 2-h intervals from 1200 h on the following day (proestrus) until 0600 h on estrus morning. After the first sample the animals were injected s.c. with 0.2 mg/kg MK801, a noncompetitive NMA receptor antagonist, or with saline. Four of the 5 saline-treated animals exhibited surges of LH and FSH as well as elevated progesterone levels, with LH and progesterone peaking at 2000 h. Five of 5 MK801-treated animals failed to have elevated LH, FSH, or progesterone levels at any time point. These data demonstrate that LHRH mediates the LH response to NMA in rats and that endogenous NMA receptor binding may be necessary for the preovulatory gonadotropin surges. The lack of FSH responses to NMA during periods of low-level gonadotropin secretion suggests that physiological increments in endogenous LHRH secretion sufficient to induce a pulse of LH are insufficient to stimulate pulse-like FSH release.(ABSTRACT TRUNCATED AT 400 WORDS)