Mice carrying a null mutation for either of the two cyclooxygenase (COX) isoenzymes, necessary for prostanoid production, exhibit several isotype-specific reproductive abnormalities. Mice deficient in COX-1 are fertile but have decreased pup viability, whereas mice deficient in COX-2 fail to ovulate and have abnormal implantation and decidualization responses. The present study identifies the specific contribution of each COX isoenzyme in hypothalamic, pituitary, and ovarian function and establishes the pathology and rescue of the anovulatory syndrome in the COX-2-deficient mouse. In both COX-1- and COX-2-deficient mice, pituitary gonadotropins were selectively increased, whereas hypothalamic LHRH and serum gonadotropin levels were similar to those in wild-type animals (+/+). No significant differences in serum estrogen or progesterone were noted among the three genotypes. Exogenous gonadotropin stimulation with PMSG and hCG produced a comparable 4-fold increase in ovarian PGE2 levels in wild-type and COX-1(-/-) mice. COX-2(-/-) mice had no increase in PGE2 over PMSG-stimulated levels. Wild-type and COX-1(-/-) mice ovulated in response to PMSG/hCG; very few COX-2(-/-) animals responded to this regimen. The defect in ovulation in COX-2 mutants was attributed to both an abnormal cumulus oophorum expansion and subsequent stigmata formation. Gonadotropin stimulation and concurrent treatment with PGE2 or interleukin-1beta resulted in ovulation of COX-2(-/-) mice comparable to that in COX-2(+/+), whereas treatment with PGF2alpha was less effective. Collectively, these data demonstrate that COX-2, but not COX-1, is required for the gonadotropin induction of ovarian PG levels; that COX-2-related prostanoids are required for stabilization of the cumulus oophorum during ovulation; and that ovulation can be restored in the COX-2(-/-) animals by simultaneous treatment with gonadotropins and PGE2 or interleukin-1beta.

We have recently reported that a subpopulation of galanin (GAL)-immunoreactive perikarya in the preoptic area near the organum vasculosum of the lamina terminalis (OVLT) has morphological characteristics similar to those of LHRH-containing neurons. In fact, both peptides are colocalized in those neurons in the male rat brain. In these studies we describe sexual differences in the incidence of neurons colocalizing GAL and LHRH in this region. In male rats, about 20% of LHRH-immunoreactive cells in the diagonal band of Broca and the medial preoptic area are also immunoreactive for GAL. In contrast, in female rats, about 65% of LHRH-containing perikarya near the OVLT are immunostained for GAL. In addition, GAL and LHRH levels were measured in tissue extracts containing either the OVLT and surrounding areas or the median eminence. The data indicate that the preoptic area, including the OVLT, contains a significantly higher amount of GAL in females killed in proestrus than in those killed in estrus. The amount of GAL measured in males is lower than that in the proestrous females, but somewhat greater than that present in the estrous females. There were no significant differences among the three groups in LHRH content in this region. The GAL content of the median eminence was the highest in proestrous females, followed by estrous females and males. The LHRH content in the median eminence was not significantly different among the three groups. In conclusion, these observations indicate that the coexpression of GAL with LHRH in a discrete subpopulation of LHRH-producing neurons is a sex-related phenomenon. The fact that changes in GAL levels in specific regions can be seen at different times during the estrous cycle suggests that gonadal steroids, possibly estrogens, may physiologically regulate the expression of GAL in this subpopulation of LHRH neurons.

The incidence of colocalization of galanin (GAL) in luteinizing hormone-releasing hormone (LHRH) neurons is 4-to 5-fold higher in female than male rats. This fact and the finding that the degree of colocalization parallels estradiol levels during the estrous cycle suggest that GAL is an estrogen-inducible product in a subset of LHRH neurons. To analyze further this paradigm we evaluated the effects of gonadectomy and steroid replacement therapy in male and female rats. Ovariectomy resulted in a significant decrease in the number of cells colocalizing LHRH and GAL, whereas estradiol replacement to such animals restored the incidence of colocalization to that observed in controls. In males, however, estradiol treatment failed to enhance the incidence of colocalization of GAL and LHRH, indicating, therefore, that the colocalization of these peptides is gender-determined. This possibility-i.e., gender-specific determination of LHRH neurons coexpressing GAL-was evaluated by neonatal manipulation of hypothalamic steroid imprinting. As mentioned above, male rats did not respond to estrogen or testosterone by increasing GAL/LHRH colocalization as females did. Neonatally orchidectomized rats, whose hypothalami have not been exposed to testosterone during the critical period, when treated with estrogen in adulthood showed an increase in colocalization of GAL and LHRH similar to that seen in female animals. These observations indicate that the colocalization of LHRH/GAL is neonatally determined by an epigenetic mechanism that involves the testis. In summary, this sex difference in the incidence of colocalization of GAL and LHRH represents a unique aspect of sexual differentiation in that only certain phenotypic characteristics of a certain cellular lineage are dimorphic. The subpopulation of LHRH neurons that also produces GAL represents a portion of the LHRH neuronal system that is sexually differentiated and programed to integrate, under steroidal control, a network of LHRH neurons that could synchronize their activity to control the estrous cycle in rats.Recent observations from our laboratory indicate that a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons in the rat brain also expresses galanin (GAL) (1). The incidence of colocalization exhibits a striking sex difference-i.e., in females =80% of the LHRH neurons contain GAL, whereas in males only 10-15% of these neurons synthesize this peptide (2). In situ hybridization studies have corroborated our earlier findings since GAL mRNA is colocalized with the decapeptide LHRH in LHRH neurons (3). It has been reported that estradiol has a profound effect on GAL gene expression in the pituitary and the hypothalamus (see ref. 4 for a review). In this respect, we have observed differential coexpression ofGAL in LHRH neuronsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.during the estrous c...

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Corticotropin-releasing hormone (CRH) is the major regulator of the pituitary-adrenal axis. CRH-immunoreactive perikarya are widely distributed in the central nervous system; however, only those which participate directly in the regulation of adrenocorticotropin are connected to the portal circulation in the external zone of the median eminence. The present study describes the identification of these hypophysiotropic neurons using retrograde labeling and CRH immunocytochemistry. Fluoro-Gold was injected peripherally then, 5 days later, the animals were treated with colchicine. Twenty-four hours later the animals were sacrificed, and their brains were immunostained for CRH with the indirect immunofluorescence technique. The results indicate that the vast majority of the Fluoro-Gold-accumulating and CRH-immunopositive perikarya (hypophysiotropic neurons) are located in the medial parvicellular subdivision of the paraventricular nucleus (PVN). However, not each CRH-immunoreactive neuron contains Fluoro-Gold, i.e. a small portion of these neurons project to areas of the brain other than the median eminence. The anterior, lateral and periventricular subdivisions of the PVN also contain hypophysiotropic CRH-immunoreactive perikarya, however, their number is much less than in the medial parvicellular subdivision. Scattered double-labeled cells are also present in the medial preoptic area and the dorsal hypothalamus, just behind the PVN. These results support previous observations that the PVN, particularly the medial parvicellular subdivision, is the predominant source of the hypophysiotropic CRH neurons.

The present study describes the distribution of hypophysiotropic neurotensin-immunoreactive (NTi) perikarya in the rat hypothalamus. After peripheral administration of the retrograde tracer Fluoro-Gold, NT immunoreactivity was demonstrated with fluorescence immunocytochemistry using Texas red-labeled avidin. The results indicate that approximately 70% of all NTi neurons that are connected to the hypophysial portal system are located in the arcuate nucleus. Approximately 30% of these hypophysiotropic NTi neurons reside in the parvocellular subdivisions of the paraventricular nucleus. Our results also show that both the arcuate and the paraventricular nuclei contain NTi perikarya that do not project to the median eminence.

The observation that tuberoinfundibular dopaminergic (TIDA) neurons of pregnant, pseudopregnant, lactating, and aged rats express enkephalins suggested that chronically elevated PRL levels, which are characteristic for these animals, are essential for the induction of proenkephalin gene expression in TIDA neurons. The present studies investigated further the role of PRL in this phenomenon. Elevated PRL levels were achieved either experimentally by implanting anterior pituitaries under the kidney capsule of intact or hypophysectomized female rats or by using lactating rats. For controls, the elevated PRL levels were reduced with bromocryptine, a dopamine receptor agonist. The role of sex steroids in PRL-induced enkephalin gene expression was also studied in cycling, sex hormone-treated, hypophysectomized or ovariectomized rats, pituitary-implanted/sex hormone-treated rats, and ovariectomized mothers. Enkephalin immunoreactivity was detected by immunocytochemistry and enkephalin messenger RNA with in situ hybridization histochemistry using 35S- or digoxigenin-labeled riboprobes. Enkephalin or its messenger RNA was present in TIDA neurons in all experimental animals with elevated PRL levels. Although estradiol had no or only a minor effect on PRL-induced enkephalin gene expression, progesterone supported the effect of PRL. The present observations suggest that the expression of enkephalin in TIDA neurons is PRL dependent and supported by sex steroids, primarily progesterone.

Ethylene glycol monomethyl ether (EGME) and its proximate metabolite, 2-methoxyacetic acid (MAA), increase ovarian luteal cell progesterone production in the female rat in vivo and in cultured rat luteal cells in vitro, respectively. In order to better assess the potential hazard of EGME and MAA to women, these studies were conducted to determine whether the same concentrations of MAA increase progesterone in human luteinized granulosa cells as in rat luteal cells. Human cells were collected from healthy anonymous oocyte donors, washed, plated 25,000 viable cells per well, and treated with 10 IU hCG and 0-5 mM MAA for 6-48 hr. Progesterone in media was significantly elevated after 24 hr incubation at 3=1 mM MAA. MAA had no effect on ATP levels at 6 or 24 hr. Thus, MAA increased progesterone production in cultured human luteal cells at the same concentration as MAA increased progesterone in rat luteal cells. The implication is that EGME has the potential to alter ovarian luteal function in women. These data should be useful for determining the real health hazards and potential risks of EGME exposure, e i»7 society of To Ethylene glycol monomethyl ether (EGME) is a commonly used solvent that has been identified as a reproductive toxicant in humans and in animal models of human disease (reviewed in National Institute for Occupational Safety and Health, 1983, 1991). Recently published epidemiological studies have associated ethylene glycol-based solvent exposure in women with increased risks of spontaneous abortion, menstrual cycle dysfunction, and subfertility (Pastides et al., 1988;Gold et al., 1995;Correa et al, 1996). Specifically, solvent-exposed women had a 4.6-fold increase in risk of infertility (95% confidence interval 1.6-13.3) and a 2.8-fold increase in risk of spontaneous abortion (95% confidence interval 1.4-5.6) (Correa et al., 1996). A dose-response relationship between estimated ethylene glycol exposure and each of these outcomes has also been found.Concurrent with the publication of these studies, we determined that the luteal cell is a target for EGME and its proximate toxic metabolite, 2-methoxyacetic acid (MAA), in the female rat (Davis et al., 1997). Specifically, in vivo dosing with EGME (300 mg/kg) elevated serum progesterone and suppressed cyclicity, inhibited ovulation, and increased corpora lutea size by inhibiting luteolysis. In vitro, 1-10 mM MAA elevated progesterone after 24 hr of treatment, but had no effect on cAMP at 1, 3, 24, or 48 hr or ATP levels at 24 hr. Based on these data, we concluded that EGME and MAA increase ovarian luteal cell progesterone production independent of LH-stimulated cAMP.The question remains as to how the reproductive toxicity of EGME and MAA in the rat relates to the potential reproductive toxicity of EGME and MAA in women. To begin to bridge the knowledge gap, this study was designed to determine whether MAA affects progesterone production in cultured human luteinized granulosa cells at concentrations similar to those in the rat and provide data to allow d...