Ovulation is a major target for controlling fertility and treating infertility. Natural ovulation is initiated by a surge release of hypothalamic gonadotropin-releasing hormone (GnRH), which is triggered via interaction of other neuropeptides and neurotransmitters (norepinephrine, neuropeptide Y, -endorphin, galanin and gamma aminoabutyric acid). These neural chemicals exist in the hypothalamus and/or brainstem, but their exact locations and mechanistic steps in regulating GnRH secretion is currently unknown. We hypothesize that during the GnRH surge, the magnitude of gene transcription and translation in neurons expressing these chemicals should also increase. To test this hypothesis, we utilized the expression of the early oncogene, c-fos, as a marker for the onset of gene transcriptional processes. We reasoned that during the preovulatory GnRH surge, those brainstem areas that show an increase in c-fos mRNA expression probably contain neurons that secrete, or influence secretion of, GnRH surge-inducing chemicals. The female rabbit is an ideal model to test this hypothesis because the exact timing of the ovulatory GnRH surge can be controlled by coitus with a male. We have shown that the coitally induced secretion of both norepinephrine (NE) and GnRH begins within 10-30 min and is complete by 240 min after copulation. An experiment was initiated, therefore, in which female rabbits were killed and their brains fixed by paraformaldehyde infusion at times before and 30 min, 60 min, 120 min and 240 min after coitus. Mated male brains were also collected at similar intervals for use as controls because coitus does not induce NE/GnRH increases in the buck. Expression of c-fos mRNA is being examined by the in situ hybridization technique in 20 m sections of the hypothalamus and brainstem. In the female brainstem at 60 min after coitus (tissues at other times have not yet been examined), several noradrenergic neuronal areas, including the lateral tegmentum (A1), the nucleus of the solitary tract (A2) and the locus coeruleus (A6), exhibited enhanced c-fos mRNA expression. Moreover, the nucleus of prepositus hypoglossy and the paragigantocellular nucleus, the only two caudal nuclei that innervate the locus coeruleus, also expressed c-fos in higher content in the mated female than in the mated male. Several areas in the hypothalamus, including the arcuate and paraventricular nuclei where neuropeptide Y, GnRH and galanin neurons are located, also exhibited c-fos expression, although differences in the magnitude of c-fos expression in these areas between males and females were not apparent. These preliminary results suggest that c-fos containing neurons may be useful in identifying cells and chemicals involved in early activation of the coitus-induced preovulatory GnRH surge. Ultimately, we hope to extend these studies to primates because we have observed in the monkey (Macaca mulatta) that a 10-fold increase in norepinephrine gene expression in the brainstem locus coeruleus and a massive increase in hypothalamic norepinephrine release occur immediately before and during the estrogen-driven preovulatory GnRH surge. We speculate that disrupted ovulation and reduced reproductive performance in patients under chronic stress or in menopausal women may reflect a lesion in noradrenergic gene expression and activity.
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