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Growth-Associated Protein-43 Messenger Ribonucleic Acid Expression in Gonadotropin-Releasing Hormone Neurons during the Rat Estrous Cycle

INSERM, U-422, Institut Fédératif de Recherches 22, Unité de Neuroendocrinologie et de Physiopathologie Neuronale (V.P., S.B., D.C., V.M., J.-C.B.), 59045 Lille, France; and INSERM U-336, University of Montpellier II (G.A.), 34095 Montpellier, France; University of Kentucky College of Medicine (L.J.), Lexington, Kentucky 40536; and Cresap Neuroscience Laboratory, Northwestern University (A.R.), Evanston, Illinois 60208

Address all correspondence and requests for reprints to: Dr. Vincent Prevot, INSERM, U-422, Unité de Neuroendocrinologie et de Physiopathologie Neuronale, 59045 Lille Cedex, France. E-mail: prevot{at}lille.inserm.fr

We have shown previously at the ultrastructural level that morphological changes occur in the external zone of the median eminence allowing certain GnRH nerve terminals to contact the pericapillary space on the day of proestrus. The present study was designed to determine whether the intrinsic determinant of neuronal outgrowth, growth-associated protein-43 (GAP-43), was expressed in GnRH neurons of adult female rats, and whether its expression varied throughout the estrous cycle. To accomplish this, we perfusion-fixed groups of adult female rats at 0800 and 1600 h on diestrous day 2 (diestrous II), at 0800 h and 1600 h on proestrus, and at 0800 and 1600 h on estrus (n = 4 rats/group) and used double labeling in situ hybridization and quantification to compare the levels of GAP-43 messenger RNA (mRNA) in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense complementary RNA (cRNA) probe labeled with the hapten digoxigenin, whereas the GAP-43 cRNA probe was labeled with ³⁵S and detected by autoradiography. In addition, GAP-43 protein was identified with immunohistochemistry in the median eminence. The results show that many GnRH neurons expressed GAP-43 mRNA and that GAP-43 protein was present in many GnRH axon terminals in the outer layer of the median eminence. The number of GnRH neurons expressing GAP-43 mRNA was significantly higher on proestrus (64 ± 5%) than on diestrous II (40 ± 2%; P < 0.001) or on estrus (45 ± 8%; P < 0.05), and the GAP-43 mRNA levels in GnRH neurons also varied as a function of time of death during the estrous cycle. The GAP-43 mRNA levels in GnRH neurons were higher on proestrus and estrus than on diestrous II (P < 0.05). These data show that 1) GAP-43 is expressed in adult GnRH neurons; 2) GAP-43 mRNA expression in GnRH neurons fluctuates during the estrous cycle; and 3) GAP-43 mRNA content in GnRH neurons is highest on the day of proestrus, before and during the onset of the LH surge. These observations suggest that the increased GAP-43 mRNA expression in GnRH neurons on the day of proestrus could promote the outgrowth of GnRH axon terminals to establish direct neurovascular contacts in the external zone of the median eminence and thus facilitate GnRH release into the pituitary portal blood.

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