부산대학교 도서관

RF-amide Related Peptide 3 (RFRP3) and its receptor, G Protein-coupled Receptor 147 (GPR147), have been implicated in regulating reproductive seasonality in some species. Marked changes in hypothalamic RFRP3 gene expression and peptide accumulation have been observed in response to changing photoperiod. Immunohistochemical evaluation has illustrated RFRP3-immunoreactive fibers projecting towards and in close apposition with GnRH neurons, suggesting potential effects of RFRP3 directly at the hypothalamic level. However, in humans and rodents, GPR147 mRNA is also present in the anterior pituitary. Work in the ovariectomized female rat and ewe, as well as in pituitary cell cultures, indicates that RFRP3 plays a functional role at the level of the hypophysis. This is further supported by evidence from anterograde tracing in the ewe that demonstrates RFRP3-immunoreactive cells project to the neurosecretory zone of the median eminence. Further, reduced LHβ and FSHβ gene expression have been reported in GnRH-stimulated ovine pituitary cells treated with RFRP3. In the equine, GnIH (avian homolog of RFRP3) -immunoreactive neurons have been localized to the DMH, with GnIH-containing fibers in close proximity to GnRH dendrites and cell bodies, as well as projecting towards the median eminence. However, in our laboratory, RFRP3 has been shown to have no effect on GnRH-mediated release of LH in primary equine adenohypophyseal cell culture or when administered peripherally in the mare. In the current study, our objectives were to evaluate the hypothalamic mRNA expression pattern of RFRP3 and adenohypophyseal expression of GPR147 during the winter anovulatory season (December-January; n = 4), and in the natural breeding season (May-July) during the follicular (n = 4) and luteal (n = 4) phases of the estrous cycle. Following euthanasia, adenohypophyses and hypothalami were snap frozen and stored at −80oC. Hypothalami were cut in coronal sections (20 μm) and a series of sections 200μm apart were used for detection of RFRP3 mRNA by isotopic in situhybridization. Total RNA was isolated from adenohypophyseal tissue and reverse-transcribed to cDNA. Real-time RT-PCR was performed to quantify GPR147 gene expression in the adenohypophysis and data were analyzed after normalizing mean gene expression to a reference gene (RPS20). The effects of season and reproductive phase on GPR147 gene expression was compared between groups by analysis of variance. In agreement with observations in other species, hypothalamic RFRP3 mRNA was detected within the dorsomedial nucleus (DMH) and paraventricular nucleus (PVN). In the PVN, RFRP3-expressing cells were distributed sparsely. A greater number of RFRP3-expressing cells were observed throughout the rostral-caudal extension of the DMH. Adenohypophyseal expression of GPR147 during the winter anovulatory season did not differ (0.74 ± 0.09; P= 0.60) from that during either the follicular (1.11 ± 0.34) or luteal (0.88 ± 0.26) phases of the estrous cycle when normalized to a pooled reference sample. Taken together, these initial results indicate that the cellular machinery associated with RFRP3 function is present in the equine hypothalamus and adenohypophysis. However, the functional role of this neuroendocrine system during different reproductive states in the mare is still uncertain. Supported by Texas H-9137 and the Link Equine Research Fund.