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Abstract
The excitatory amino acids (EAA)s, glutamate and aspartate, are considered major endogenous neuroactive substances involved in excitatory neurotransmission. The N-methyl-D-aspartate (NMDA) receptor, one of the major EAA receptors, has been implicated in the control of luteinizing hormone (LH) secretion through facilitation of luteinizing hormone releasing-hormone (LHRH). Interestingly, the ability of NMDA receptors to stimulate LHRH release is significantly compromised in castrated animals as compared to intact animals. The present study had two aims. First, to determine whether a difference exists in the distribution of NMDA receptor subtype-1 (NMDAR-1) in different hypothalamic regions in intact peripubertal male sheep. Second, to determine if testicular steroid hormones were involved in NMDA receptor expression in the hypothalamus. Messenger RNA and protein levels of NMDAR-1 were measured in the four quadrants of the hypothalamus (dorsal-rostral, ventral-rostral, dorsal-caudal, and ventral-caudal quadrant) of intact, castrated, and (or) testosterone implanted castrate male sheep by Northern and Western blot analysis, respectively. The NMDA receptor PCR product was cloned, sequenced and submitted in NCBI as accession number AY434689. This sequence has 94–97% homology with homo sapiens, 94–100% homology with sus scrofa and 88–90% homology with rattus norvegicus NMDAR-1 mRNA sequence. In Exp. 1, there was no effect of quadrant on mRNA for the receptor in the hypothalamus of the intact male sheep. There was a tendency (P < 0.10) for increased receptor mRNA in the ventral-rostral quadrant compared to mRNA in the dorsal-rostral quadrant. However, there was an effect of quadrant on the protein expression of receptor for NMDAR-1 (P < 0.05). In particular, dorsal-rostral quadrant (31.5567 ± 13.0831) expressed more receptor than ventral-caudal quadrant (17.1517 ± 13.0831; P < 0.05) and ventral-rostral quadrant (16.4683 ± 13.0831; P < 0.05). In Exp. 2, castration reduced (P < 0.01) the expression of NMDA receptor mRNA compared to expression in intact males (0.6171 ± 0.0719 vs. 0.3390 ± 0.0731). Individual quadrants that were affected by castration were dorsal-rostral (0.6291 ± 0.1078 vs. 0.3372 ± 0.1101; P < 0.10) and ventral-rostral (0.7245 ± 0.1096 vs. 0.2457 ± 0.1101; P < 0.01). The protein expression of NMDA receptors was unchanged by castration. In Exp. 3, replacement of testosterone via subcutaneous implants did not affect expression of mRNA for the receptor or the expression of the receptor. Taken together, these data suggest that NMDAR-1 has greater expression in the rostral area of the hypothalamus of male sheep. Furthermore, NMDA receptors expressed in the ventral-rostral area of the hypothalamus of sheep were affected by testicular androgens. The rostral area includes the anteroventral-periventricular nucleus (AVPV) of the preoptic area (POA) of the hypothalamus, an essential part of the neural pathways mediating hormonal feedback on gonadotropin secretion. These experiments also suggest that testosterone alone may not alter NMDAR-1 expression in male sheep. And last, it is suggested that testicular steroid hormones are involved in the regulation of NMDAR-1 expression at the transcriptional and posttranscriptional levels.
