Post-Natal Developmental Changes Of Gnih And Its Receptors In Male Xiaomeishan Pig Reproductive Axis

Gonadotropin-inhibitory hormone (GnIH), a key regulator of vertebrate reproduction and a neuropeptide capable of inhibiting gonadotropin release, was recently identified in the Japanese quail. GnIH was reported to have many important physiological roles mediated by its receptors GPR147 and GPR74 in reproduction, behavior, feeding and stress etc. Yet the potential role of GnIH in regulating reproductive axis was few reported, especially in domestic animals, and there was no research about reproductive regulation of GnIH to male pigs in literature. Thus, we studied post-natal developmental changes of GnIH and its receptors in male Xiaomeishan pig reproductive axis by means of semiquantitative RT-PCR, immunohistochemistry (IHC), cell culture and radioactive immunoassay analysis (RIA).1. Expression of GnIH and its receptors mRNAs in the pig hypothalamus-pituitary-testis axis during the postnatal developmentThe aim of this experiment was to explore the expression patterns of GnIH and its receptors in the hypothalamus-pituitary-testis axis of male pigs at different stages of development (3 d,30 d,60 d,90 d, and 120 d) using semiquantitative RT-PCR. The results were as follows:(1) In the hypothalamus, the expression patterns of GnIH, GPR147, and GPR74 mRNAs were similar. The highest level of GnIH, GPR147, and GPR74 expressions was found on 3 d, followed by a significant decrease on 30 d and then increase on 60 d and finaly declined from day 60 d to 120 d, with the lowest expression level on 120 d.(2) In the pituitary, the expression patterns of GnIH, GPR147, and GPR74 mRNAs were basically similar. GnIH and GPR147 mRNAs expression levels, increased from 3 d to 60 d, and declined from 60 d to 120 d, with the highest expression level on 60 d and the lowest expression level on 3 d. GPR74 mRNA expression level declined from day 3 d to 120 d, the highest expression level on 3 d.(3) In the testis, the expression patterns of GnIH, GPR147, and GPR74 mRNAs were deferent. The GnIH expression was declined from 3 d to 30 d and increased from 30 d to 120 d, with the lowest expression level was observed on 30 d. However, the inverse was observed for GPR147 mRNA levels, the highest expression level on 30 d. GPR74 mRNA level declined from day 3 d to 60 d, then increased to the highest expression level on 90 d and finally decreased to the lowest expression level on 120 d.2. Distribution of GnIH in the pig hypothalamus, pituitary, and testis during sexual developmentIn this experiment, we researched the distribution of GnIH in male Xiaomeishan pig hypothalamus, pituitary, and testis during sexual development by immunohistochemistry. The results were as follows:(1) In the hypothalamus, GnIH positive neurons in the paraventricular nucleus (PVN), were seen in spindle and polygon.(2) In the pituitary, GnIH positive cells were localized in the adenohypophysis and neurohypophysis.(3) In the testis, GnIH was mainly localized in the Sertoli cells and spermatogenic cell of the testis throughout sexual development and in the Leydig cells (LC) from 60 d to 90 d.3. The effect of GnIH on pig hypothalamus-pituitary-testis axis in vitroIn order to study the effect of (10-6 to 10-12 M) GnIH on reproduction, the secretion of testosterone and the expression of related genes (3?-HSD and P450) in the leydig cells were analysed by using cell immunohistochemistry, semiquantitative RT-PCR, and radioimmunoassy. The results were as follows:Radioimmunoassay analysis indicated that testosterone concentrations decreased significantly when the Leydig cells were treated with 10-8M GnIH. However, there was no significant decrease in testosterone concentration when the cells were treated with any other doses of GnIH (i.e.10-6,10-10, and 10-12 M). Semiquantitative RT-PCR analysis indicated that the expression of 3?-HSD decreased significantly when the cells were treated with 10"8, 10-10 and 10-12 M GnIH, while 10-8 and 10-10 M GnIH inhibited the expression of P450 significantly.The studies above provide basis for research on the regulation mechanism of GnIH fertility on Xiaomeishan pig in gene, protein, and cells levels.