The Expression Of Kisspeptin/GPR54and Leptin Receptor In Hypothalamus And Their Effect On Testicular Function Of Wild Male Ground Squirrel(Citellus Dauricus Brandt)

To adapt to the environment and ensure their offspring survive, wildlife formed their unique reproductive strategies in the long-term evolutionary process, such as seasonal breeding. The environmental factors affecting wildlife seasonal reproduction include photoperiod (day length), food supply, ect. However, the action mechanism of these environmental factors on reproduction is yet unclear. Recent studies have shown that kisspeptin and leptin may mediate the influence of photoperiod and energy state on reproduction, respectively. Therefore, the aim of this study is to elucidate the role of hypothalamic kisspeptin and adipose-secreting leptin on the hypothalamus-pituitary-gonadal axis in the wild male ground squirrel (Citellus dauricus Brandt) during the physiological process of seasonal changes in testis function. Wild male ground squirrels were collected in different months. The hypothalamus and testicular tissue were used for morphological and histological observation, immunohistochemistry, Western blotting and real-time PCR experiments and serum samples were uesd for hormone determination. Morphological and histological results showed that the testis size, weight and spermatogenesis score of wild ground squirrel decreased significantly from the breeding season to the non-breeding season, while a significant increase before the period of hibernation,(i) The immunohistochemical results of Bcl-2, Bax and PCNA in the wild ground squirrel testes showed the positive stainings of Bcl-2were immunolocalized in the spermatocytes; the positive stainings of Bax were immunolocalized in the spermatogonia and spermatocytes; the positive stainings of PCNA were immunolocalized in the Leydig cells, spermatogonia and spermatocytes. Western blotting analysis showed that the immunoactivities of Bcl-2and PCNA in the testis of the breeding season and pre-hibernation were significantly higher than non-breeding season; and Bax in the immunological activity of the non-breeding season were significantly higher than breeding season and pre-hibernation.(ii) The immunoactivies of AR and ERαin the hypothalamus were no significant changes in different periods. The immunoactivities of ERβ in the hypothalamus significantly reduced in the non-breeding season. The immunoactivities of kisspeptin in the the AVPV area increased significantly pre-hibernation. The expression of kisspeptin (including mRNA and protein) in the whole hypothalamus decreased significantly from the breeding season to the non-breeding, and then increased significantly in the pre-hibernation. There was a positive correlation between the expression of GPR54and leptin receptor in the ARC aera and the entire hypothalamus, which showed a significant reduction during the non-breeding season,(ⅲ) The body weight and fat content of wild male ground squirrel increased significantly from the non-breeding season to the pre-hibernation. The immunohistochemical results of leptin receptor in testis showed that, leptin receptors were immunolocalized in the Leydig cells, Sertoli cells and different stages of germ cells during the breeding season;, leptin, receptors are mainly immonulocalized in the Leydig cells and spermatocytes during the non-breeding season and pre-hibernation. The Western blotting results of leptin receptor in testes of wild male ground squirrel showed that the immnoactivies of leptin receptor decreased significantly from the breeding season to the non-breeding season, and then increased significantly during the pre-hibernation.(ⅳ) The testosterone concentration of wild male ground squirrel in serum decreased significantly from the breeding season to the non-breeding season, and then increased significantly during the pre-hibernation. There was a moderate increase in the leptin concentration during the non-breeding season, and a peak during the pre-hibernation in the serum of wild male ground squirrel. These results of this study suggest that kisspeptin and leptin, as a sign of the light cycle and energy staterespectively, influence the hypothalamus-pituitary-gonad axis activity, causing seasonal changes of cells proliferation and apoptosis in the testis of wild male ground squirrel, resulting in periodic change of testicular weight and volume in wild male ground squirrel.