The functional study of KiSS-1 and GPR54 expression in mouse Leydig cell

The function of animal reproduction is mainly regulated by the hypothalamic-pituitary-gonadal (HPG) axis. As animals reach puberty, the reproductive hormones start to work. The gonadotropin releasing hormone (GnRH) from hypothalamus would provoke to increase gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH) which are released from anterior pituitary. Gonadotropins stimulate gonads via the blood circulatory system to regulate the development and functions of sexual gonads. In recent years, researchers have found that GPR54, which is located in hypothalamus of human and mice can stimulate GnRH neuron to secret GnRH and subsequently activate HPG axis. KiSS-1 gene is a cancer cell metastasis suppressor. Recently, KiSS-1 has been identified as the upregulatory gene for the HPG axis. The KiSS-1 product, kisspeptin, has strong affinity to GPR54 receptor on the membrane of GnRH neuron. It can tightly bind to GPR54, and stimulate GnRH neurons to release GnRH and then activate downstream genes regulating the onset of puberty and reproductive function. So far kisspeptin and GPR54 have been identified expressing in various tissues, but researchers were devoted to understand their role in upstream HPG axis. Therefore, their roles need to be identified in other tissues, especially in sex organ. The purpose of this study was to elucidate the expression of KiSS-1 and GPR54 in testis of mice. For this reason, thirteen-week old ICR strain mice were used in this study. The tissue samples were collected from these mice and RNA were isolated. The reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) staining and Western blotting were used in this study. The results showed that Kiss-1 and GPR54 gene were highly expressed in hypothalamus, kidney and testis. KiSS-1 and GPR54 were also expressed in intestine, pancreas, ovary and adipose tissue. This mRNA expression data was confirmed by Western blot analysis. Due to steroidogenic and spermatogenic properties of testis, the testis was chosen for the further study. In order to identify the location of kisspeptin and GPR54 in testis, the following experiment was designed to identify the localization of kisspeptin and GPR54. From the IHC staining results, we found that both kisspeptin and GPR54 were highly expressed in interstitial cells (Leydig cells) of testis. We speculated that Leydig cells might secret kisspeptin and have autocrine characteristic. Besides, four-week old juvenile mice were taken to do the same experiments. However, less kisspeptin and GPR54 expression were found via IHC analysis. In conclusion, the HPG axis is very important in maintenance of reproduction function in mammals. The KiSS-1 and kisspeptin have been identified involved in this system. Although kisspeptin may have direct effects on the testis through autocrine route, the precise physiological role of the kisspeptin-GPR54 system in the regulation of reproductive function remains to be elucidated.