Samir Haney, Nagaoka Kentaro, Watanabe Gen
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan; Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt.
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan.
Theriogenology. 2018 Nov;121:134-140. doi: 10.1016/j.theriogenology.2018.07.038. Epub 2018 Aug 14.
Kisspeptin is the protein product of Kiss1 gene. In contrast to its fundamental role as GnRH Secretagogue and puberty initiation, its functions in extrahypothalmic tissues such as the testes remain unknown. The objective of this study was to investigate the potential local role of kisspeptin in the Leydig cell culture in goats. Five male Shiba goats were castrated to isolate Leydig cells from the testes. The isolated Leydig cells were then purified and cultured with different concentration of kisspeptin antagonist (0, 5, and 10 nmol; 0KPA, 5KPA, and 10KPA; respectively). After 12 h, 20 IU of hCG was added to some wells (0KPA + hCG, 5KPA + hCG, and 10KPA + hCG; respectively); other wells did not receive hCG (0KPA + Con, 5KPA + Con, and 10KPA + Con; respectively). Media of Leydig cell culture was harvested at 2, 6, 12, and 24 h after addition of hCG for testosterone (T) and estradiol (E2) assays. The contents of some non KPA treated wells (0KPA + Con and 0KPA + hCG wells) were collected and stored at -80 C for further identification the expression of mRNA encoding Kiss1 and its receptor (GPR54) using real time PCR. Results revealed high expressions of Kiss1/GPR54 mRNAs in the hCG treated (0KPA + hCG) wells compared to non-hCG treated ones (0KPA + Con). KPA significantly reduced the basal T production at 6 h incubation as well as hCG-induced T production in 2, 6, and 24 h incubation. Basal E2 concentrations were significantly lower in the KPA treated wells at 6, and 24 h likewise, KPA significantly attenuated induced E2 production at 12 h compared with its values in non KPA treated wells. In conclusion, kisspeptin antagonist significantly attenuated both basal and hCG-activated T and E2 production by purified Leydig cells in goats. Therefore, involvement of kisspeptin in the steroidogenic capability of Leydig cells is possible in goats.
kisspeptin是Kiss1基因的蛋白质产物。与其作为促性腺激素释放激素（GnRH）分泌促进因子和启动青春期的基本作用不同，其在睾丸等下丘脑外组织中的功能尚不清楚。本研究的目的是探讨kisspeptin在山羊睾丸间质细胞培养中的潜在局部作用。对5只雄性柴山羊进行去势，以从睾丸中分离间质细胞。然后将分离出的间质细胞纯化，并用不同浓度的kisspeptin拮抗剂（分别为0、5和10 nmol；0KPA、5KPA和10KPA）进行培养。12小时后，向一些孔中加入20 IU的人绒毛膜促性腺激素（hCG）（分别为0KPA + hCG、5KPA + hCG和10KPA + hCG）；其他孔不添加hCG（分别为0KPA + Con、5KPA + Con和10KPA + Con）。在添加hCG后的2、6、12和24小时收集睾丸间质细胞培养液，用于检测睾酮（T）和雌二醇（E2）。收集一些未用KPA处理的孔（0KPA + Con和0KPA + hCG孔）的内容物，并储存在-80℃，以便使用实时PCR进一步鉴定编码Kiss1及其受体（GPR54）的mRNA的表达。结果显示，与未用hCG处理的孔（0KPA + Con）相比，用hCG处理的孔（0KPA + hCG）中Kiss1/GPR54 mRNA的表达较高。KPA在孵育6小时时显著降低基础T分泌，以及在孵育2、6和24小时时降低hCG诱导的T分泌。同样，在孵育6和24小时时，KPA处理的孔中基础E2浓度显著降低，与未用KPA处理的孔相比，KPA在孵育12小时时显著减弱诱导的E2分泌。总之，kisspeptin拮抗剂显著减弱了山羊纯化睾丸间质细胞的基础和hCG激活的T和E2分泌。因此，kisspeptin可能参与了山羊睾丸间质细胞的类固醇生成能力。
