Fujii Takashi, Hirayama Hiroki, Fukuda Shigeo, Kageyama Soichi, Naito Akira, Yoshino Hitomi, Moriyasu Satoru, Yamazaki Takashi, Sakamoto Kozo, Hayakawa Hiroyuki, Takahashi Kenichi, Takahashi Yoshiyuki, Sawai Ken
Animal Biotechnology Group, Animal Research Center, Hokkaido Research Organization, Hokkaido 081-0038, Japan.
United Graduate School of Agricultural Science, Iwate University, Iwate 020-8550, Japan.
J Reprod Dev. 2018 Aug 20;64(4):327-335. doi: 10.1262/jrd.2017-166. Epub 2018 May 24.
Artificial insemination with cryopreserved semen is a well-developed technique commonly used for controlled reproduction in cattle. However, despite current technical advances, cryopreservation continues to damage bull spermatozoa, resulting in a loss of approximately 30 to 50% of viable spermatozoa post thawing. To further improve the efficiency of cryopreservation of bull spermatozoa, understanding the molecular mechanisms underlying the cryobiological properties that affect cryoinjuries during cryopreservation process of bull spermatozoa is required. In this study, we examined the expression and localization of aquaporin (AQP) 3 and AQP7 in fresh, cooled, and frozen-thawed bull spermatozoa. Furthermore, we investigated the relevance of AQP3 and AQP7 to motility and to membrane integrity in frozen-thawed bull spermatozoa. Western blotting against AQP3 and AQP7 in bull spermatozoa revealed bands with molecular weights of approximately 42 kDa and 53 kDa, respectively. In immunocytochemistry analyses, immunostaining of AQP3 was clearly observed in the principal piece of the sperm tail. Two immunostaining patterns were observed for AQP7 -pattern 1: diffuse staining in head and entire tail, and pattern 2: diffuse staining in head and clear staining in mid-piece. Cooling and freeze-thawing did not affect the localization pattern of AQP7 and the relative abundances of AQP3 and AQP7 evaluated by Western blotting. Furthermore, we demonstrated that the relative abundances of AQP3 and AQP7 varied among ejaculates, and they were positively related to sperm motility, particularly sperm velocity, post freeze-thawing. Our findings suggest that AQP3 and AQP7 are possibly involved in the tolerance to freeze-thawing in bull spermatozoa, particularly in the sperm's tail.
使用冷冻精液进行人工授精是一项成熟的技术，常用于牛的可控繁殖。然而，尽管目前技术有所进步，但冷冻保存仍会损害公牛精子，导致解冻后约30%至50%的活精子丧失。为了进一步提高公牛精子冷冻保存的效率，需要了解影响公牛精子冷冻保存过程中冷冻损伤的低温生物学特性的分子机制。在本研究中，我们检测了水通道蛋白（AQP）3和AQP7在新鲜、冷却和冻融公牛精子中的表达和定位。此外，我们研究了AQP3和AQP7与冻融公牛精子活力和膜完整性的相关性。针对公牛精子中AQP3和AQP7的蛋白质免疫印迹分析显示，条带分子量分别约为42 kDa和53 kDa。在免疫细胞化学分析中，在精子尾部的主段清晰观察到AQP3的免疫染色。观察到AQP7有两种免疫染色模式——模式1：头部和整个尾部弥漫性染色，模式2：头部弥漫性染色，中段清晰染色。冷却和冻融不影响AQP7的定位模式以及通过蛋白质免疫印迹评估的AQP3和AQP7的相对丰度。此外，我们证明AQP3和AQP7的相对丰度在不同射精样本中有所不同，并且它们与冻融后的精子活力，特别是精子速度呈正相关。我们的研究结果表明，AQP3和AQP7可能参与公牛精子对冻融的耐受性，特别是在精子尾部。
