Cloning of Yak (Bos grunniens) AQP1 Gene and Its Expression in Yak Testis of Different Ages
LIU Min-Qing, WANG Ya-Ying, HE Hong-Hong, PAN Yang-Yang, YANG Shan-Shan, GAO Ze-Chuan, ZHANG Tong-Xiang, ZHAO Ling, WANG Jing-Lei, CUI Yan, YU Si-Jiu*
Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep & Goat, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
Abstract：The aim of this study was to clone the gene sequence of Aquaporin-1 (AQP1) in yak (Bos grunniens) and study the expression of AQP1 in yak testes of different ages. Testicular samples of newborn (3 day), young (1 year old), adult (4 years old) and old (8 years old) yak were collected. The full-length cDNA sequence of AQP1 gene was amplified by RT-PCR technology and analyzed by bioinformatics. qRT-PCR, Western blott and immunohistochemistry methods were used to detect and locate at the gene and protein level. The qRT-PCR results showed that as the yak grew older, the expression level of the yak AQP1 gene showed a trend of rising first and then falling, reaching the highest in the young testis; the expression level in the young group was significantly different from other groups (P<0.05), while the difference between the middle-aged group and the elderly group was not significant (P>0.05). Western blot results showed that as the yak grew older, the expression level of AQP1 protein also showed a trend of first rising and then falling, reaching the highest in the young testis; the expression difference between the young group and other groups was significant (P<0.05), while the differences between the newborn group, middle-aged group and old group were not significant (P>0.05). Immunohistochemical results showed that AQP1 protein is positively expressed in yak testes of different ages, and the expression sites are basically same, mainly expressed in sperm, sperm cells, spermatogenic cells, testicular stromal cells, supporting cells, and peritubular muscles like cells and seminiferous tubules in the basement membrane. The results of showed that the expression of AQP1 in the young yak group was significantly higher than that in the other groups (P<0.05). It is speculated that AQP1 plays an important role in the young yak testis. This study provides theoretical basis for further research on the mechanism of AQPs in the yak testis.
刘敏清, 王亚营, 何翃闳, 潘阳阳, 杨珊珊, 高泽川, 张同享, 赵凌, 王靖雷, 崔燕, 余四九. 牦牛AQP1基因克隆及其在不同年龄牦牛睾丸中的表达[J]. 农业生物技术学报, 2021, 29(5): 933-942.
LIU Min-Qing, WANG Ya-Ying, HE Hong-Hong, PAN Yang-Yang, YANG Shan-Shan, GAO Ze-Chuan, ZHANG Tong-Xiang, ZHAO Ling, WANG Jing-Lei, CUI Yan, YU Si-Jiu. Cloning of Yak (Bos grunniens) AQP1 Gene and Its Expression in Yak Testis of Different Ages. 农业生物技术学报, 2021, 29(5): 933-942.
[1] 陈世彪. 2002. 青海牦牛业的现状及可持续发展的对策[J]. 青海大学学报(自然科学版), (06):47-49. (Chen S B. 2002.The present situation of Qinghai Yaks industry and countermeasure of sustainable development[J]. Journal of Qinghai University. (06):47-49.) [2] 杜锦梅. 2019. 水通道蛋白在中华绒螯蟹生精细胞的表达及分布特征[D]. 河北大学, 导师: 穆淑梅, pp. 6-7. (Du J M.2019. Expression and distribution characteristics of Aquaporins in spermatogenic cells of eriocheir sinensis[D]. Hebei University. Supervisor: Mu S M, pp. 6-7.) [3] 马悦, 樊江峰, 何翃闳, 等. 2020. 牦牛胎盘组织细胞凋亡相关蛋白的表达[J]. 农业生物技术学报, 28(01): 72-83. (Ma Y, Fan J F, He H H,et al.2020.Expression of apoptosis-related proteins in placenta tissue of Yak (Bos grunniiens)[J].Journal of Agricultural Biotechnology, 28(01):72-83.) [4] 屈春凤, 李胜, 李卉, 等. 2015. 水牛水通道蛋白9基因克隆及其表达分析[J]. 中国畜牧兽医, 42(07): 1621-1629. (Qu C F, LI S, LI H, et.al.2015. Cloning of Buffalo AQP9 gene and analysing its expression in the Buffalo tissue[J]. China Animal Husbandry ＆ Veterinary Medicine. 42(07):1621-1629.) [5] 万茜, 张扬, 张跃环, 等. 2015. 香港牡蛎(Crassostrea hongkongensis)水通道蛋白基因AQP1的克隆, 分子特性和表达分析[J]. 海洋与湖沼, 46(05): 1078-1087. (Wan Q, Zhang Y, Zhang Y H, et.al. 2015. Molecular cloning, characterization, and expression of aquaporin1 gene in Crassostrea Hongkongensis[J]. Oceanologia et Limnologia Sinica, 2015, 46(05):1078-1087.) [6] Agre P, Brown D, Nielsen S.1995. Aquaporin water channels: Unanswered questions and unresolved controversies[J]. Current Opinion in Cell Biology, 7(4): 472-483. [7] Agre P, Preston G M, Smith B L, et.al.1993. Aquaporin chip: The archetypal molecular water channel[J]. American Journal of Physiology Renal Physiology, 265(4), F463-F476. [8] Bai S, Li T, Qin W, et al.2016. Effects of high fluoride on the expression of AQP4 and AQP1 in testis of C57BL/6J mice[J]. Chinese Journal of Andrology, 30(04): 3-6+12. [9] Boj M, Chauvigné F, Cerdà J.2015. Coordinated action of Aquaporins regulates sperm motility in a marine teleost[J]. Biology of reproduction, 93(2): 401-411. [10] Brown D, Katsura T, Gustafson C E.1998. Cellular mechanisms of aquaporin trafficking[J]. The American Journal of Physiology, 275(3 Pt 2) [11] Brown D, Verbavatz J M, Valenti G.1993. Localization of the CHIP28 water channel in reabsorptive segments of the rat male reproductive tract[J]. European Journal of Cell Biology, 61(2): 264-273. [12] Connolly D L, Shanahan C M, Weissberg P L.1998. The aquaporins. A family of water channel proteins[J]. International Journal of Biochemistry & Cell Biology, 30(2): 169-172. [13] Denker B M, Smith B L, Kuhajda F P.1988. Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules[J]. Journal of Biological Chemistry, 263(30): 15634-15642. [14] Gena P, Pellegrini-Calace M, Biasco A.2011. Aquaporin membrane channels: Biophysics, classification, functions, and possible biotechnological applications[J]. Food Biophysics, 6(2): 241-249. [15] Hattori M, Tonooka A, Zaitsu M, et al.2015. Overexpression of aquaporin 1 in the tunica vaginalis may contribute to adult-onset primary hydrocele testis[J]. Advances in Urology, 202434, DOI: 10.1155/2014/202434. [16] Hermo L, Krzeczunowicz D, Ruz R.2004. Cell specificity of Aquaporins 0, 3, and 10 expressed in the testis, efferent ducts, and epididymis of adult rats[J]. Journal of Andrology, 25(4): 494-505. [17] Huang H F, He R H, Sun C C, et al.2006. Function of aquaporins in female and male reproductive systems[J]. Human Reproduction Update, 12(6): 785-795. [18] Ishibashi K, Kuwahara M, Kageyama Y, et al.1997. Cloning and functional expression of a second new aquaporin abundantly expressed in testis[J]. Biochemical & Biophysical Research Communications, 237(3): 714-718. [19] Ito J, Kawabe M, Ochiai H.2008. Expression and immunodetection of aquaporin 1 (AQP1) in canine spermatozoa[J]. Cryobiology, 57(3): 312-314. [20] Kozono D, Yasui M, King L S, et al.2002. Aquaporin water channels: Atomic structure molecular dynamics meet clinical medicine[J]. Journal of Clinical Investigation, 109(11): 1395-1399. [21] Liu C, Gao D, Preston G M, et al.1995. High water permeability of human spermatozoa is mercury-resistant and not mediated by CHIP28[J]. Biology of Reproduction, 52(4): 913. [22] Rossato M, Galeazzi C, Ferigo M, et al.2004. Antisperm antibodies modify plasma membrane functional integrity and inhibit osmosensitive calcium influx in human sperm[J]. Human Reproduction, (8): 1816-1820. [23] Rossato M, Virgilio F D, Foresta C.1996. Involvement of osmo-sensitive calcium influx in human sperm activation[J]. Molecular Human Reproduction, 2(12):903-909. [24] Sha X Y, Xiong Z F, Liu H S.2011. Pregnant phenotype in aquaporin 8-deficient mice[J]. Acta Pharmacologica Sinica, 32(6): 840-844. [25] Sun X L, Zhang J, Fan Y, et al.2009. Aquaporin-4 deficiency induces subfertility in female mice[J]. Fertility & Sterility, 92(5): 1736-1743. [26] Verkman A S, Mitra A K.2000. Structure and function of aquaporin water channels[J]. American Journal of Physiology-Renal Physiology, 278(1): F13-F28. [27] Umberto L, Giorgia P, Anna M, et al.2016. Aquaporin-Mediated water and hydrogen peroxide transport is involved in normal human spermatozoa functioning[J]. International Journal of Molecular Sciences, 18(1): 66. [28] Watson A J.1992. The cell biology of blastocyst development[J]. Molecular Reproduction & Development, 33(4): 492-504. [29] Willoughby C E.1996. Osmotic tolerance limits and properties of murine spermatozoa[J]. Biology of Reproduction, 55(3): 715-727. [30] Yu H M, Sun B M, Bai Q, et al.2002. Influence of acetazolamide on aqp1 gene expression in testis and on sperm count/motility in epididymis of rats[J]. Archives of Andrology, 48(4): 281-294. [31] Zanetti N, Mayorga L S.2009. Acrosomal swelling and membrane docking are required for hybrid vesicle formation during the human sperm acrosome reaction[J]. Biology of Reproduction, 81(2): 396-405.
