Kern Chandlar H, Feitosa Weber B, Liu Wan-Sheng
Department of Animal Science, Center for Reproductive Biology and Health, College of Agricultural Sciences, The Pennsylvania State University, University Park, PA, United States.
Front Genet. 2022 Mar 21;13:846345. doi: 10.3389/fgene.2022.846345. eCollection 2022.
The preferentially expressed antigen in melanoma, Y-linked (PRAMEY) is a cancer/testis antigen expressed predominantly in bovine spermatogenic cells, playing an important role in germ cell formation. To better understand PRAMEY's function during spermatogenesis, we studied the dynamics of PRAMEY isoforms by Western blotting (WB) with PRAMEY-specific antibodies. The PRAMEY protein was assessed in the bovine testicular and epididymal spermatozoa, fluid and tissues, and as well as in ejaculated semen. The protein was further examined, at a subcellular level in sperm head and tail, as well as in the subcellular components, including the cytosol, nucleus, membrane, and mitochondria. RNA expression of PRAMEY was also evaluated in testis and epididymal tissues. Our WB results confirmed the previously reported four isoforms of PRAMEY (58, 30, 26, and 13 kDa) in the bovine testis and spermatozoa. We found that testicular spermatozoa expressed the 58 and 30 kDa isoforms. As spermatozoa migrated to the epididymis, they expressed two additional isoforms, 26 and 13 kDa. Similarly, the 58 and 30 kDa isoforms were detected only in the testis fluid, while all four isoforms were detected in fluid from the cauda epididymis. Tissue evaluation indicated a significantly higher expression of the 58 and 13 kDa isoforms in the cauda tissue when compared to both the testis and caput tissue ( < 0.05). These results indicated that testis samples (spermatozoa, fluid, and tissue) expressed predominantly the 58 and 30 kDa PRAMEY isoforms, suggesting their involvement in spermatogenesis. In contrast, the 26 kDa isoform was specific to epididymal sperm and the 13 kDa isoform was marked in samples derived from the cauda epididymis, suggesting their involvement in sperm maturation. Results from the sperm head and tail experiments indicated that the 13 kDa isoform increased 4-fold in sperm tails from caput to cauda, suggesting this isoform may have a significant role in tail function. Additionally, the 13 kDa isoform increased significantly ( < 0.05) in the cytosol during epididymal passage and tended to increase in other subcellular components. The expression of PRAMEY in the sperm subcellular components during epididymal maturation suggests the involvement of PRAMEY, especially the 13 kDa isoform, in sperm motility.
黑色素瘤优先表达抗原Y连锁型（PRAMEY）是一种癌胚抗原，主要在牛的生精细胞中表达，在生殖细胞形成中发挥重要作用。为了更好地了解PRAMEY在精子发生过程中的功能，我们使用PRAMEY特异性抗体通过蛋白质免疫印迹法（WB）研究了PRAMEY亚型的动态变化。在牛的睾丸、附睾精子、液体和组织以及射出的精液中评估了PRAMEY蛋白。还在精子头部和尾部以及包括细胞质、细胞核、细胞膜和线粒体在内的亚细胞组分的亚细胞水平上进一步检测了该蛋白。在睾丸和附睾组织中也评估了PRAMEY的RNA表达。我们的WB结果证实了先前报道的牛睾丸和精子中的四种PRAMEY亚型（58、30、26和13 kDa）。我们发现睾丸精子表达58和30 kDa亚型。当精子迁移到附睾时，它们表达另外两种亚型，26和13 kDa。同样，仅在睾丸液中检测到58和30 kDa亚型，而在附睾尾部的液体中检测到所有四种亚型。组织评估表明，与睾丸和附睾头部组织相比，附睾尾部组织中58和13 kDa亚型的表达显著更高（<0.05）。这些结果表明，睾丸样本（精子、液体和组织）主要表达58和30 kDa的PRAMEY亚型，表明它们参与精子发生。相比之下，26 kDa亚型是附睾精子特有的，13 kDa亚型在附睾尾部来源的样本中显著，表明它们参与精子成熟。精子头部和尾部实验的结果表明，从附睾头部到尾部，精子尾部的13 kDa亚型增加了4倍，表明该亚型可能在尾部功能中起重要作用。此外，在附睾通道中，13 kDa亚型在细胞质中显著增加（<0.05），并且在其他亚细胞组分中趋于增加。附睾成熟过程中PRAMEY在精子亚细胞组分中的表达表明PRAMEY，尤其是13 kDa亚型，参与精子运动。
