A genetic method for sex determination in Ovis spp. by interruption of the zinc finger protein, Y-linked (ZFY) gene on the Y chromosome.

The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene on the Y chromosome have not been completely elucidated, due, in part, to difficulties in gene targeting analysis of the Y chromosome. The zinc finger protein, Y-linked (ZFY) gene was first proposed to be a sex determination factor, although its function in spermatogenesis has recently been elucidated. Nevertheless, ZFY gene targeting analysis has not been performed to date. In the present study, RNA interference (RNAi) was used to generate ZFY-interrupted Hu sheep by injecting short hairpin RNA (shRNA) into round spermatids. The resulting spermatozoa exhibited abnormal sperm morphology, including spermatozoa without tails and others with head and tail abnormalities. Quantitative real-time polymerase chain reaction analysis showed that ZFY mRNA expression was decreased significantly in Hu sheep with interrupted ZFY compared with wild-type Hu sheep. The sex ratio of lambs also exhibited a bias towards females. Together, the experimental strategy and findings of the present study reveal that ZFY also functions in spermatogenesis in Hu sheep and facilitate the use of RNAi in the control of sex in Hu sheep.

Sex control by Zfy siRNA in the dairy cattle.

Zinc-finger Y is located in the short arm of the Y-chromosome and is a highly conserved gene that plays an important role in spermatogenesis. The objective of this study was to investigate the influence of silencing the Zfy gene during spermatogenesis on Y-sperm formation and offspring sex determination in Bos taurus cattle. Three recombinant expression vectors pLL3.7/a, pLL3.7/b and pLL3.7/c were evaluated and only pLL3.7/a effectively silenced the Zfy gene. The pLL3.7/a recombinant expression vector was injected into bull testes, using three injections. Semen was collected and preserved by extending and freezing. The frozen semen was subsequently used in artificial insemination of cows during a breeding season in accordance with the production plan on the farm where the experiment was conducted. Results showed that, after exposure to pLL3.7/a, sperm motility decreased (P < 0.01), but the sperm density was similar (p > 0.05) to the non-treated control semen. Injection of pLL3.7/a resulted in 72.0% female offspring, and was greater than the 49.4% female calves in the control (P ＜ 0.01), Results from this research suggests that the Zfy gene plays a role in the process of Y-sperm formation, and Zfy siRNA is a potential useful approach to control sex of offspring in cattle.