RNAi as a tool to control the sex ratio of mouse offspring by interrupting Zfx/Zfy genes in the testis

Abstract

The objective of this study was to explore a novel method to alter the sex-ratio balance of mouse offspring by silencing the paralogous genes Zfx/Zfy (Zinc finger X/Y-chromosomal transcription factor gene) during spermatogenesis. Four recombined vectors PRZ1, PRZ2, PRZ3, and PRZ4 (RNAi-Ready-pSIREN-RetroQ-ZsGreen) were constructed for interrupting the Zfx gene. Additionally, a recombined vector Psilencer/Zfy-shRNA was constructed for interrupting the Zfy gene. Male mice were randomly divided into 8 groups, with 20 animals per group. Five groups of mice were injected with PRZ1, PRZ2, PRZ3, PRZ4, and Psilencer/Zfy-shRNA vectors, respectively. The three control groups were injected with an equal volume of physiological saline, empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector, and empty Psilencer/Zfy-shRNA vector, respectively. All groups were injected every 7 days for a total of four injections. Fourteen days after the fourth injection, 10 male mice from each group were mated individually with 10 females. Testicular tissue of 10 male mice in each group was collected, and the expression level of Zfx/Zfy mRNA was determined by qRT-PCR. Results showed that, compared with the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and the physiological saline group, expression of Zfx mRNA decreased significantly after injection of PRZ1 (p < 0.01), PRZ3 (p < 0.01), and PRZ4 (p < 0.01), and 78.75 ± 7.50% of the offspring were male in PRZ4 group, significantly higher than the offspring derived from the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and physiological saline group (p < 0.01). In the PRZ1 group, the expression of Zfx mRNA was also significantly lower (p < 0.01), but the male rate of offspring was not different (p > 0.05). Conversely, the expression of Zfy mRNA decreased significantly after injection of Psilencer/Zfy-shRNA (p < 0.01) and 31.00 ± 11.00% of the offspring were male, significantly lower than in the physiological saline group (p < 0.01). In conclusion, our findings show that RNAi-mediated disruption of Zfx/Zfy in mouse testis affected X/Y spermatogenesis. Additionally, results suggest that the paralogous genes Zfx/Zfy play an important role in the process of X and Y sperm development. The individual interference of Zfx/Zfy may predict the outcome of X and Y haploid sperms. Presented herein is an advanced method developed to control mouse X/Y spermatogenesis and sex ratio of offspring.

The RNAi vecter construction and verification of the functions in sexual control of Cervus elaphus Zfx gene

“…The formation of X or Y sperm during spermatogenesis plays an important role in sex determination of offspring [19]. Previous studies in our laboratory found that Zfx or Zfy siRNA can change the sex ratio of animal offspring, such as cows, mouse, sheep, swine [14,17,20]. Cervus elaphus is a special economic animal and its antler has great market economic value.…”

“…At present, there are only a little research on Zfx gene silencing to make the sex shift of offspring, so the mechanism of action of Zfx in sperm is not clear. Some studies have shown that reducing the level of Zfx/Zfy mRNA expression will make the physiological function of X/Y sperm worse, which can influence offspring gender [14,17,[20][21][22]. Zfy is closely related to sperm head and tail formation and neck development [23], which is highly expressed between meiosis I and II.…”

“…Zfx is concerned with X spermatogenesis, can serve as a strong transcription activation factor, and guides the target genes positioning in X sperm nuclei through the nuclear membrane, it may also have a transcription activation function for some genes during X spermatogenesis [13]. Silencing the expression of Zfx may interfere with the normal function of these sex-specific genes after meiosis I [14]. Therefore, simple methods of identifying Zfx at the level of RNA alone may allow for directional manipulation of X or Y sperm, respectively.…”

“…The RNAi technology has the advantages of simple operation, low cost, quick effect, high accuracy and good repeatability. RNAi is an effective method of silencing Zfx/Zfy mRNA expression [14][15][16][17].…”

Host genes regulate transcription of sperm-introduced hepatitis B virus genes in embryo

“…It has been demon-strated that hamster and human sperm have a strong tendency to interact with exogenous DNA and are able to transfer DNA to oocytes [37]. Zhang et al injected plasmid RNAi-Ready-pSIREN-RetroQ-ZsGreen, which was constructed for interrupting the Zfx gene, into testis in the test group mice, and then the male mice were mated individually with females, resulting in 78.75 ± 7.50% of the male offspring, significantly higher than the offspring derived from the control groups (p < 0.01), suggesting that RNAi could be as a tool to control the sex ratio of mouse offspring by interrupting Zfx/Zfy genes in sperm cells [38]. In the current study, the two-cell embryos derived from sperm co-transfected with plasmids containing the full-length HBV genome and gene-specific shRNA/siRNA for interrupting the target genes were as the test group, and those derived from sperm transfected with plasmid containing the full-length Each sperm sample from a donor was divided into two groups: a test group (T), which was co-transfected with plasmids containing the full-length HBV genome and a non-interfering scrambled oligonucleotide (NISO); and a control group (C), which was transfected with plasmid containing the full-length HBV genome alone.…”