Overexpression of bmp4, dazl, nanos3 and sycp2 in Hu Sheep Leydig Cells Using CRISPR/dcas9 System Promoted Male Germ Cell Related Gene Expression

SIMPLE SUMMARY: Male germ cell development plays a crucial role in male reproduction, and gene expression also presents an essential regulatory role in its development. Many studies have been devoted to the induction and differentiation of pluripotent stem cells into germ cells in vitro. However, the culture system for pluripotent stem cells from domestic animals is not stable, especially in sheep. Our study attempted to transdifferentiate sheep somatic cells into germ cells in vitro by the overexpression of key germ cell related genes, with the aim of perfecting the construction of germ cell research models in vitro. Therefore, we explored the expression pattern of four crucial genes, bmp4, dazl, nanos3 and sycp2, in Hu sheep testicular development, and investigated the potential efficiency of overexpression of the four candidate genes using the CRISPR/dcas9 system in Leydig cells. We revealed that the overexpression of bmp4, dazl, nanos3 and sycp2 can promote the expression of male germ cell related genes. To the best of our knowledge, this is the first study to construct an overexpression induction system using CRISPR/dcas9 technology, and to induce sheep somatic cells into germ cells in vitro. ABSTRACT: Male germ cells directly affect the reproduction of males; however, their accurate isolation and culture in vitro is extremely challenging, hindering the study of germ cell development and function. CRISPR/dcas9, as an efficient gene reprogramming system, has been verified to promote the transdifferentiation of pluripotent stem cells into male germ cells by editing target genes. In our research, we explored the expression pattern of the germ cell related genes bmp4, dazl, nanos3 and sycp2 in Hu sheep testicular development and constructed the overexpression model using the CRISPR/dcas9 system. The results indicated that four genes showed more expression in testis tissue than in other tissues, and that bmp4, dazl and sycp2 present higher expression levels in nine-month-old sheep testes than in three-month-olds, while nanos3 expressed the opposite trend (p < 0.05). In addition, the expression of four potential genes in spermatogenic cells was slightly different, but they were all expressed in sheep Leydig cells. To verify the potential roles of the four genes in the process of inducing differentiation of male germ cells, we performed cell transfection in vitro. We found that the expression of the germ cell related genes Prdm1, Prdm14, Mvh and Sox17 were significantly increased after the overexpression of the four genes in Leydig cells, and the co-transfection effect was the most significant (p < 0.05). Our results illustrate the crucial functions of bmp4, dazl, nanos3 and sycp2 in Hu sheep testis development and verified the effectiveness of the overexpression model that was constructed using the CRISPR/dcas9 system, which provided a basis for further male germ cell differentiation in vitro.