Spermatogenesis is an extremely complex process, and any obstruction can cause male infertility. RhoGDIα has been identified as a risk of male sterility. In this study, we generate RhoGDIα knockout mice, and find that the males have severely low fertility. The testes from RhoGDIα−/− mice are smaller than that in WT mice. The numbers of spermatogonia and spermatocytes are decreased in RhoGDIα−/− testis. Spermatogenesis is compromised, and spermatocyte meiosis is arrested at zygotene stage in RhoGDIα−/− mice. Acrosome dysplasia is also observed in sperms of the mutant mice. At the molecular level, RhoGDIα deficiency activate the LIMK/cofilin signaling pathway, inhibiting F-actin depolymerization, impairing testis and inducing low fertility in mouse. In addition, the treatment of RhoGDIα−/− mice with Rac1 inhibitor NSC23766 alleviate testis injury and improve sperm quality by inhibiting the LIMK/cofilin/F-actin pathway during spermatogenesis. Together, these findings reveal a previously unrecognized RhoGDIα/Rac1/F-actin-dependent mechanism involved in spermatogenesis and male fertility.

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Spermatogenesis and sperm maturation are complex and highly ordered biological processes. Any failure or disorder in these processes can cause defects in sperm morphology, motility, and fertilization ability. Cathepsin B (CTSB) is involved in the regulation of a variety of pathological processes. In the present study, we found that CTSB was abundantly expressed in the male reproductive system, however, the specific role of CTSB in regulating spermatogenesis and sperm maturation remained elusive. Hence, we generated Ctsb-/- mice using CRISPR/Cas9 technology. In Ctsb-/- mice, sperm count was significantly decreased while the level of morphologically abnormal sperm was markedly increased. Additionally, these mice had significantly lower levels of progressive motility sperm and elevated levels of immobilized sperm. Histological analysis showed slight vacuolization in the testis epithelium, as well as the loss of epididymal epithelium cells. Further investigation showed that autophagic activity was inhibited and apoptotic activity was increased in both the testis and epididymis of Ctsb-/- mice. Together, our findings demonstrate that CTSB plays an important role in spermatogenesis and sperm maturation in mice.

The process of spermatogenesis is a complex and delicate process that is still not fully understood. In this study, we examined the role of fatty acid oxidase 3-hydroxy acyl CoA dehydrogenase (HADH) in maintaining normal spermatogenesis in mice. In male mice, ablation of the Hadh gene using CRISPR/Cas9 technology arrested spermatocyte meiosis, increased multinucleated giant germ cells and vacuoles in seminiferous tubules, and accompanied with acrosomal dysplasia. Hadh −/− male mice showed the typical features of oligoasthenoteratozoospermia (OAT), including decreased sperm concentration and motility and increased sperm abnormalities. Next, we explored the molecular events in the testes of the mutant mice. We found fatty acids accumulated in the testis of Hadh −/− mice. And also, inflammatory factors TNF-α, IL-1β, and IL-6 were significantly increased, apoptosis-related protein Bcl-2 was decreased, and Bax and cleaved-Caspase3 were increased in Hadh −/− male mice testis. After using etanercept, a specific inhibitor of TNF-α, testis injury caused by Hadh knockout was significantly alleviated, the sperm quality and motility were improved, and germ cell apoptosis was reduced. So our study demonstrated that Hadh deletion caused an increase in fatty acids. The accumulated fatty acids further induced testicular inflammation and germ cell apoptosis through the TNF-α/Bcl-2 signaling pathway, finally resulting in OAT in the Hadh −/− mice. Inhibiting TNF-α may be used as a new treatment approach for testicular inflammation and OAT.