The Cre-LoxP system: Redefining genetic inactivation of SUMOylation to successfully study male infertility in mice models

Abstract

Infertility affects approximately 15% of couples worldwide, with male infertility contributing to half of all infertility cases. Despite known causes of male infertility, 40% of all male infertility cases remain idiopathic. A potential factor involved in idiopathic male infertility is impairment of the proteins or posttranslational modifications that regulate spermatogenesis. SUMOylation (covalent modification by small ubiquitin-like modifiers; SUMO proteins) has recently been identified as a crucial regulatory mechanism in various cell types. When in vitro inhibition of SUMOylation in type B spermatogonia-derived GC1 cell lines was performed using Ginkgolic Acid, UBC-9 SiRNA, and KAP-1 SiRNA, cells displayed reduced spermatocytic chromosome condensing, increased synaptonemal complex disassembly, and the downregulation of proteins integral to the cell cycle such as nucleophosmin. Since meiosis cannot be accurately represented in vitro, in vivo analysis was necessary to confirm that inhibiting SUMOylation causes spermatogenic arrest. Due to its vital role in the cell cycle, SUMOylation could not be inhibited using traditional transgenic methods without resulting in embryonic lethality. Stra8-Cre was therefore employed to excise Uba2, a gene encoding for a key enzyme of the SUMOylation cycle, exclusively in the testicular germ cells of mice models. The testicular weight of the experimental group showed a statistically significant reduction compared to the control group, while no statistically significant difference was observed between the body weight without testicles of the two groups. Furthermore, histological analysis of the seminiferous tubules further suggested spermatogenic arrest in the experimental group, which displayed only spermatocytes, while the control group also displayed round spermatids and spermatozoa. Future prospects include analyzing the molecular signaling and specific markers of spermatogenesis to understand its key meiotic pathways and the relevant kinases that are affected by inhibiting SUMOylation.