Characterizing Spermatogenesis through the Inactivation of Uba2 in the SUMOylation Cycle and its Meiotic Arrest
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Abstract
Between 8-12% of couples struggle with infertility due to various male or female factors. However, 33% of infertility cases are idiopathic. To identify a potential cause of male infertility, this research examined the relationship between male fertility and the inhibition of the SUMOylation cycle within the testes. In vivo experimentation utilized the Cre-LoxP mechanism in mice to excise exon 3 of Uba2, the first enzyme used in the SUMOylation cycle, through the expression of Stra-8 promoter. Ex vivo spermatogenesis analysis in the transgenic tubules through counting and statistical analysis of sperm cells indicated SUMOylation inhibition caused by a dramatic decrease in the progression of germ cells through spermatogenesis. H1T antibody was used as a meiotic marker, indicating that spermatocytes reached mid-late meiosis before arrested development. The efficiency of Uba2 inactivation was assessed via analysis of the transgenic testicular cells with a Uba2 antibody. The results indicate that the Cre-LoxP excision of exon 3 was not 100% successful. RNA BaseScope microscopy was used to further confirm the presence or absence of exon 3 in control and transgenic cells. The results supported the antibody results regarding the recombination efficiency and suggest that this type of analysis is highly affected by RNA instability due to slide preparation. Western Blot analysis confirmed the decrease of SUMOylation in vivo through the increase of free SUMO and decrease of SUMO-bound proteins within the transgenic tubules. Although about a half of the germ cells still expressed Uba2, the presence of the significant numbers of Uba2-negative ells was sufficient to significantly affect testicular metabolism, disturbing the germ-somatic cell crosstalk, and causing complete male infertility.