Spermiogenesis is the step during which post-meiotic cells, called spermatids, undergo numerous morphological changes and differentiate into spermatozoa. Thousands of genes have been described to be expressed at this stage and could contribute to spermatid differentiation. Genetically-engineered mouse models using Cre/LoxP or CrispR/Cas9 are the favored approaches to characterize gene function and better understand the genetic basis of male infertility. In the present study, we produced a new spermatid-specific Cre transgenic mouse line, in which the improved iCre recombinase is expressed under the control of the acrosomal vesicle protein 1 gene promoter (Acrv1-iCre). We show that Cre protein expression is restricted to the testis and only detected in round spermatids of stage V to VIII seminiferous tubules. The Acrv1-iCre line can conditionally knockout a gene during spermiogenesis with a > 95% efficiency. Therefore, it could be useful to unravel the function of genes during the late stage of spermatogenesis, but it can also be used to produce an embryo with a paternally deleted allele without causing early spermatogenesis defects.

Generation and Characterization of a Transgenic Mouse That Specifically Expresses the Cre Recombinase in Spermatids

39 40 Spermatozoa have a unique genome organization: their chromatin is almost completely devoid of 41 histones and is formed instead of protamines which confer a higher level of compaction than 42 nucleosomes and preserve paternal genome integrity until fertilization. Histone-to-protamine 43 transition takes place in postmeiotic male germ cells called spermatids, and is indispensable for the 44 production of functional spermatozoa and thus for male fertility. Here we show that the H3K79 45 (histone 3 lysine 79) methyltransferase DOT1L controls spermatid chromatin remodelling and 46 subsequent reorganization and compaction of spermatozoon genome. Using a mouse model in which 47 Dot1l is knocked-out in adult male germ cells, we found that the chromatin of Dot1l -knockout (KO) 48 spermatozoa is less compact and characterized by a higher level of retained histones and of immature 49 forms of protamine 2. Proteomic analyses performed on differentiating male germ cells reveal that 50 Dot1l KO extensively modifies the spermatid chromatin prior to histone removal. By transcriptomics, 51 we also show that Dot1l KO deregulates the expression of ~1500 genes, many of which are involved in 52 other essential aspects of spermatid differentiation such as flagellum formation. As a consequence of 53 all these chromatin and gene expression defects, Dot1l -KO spermatozoa have misshapen and less 54 compact heads, and are less motile, which results in impaired fertility. 55

The histone methyltransferase DOT1L regulates chromatin reorganization and gene 1 expression during the postmeiotic differentiation of male germ cells

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Papers

The histone methyltransferase DOT1L regulates chromatin reorganization and gene 1 expression during the postmeiotic differentiation of male germ cells

Acknowledgment to Reviewers of Pathogens in 2021