Abstract
BackgroundMll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis.Methodology/Principal FindingsMll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated.Conclusions/SignificanceOur data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects.
Highlights
Spermatogenesis occurs in most male mammals throughout their lifetime
We generated Mll5tm1Apa by insertion of a b-galactosidase (b-Gal) reporter cassette and neomycin resistance cassette under an independent promoter (MC1) in coding exon 3 of the murine Mll5 locus in 129S6 (129SvEv) embryonic stem cells resulting in the deletion of 180 bp of coding sequence
Heterozygous Mll5tm1Apa intercrosses result in Mendelian ratios of homozygous, heterozygous and wild type pups at embryonic day 16.5, non-Mendelian ratios were observed later in development, likely due to loss of homozygous pups between birth and weaning secondary to an immune defect [6]
Summary
Spermatogenesis occurs in most male mammals throughout their lifetime. Successful self-renewal of the spermatogonial stem cells underpins this process [1], which comprises three distinct phases [2]. The round haploid spermatids elongate, condense their chromatin by replacing histones with protamines [3], develop an acrosomal cap, a tail assembly packed with mitochondria, and shed excess cytoplasm, all in the third and final phase of differentiation, termed spermiogenesis [4]. This phase culminates in the release of spermatozoa into the lumen of the seminiferous tubules. As a by-product of spermiogenesis, residual bodies containing excess cytoplasm from late stage spermatids are released into the lumen of the seminiferous tubules These must be removed by phagocytosis by the Sertoli cells [5]. Mll deficient male mice are infertile and here we analyse the consequences of Mll deficiency for spermatogenesis
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