Tsga8 is required for spermatid morphogenesis and male fertility in mice

概要

Introduction
Proper epigenetic programs are crucial for differentiation of germ cells in mammals. During spermatogenesis, a lysine (K)-specific methyltransferase 2B (KMT2B), which conveys H3K4me3 at a subset of promoters, is essential for spermatogonial stem cell (SSC) differentiation. In SSCs, KMT2B targets over 1,000 genes including many uncharacterized genes highly activated later during spermiogenesis. Based on this finding, a bioinformatic search for novel genes important for spermatogenesis was conducted. Here, I demonstrate that testis-specific gene A8 (Tsga8) is one of the targets of KMT2B and is specifically upregulated in round and elongating spermatids. Tsga8 is encoded on the X chromosome in the mouse, and possesses no known protein domains except for two alanine-rich repeat motifs (Uchida et al., 2000). Thus, the function of Tsga8 has not been elucidated yet. In this study, generation and analysis of Tsga8 knockout mice using CRISPR-Cas9 led to the finding that Tsga8 is essential for spermatogenesis and male reproduction. Furthermore, loss of Tsga8 causes dysregulation of thousands of genes, including X-chromosome escapee genes, in spermatids (Sassone-Corsi, 2002). Knockout of Tsga8 caused reduced levels of transition protein 1 (TNP1) and protamine 1 (PRM1) proteins, which are involved in DNA condensation during spermiogenesis, and exhibited striking malformation of the heads of elongating spermatids. These results demonstrate the first example of a gene targeted by KMT2B, for its important functions for spermatogenesis and fertility.

Methods
Mouse experiments
Tsga8 knockout mice were generated by injecting a vector expressing a CRISPR- Cas9 guide RNA for Tsga8 into fertilized eggs. Two independent lines of Tsga8 knockout mice were used for further experiments including fertility tests, histological and molecular analysis.

RNA sequencing (RNA-seq)
Total RNA from 8-week-old mouse whole testis or spermatids collected by laser- capture microdissection (LCM) were used for RNA-seq experiments. Libraries were generated by using either NEBNext Ultra Directional RNA Library Prep Kit (NEB) or the Smart-seq2 method and sequenced using Illumina HiSeq 2500 or NovaSeq 6000.

Histological analysis
Mice were fixed by perfusion with 4% paraformaldehyde (PFA), and frozen testicular sections were used for staining. Antigen retrieval or permeabilization was performed as required. After blocking, the samples were incubated with a primary antibody overnight at 4°C, and treated with a secondary antibody and DAPI. Sections were mounted with ProLong Gold and observed with a confocal laser scanning microscope.
For hematoxylin and eosin (H&E) staining, testes from adult mice were fixed in Bouin's solution and processed according to a standard method.

Results and discussion
Analysis investigating the effect of Kmt2b knockout for H3K4me3 and mRNA using germline stem cells (Tomizawa et al., 2018) led to the finding that Tsga8 is one of the genes targeted by KMT2B. To investigate the role of Tsga8 for spermatogenesis, knockout mice were generated using the CRISPR-Cas9 system. Histological and microscopy experiments indicated that TSGA8 expression is specific to round-to-elongating spermatids. IHC analysis of adult mice suggested that Tsga8 depletion leads to complete male infertility due to epididymal azoospermia associated with highly malformed shapes of elongating spermatid heads.
To understand the cause of impaired spermiogenesis in the Tsga8 knockout spermatids, RNA sequencing (RNA-seq) was performed using whole testis and spermatids collected with laser-capture microdissection (LCM) from adult mice. Tsga8 knockout testis showed significant upregulation of apoptotic pathway genes, while downregulated genes contained various transcription factors and genes required for nuclear condensation in spermatids (Klaus et al., 2016). Furthermore, knockout spermatids exhibited a decrease in expression of a number of X chromosome escapee genes which are normally activated during spermiogenesis. Moreover, IHC experiments using antibodies for factors involved in nuclear condensation of spermatid heads showed reduction of TNP1 and PRM1 in elongating spermatids. The reduction of TNP1 and PRM1 was confirmed using western blot experiments using adult whole testis tissues.
Taken together, these results demonstrate that Tsga8, a target gene of KMT2B, is required for spermiogenesis and male fertility. Loss of Tsga8 causes dysregulation of multiple steps important for spermiogenesis: activation of X chromosome escapee genes in spermatids; expression of nuclear condensation factors such as TNP1 and PRM1 in spermatids; proper morphological transformation of elongating spermatids. This study highlights the importance of factors targeted by KMT2B for spermatogenesis, and brings new insights into the study of epigenetic programming and male infertility.

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