SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing
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Evaluation Summary:
This manuscript describes an extensive molecular and cellular analysis of spermatogenesis in male mice in the absence of splicing factor Srsf10; a factor known to be involved in alternative splicing. Loss of Srf10 did not prevent formation of spermatogonia in testes, but did inhibit spermatogonia from entering meiosis and producing meiotic spermatocytes. These results should be of interest to molecular, developmental, and reproductive biologists. However, the conclusions require additional experimental support and the molecular basis of the observations need to be more clearly defined.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using a germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. In the absence of SRSF10, spermatogonial stem cells can be formed, but the expansion of Promyelocytic Leukemia Zinc Finger (PLZF)-positive undifferentiated progenitors was impaired, followed by the failure of spermatogonia differentiation (marked by KIT expression) and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Notably, SRSF10 directly binds thousands of genes in isolated THY + spermatogonia, and Srsf10 depletion disturbed the alternative splicing of genes that are preferentially associated with germ cell development, cell cycle, and chromosome segregation, including Nasp , Bclaf1 , Rif1 , Dazl , Kit , Ret, and Sycp1 . These data suggest that SRSF10 is critical for the expansion of undifferentiated progenitors by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.
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Evaluation Summary:
This manuscript describes an extensive molecular and cellular analysis of spermatogenesis in male mice in the absence of splicing factor Srsf10; a factor known to be involved in alternative splicing. Loss of Srf10 did not prevent formation of spermatogonia in testes, but did inhibit spermatogonia from entering meiosis and producing meiotic spermatocytes. These results should be of interest to molecular, developmental, and reproductive biologists. However, the conclusions require additional experimental support and the molecular basis of the observations need to be more clearly defined.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This manuscript reports on a very extensive molecular and cellular study of the effect of splicing factor Srsf10 on spermatogenesis in male mice. Using Srsf10 knockout mice, the investigators determined that loss of Srsf10 specifically inhibited spermatogonia differentiation (into spermatocytes) and entrance into meiosis (essential for fertility). The deletion of Srsf10, a factor already well characterized and known to be involved in alternative splicing of mRNA (i.e, post-transcriptional), was responsible for male infertility. It had been shown previously that Srsf10 controls alternative splicing by binding to exons as well as to splicing factors during mitosis. It is of interest that spermatogonia are produced, suggesting that loss of Srsf10 with its effects on alternative splicing may not affect early …
Reviewer #1 (Public Review):
This manuscript reports on a very extensive molecular and cellular study of the effect of splicing factor Srsf10 on spermatogenesis in male mice. Using Srsf10 knockout mice, the investigators determined that loss of Srsf10 specifically inhibited spermatogonia differentiation (into spermatocytes) and entrance into meiosis (essential for fertility). The deletion of Srsf10, a factor already well characterized and known to be involved in alternative splicing of mRNA (i.e, post-transcriptional), was responsible for male infertility. It had been shown previously that Srsf10 controls alternative splicing by binding to exons as well as to splicing factors during mitosis. It is of interest that spermatogonia are produced, suggesting that loss of Srsf10 with its effects on alternative splicing may not affect early steps in spermatogenesis. The extensive analysis of alternative splicing was carried out in mouse testes and accounts primarily for the novelty of the research. This manuscript should be of interest to molecular, developmental, and reproductive biologists.
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Reviewer #2 (Public Review):
SRSF10, also known as SRp38, is an atypical serine/arginine-rich splicing factor that regulates the generation of isoforms of messenger RNAs (mRNAs) from common precursor pre-mRNAs, so that cells can express protein variants in need. It has been shown that SRSF10 regulates alternative splicing via binding of exons and constitutive splicing factors in response to cellular stimuli during mitotic cell cycle progression. Liu et al. now utilize genetically modified mouse model that lacks SRSF10 specifically in male germ cells to show that SRSF10 is required for spermatogenesis at a very early stage and thus male fertility by regulating alternative splicing of hundreds of genes.
Spermatogenesis encompasses a series of consecutive events to produce male gametes, sperm, including mitosis, meiosis and post-meiotic …
Reviewer #2 (Public Review):
SRSF10, also known as SRp38, is an atypical serine/arginine-rich splicing factor that regulates the generation of isoforms of messenger RNAs (mRNAs) from common precursor pre-mRNAs, so that cells can express protein variants in need. It has been shown that SRSF10 regulates alternative splicing via binding of exons and constitutive splicing factors in response to cellular stimuli during mitotic cell cycle progression. Liu et al. now utilize genetically modified mouse model that lacks SRSF10 specifically in male germ cells to show that SRSF10 is required for spermatogenesis at a very early stage and thus male fertility by regulating alternative splicing of hundreds of genes.
Spermatogenesis encompasses a series of consecutive events to produce male gametes, sperm, including mitosis, meiosis and post-meiotic cellular morphogenesis. Although alternative splicing has been known as an important step to regulate gene expression in spermatogenic cells, the underlying molecular mechanisms remain to be fully understood. Using a genetic approach, the authors created mice that carry alleles of Vasa-Cre and floxed Srsf10 (Srsf10Flox/Flox:Vasa-Cre). Since Vasa (known as Mvh in mouse, the mouse Vasa homolog) is specifically expressed in germ cells, its promoter would drive the expression of Cre recombinase specifically in germ cells and remove floxed DNA fragment, generating mutant Srsf10 gene (missing exon 3 in this case) in germ cells. To find out whether removal of functional Srsf10 gene would affect male germline development (females are not mentioned in this manuscript), the authors analyzed the fertility of male mice, testis development and spermatogenic cells using various cell biological approaches. Their results showed that Srsf10 mutants suffered severe defects in spermatogenesis and produced no spermatogenic cells beyond meiotic stage, leading to male infertility. The authors further showed, by analyzing more detailed spermatogenic steps, that mutant mice retained only earliest stage spermatogonia at decreased levels, comparing to the control counterparts that still express SRSF10.
Spermatogonial stem cells (SSCs) are the founder cells of spermatogenesis, which contain heterogenous cell populations probably due to the progressive proliferation and differentiation of self-renewing SSCs. To find out whether defected spermatogenesis of Srsf10 mutants was caused by defects in SSCs. The authors applied known marker proteins of SSCs to analyze their sub-populations in more details, using immunofluorescent staining, cell sorting and single cell RNA sequencing. The results showed that the proliferating population of SSCs expressing PLZF (PLZF+) were decreased in number, whereas the earlier stage of un-differentiated SSCs expressing GFRa1 were less affected. Consequently, meiotic spermatocytes were severely disrupted. Analysis of cell proliferation using nascent DNA labeling (Edu) supported the notion that deletion of SRSF10 impeded mitotic cell cycle of PLZF+ differentiating progenitors. This is further supported by the single cell RNA sequencing analyses. Characterization of cellular transcriptome at single cell level can not only identify changes of gene expression but also be used to classify cell types according to their similar expression patterns of typical marker genes. Bioinformatics analyses of single cell RNAseq data indeed showed that Srsf10 mutants contained a DSSC3 cell group that was not presented in the controls. In addition, they also showed that the ratios of USSC1 and USSC2 groups, two undifferentiated SSC sub-populations, are altered in mutants, comparing to the controls, supporting their cellular analyses in SSC sub-populations.
To further determine how SRSF10 affected gene expression in spermatogenic cells, especially for SSCs, the authors conducted both bulk RNA sequencing and Isoseq experiments using sorted SSCs (THY1+KIT-). They found that expression of hundreds of genes was differentially affected in Srsf10 mutant SSCs, especially for genes involved in cell cycle regulation, cellular iron ion homeostasis and spermatogenesis. The authors went on, using Isoseq data, to show that isoforms of many transcripts (mRNAs) were altered in SSCs lacking SRSF10, mainly due to exon skipping and altered first exon splicing events. Consistently, these affected genes are mostly involved in mitotic cell cycle progression and stress responses.
Overall, the authors presented convincing evidence on the defects of spermatogenesis and male sterility due to Srsf10 mutation. The RNA sequencing results support the role of SRSF10 in regulating alternative splicing of cell cycle regulators and post-translational modifiers that may impede mitotic cell cycle progression and stress responses. The RNA sequencing data also provided a rich source to further study the molecular mechanisms that underlie the SRSF10-mediated alternative splicing involved in the regulation of mouse SSCs.
However, some caveats can be seen in the manuscript that may undermine the significance of the study. For example, the authors concluded that the main causative event leading to male infertility in Srsf10 mutants is due to the defected expansion of differentiating progenitors, the progenies of un-differentiated SSCs, but not the formation of SSCs in neonatal mice. This should be further tested. Since the deletion of Srsf10 gene mediated by Mvh-Cre starts in embryonic stage of pro-spermatogonia, experiments designated to the proliferation status and population changes of pro-spermatogonia and un-differentiated SSCs should be carried out. In fact, single cell analyses and comparison of GFRa1+ cells suggested that SSCs may be altered at the beginning as well. Consequently, defects in the initiation of meiosis, as the authors concluded, may not be a causal but consequential effect due to the defective proliferation of progenitors. The manuscript also contains some in-consistency in the description of SSC sub-populations at various stages, in data presentation and interpretation, lack of sufficient introduction of research rationale, materials and methods used, as well as discussions of possibilities the current results indicate. These issues should be amenable using their current mouse models and experimental approaches. It will also be of interest to see if spermatogenic cells are maintained in adult or even aged mice in the absence of SRSF10.
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Reviewer #3 (Public Review):
The maintenance of spermatogonia stem cells is essential for fertility and a model for stem cell homeostasis. In this study the authors investigate the role of the alternative splicing factor SRSF10 in spermatogenesis, following on the discovery that germ cell-specific knock out of SRSF10 in mice caused a loss of spermatogonia and fertility in males.
This study begins by crossing SRSF10 floxed mice to those expressing Cre in the male germline. This resulted in infertile male mice with significant defects in spermatogonia differentiation. To investigate the molecular defects associated with these developmental defects, the authors carried out transcriptomic analysis in full testes at several different developmental time points. RNA-Seq of this bulk tissue revealed differential gene expression in the …
Reviewer #3 (Public Review):
The maintenance of spermatogonia stem cells is essential for fertility and a model for stem cell homeostasis. In this study the authors investigate the role of the alternative splicing factor SRSF10 in spermatogenesis, following on the discovery that germ cell-specific knock out of SRSF10 in mice caused a loss of spermatogonia and fertility in males.
This study begins by crossing SRSF10 floxed mice to those expressing Cre in the male germline. This resulted in infertile male mice with significant defects in spermatogonia differentiation. To investigate the molecular defects associated with these developmental defects, the authors carried out transcriptomic analysis in full testes at several different developmental time points. RNA-Seq of this bulk tissue revealed differential gene expression in the SRSF10-depleted testes that is consistent with reduced expansion and proliferation of spermatogonia. Subsequent single-cell RNA-Seq also revealed a gene expression profile consistent with loss of spermatogonia, while cell cycle analysis demonstrated a reduction in cell division and increase in apoptosis in the SRSF10-depleted cells. Finally analysis of alternative splicing in SRSF10-depleted cells identified several hundred impacted splicing changes, consistent with previous studies implicating SRSF10 as a splicing regulatory protein. Notably, many of the confirmed changes in splicing occurred in genes with known activities in spermatogonia development.
Together these studies provide useful physiologic and descriptive data on the impact of SRSF10 in mouse fertility. Future studies will be needed to determine which of the gene expression changes observed in the SRSF10-depleted cells drive the differentiation defects and which are a consequence of stalled development and proliferation. Moreover, the molecular mechanism by which SRSF10 impacts key splicing or gene expression events, and how many of these are direct targets of SRSF10 remains unexplored.
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