Germ Cell-Specific Targeting of DICER or DGCR8 Reveals a Novel Role for Endo-siRNAs in the Progression of Mammalian Spermatogenesis and Male Fertility

Abstract

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process.

Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development

“…Nevertheless, with reduced levels of Dicer or Drosha transcripts, although some spermatids manage to complete spermiogenesis, the spermatozoa in the cKO testes or epididymides are low in number, largely deformed and do not display normal motility, resembling human oligo-asthenoteratozoospermia (OAT). On the basis of testicular histology, sperm morphology, sperm counts and sperm motility, the phenotype of our Dicer and Drosha cKO mice appears to be less severe than that of Dicer1 and Dgcr8 cKO mice, as reported previously (Zimmermann et al, 2014). The discrepancy might well result from different Cre deletor lines (our Stra8-Cre versus their Ddx4-Cre) and different mouse strains used in these two studies.…”

MicroRNAs and DNA methylation as epigenetic regulators of mitosis, meiosis and spermiogenesis

“…Sertoli cellspecific deletion of Dicer, a central component of the RNAi machinery, severely impairs Sertoli cell competence, leading to male infertility due to the absence of mature spermatozoa and testicular degeneration (Papaioannou et al 2009), reflecting an important role of the Dicer for male germ cell development. Germ cellspecific deletion of Dicer1 leads to overexpression of genes for meiotic sex chromosome inactivation (MSCI), an increase in spermatocyte apoptosis and defects in chromatin organization and nuclear shaping of elongating spermatids (Korhonen et al 2011, Greenlee et al 2012, Zimmermann et al 2014, suggesting that Dicer1 is required for the meiotic and haploid phases of spermatogenesis. In addition to Dicer, DGCR8 has been shown to be indispensable for the biogenesis of miRNAs but not endo-siRNAs, and similar symptom occurs in the conditional DGCR8-knockout mice, although the phenotype is less severe compared to the Dicer1-knockout mice (Zimmermann et al 2014).…”

“…Germ cellspecific deletion of Dicer1 leads to overexpression of genes for meiotic sex chromosome inactivation (MSCI), an increase in spermatocyte apoptosis and defects in chromatin organization and nuclear shaping of elongating spermatids (Korhonen et al 2011, Greenlee et al 2012, Zimmermann et al 2014, suggesting that Dicer1 is required for the meiotic and haploid phases of spermatogenesis. In addition to Dicer, DGCR8 has been shown to be indispensable for the biogenesis of miRNAs but not endo-siRNAs, and similar symptom occurs in the conditional DGCR8-knockout mice, although the phenotype is less severe compared to the Dicer1-knockout mice (Zimmermann et al 2014). Moreover, the knockout study of canonical enzymes in the miRNA biogenesis process reveals that miRNAs are of great importance in meiosis and spermiogenesis phases of spermatogenesis since spermatogonial differentiation appears to be unaffected by canonical enzyme deficiency.…”

A Role of MicroRNAs in Cell Differentiation During Gonad Development

“…When Vasa/Ddx4 promoter is used to drive the expression of Cre recombinase, deletion of Dicer1 begins at 12.5 dpc and reaches~90 % by 18 dpc (Gallardo et al 2007). These Dicer1 cKO mice are non-fertile and exhibit decreased-size testes and abnormal structure of the seminiferous tubules, due to increased apoptosis that started around 15 dpp and impaired spermiogenesis (Liu et al 2012;Romero et al 2011;Zimmermann et al 2014). To evaluate whether the time of Dicer1 deletion has an impact on mice fertility, Liu et al used two promoters: Stimulated by retinoic acid 8 (Stra8) and Phosphoglycerate kinase 2 (Pgk2) to drive the expression of Cre recombinase.…”