Stable intercellular bridges are a conserved feature of gametogenesis in multicellular animals observed more than 100 years ago, but their function was unknown. Many of the components necessary for this structure have been identified through the study of cytokinesis in Drosophila; however, mammalian intercellular bridges have distinct properties from those of insects. Mammalian germ cell intercellular bridges are composed of general cytokinesis components with additional germ cell -specific factors including TEX14. TEX14 is an inactive kinase essential for the maintenance of stable intercellular bridges in gametes of both sexes but whose loss specifically impairs male meiosis. TEX14 acts to impede the terminal steps of abscission by competing for essential component CEP55, blocking its interaction in nongerm cells with ALIX and TSG101. Additionally, TEX14-interacting protein RBM44, whose localization in stabile intercellular bridges is limited to pachytene and secondary spermatocytes, may participate in processes such as RNA transport but is nonessential to the maintenance of intercellular bridge stability.

Nuage are amorphous ultrastructural granules in the cytoplasm of male germ cells as divergent as Drosophila, Xenopus, and Homo sapiens. Most nuage are cytoplasmic ribonucleoprotein structures implicated in diverse RNA metabolism including the regulation of PIWI-interacting RNA (piRNA) synthesis by the PIWI family (i.e., MILI, MIWI2, and MIWI). MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement. We find that GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) co-localizes with MILI in intermitochondrial cement. Knockout of Gasz in mice results in a dramatic downregulation of MILI, and phenocopies the zygotene–pachytene spermatocyte block and male sterility defect observed in MILI null mice. In Gasz null testes, we observe increased hypomethylation and expression of retrotransposons similar to MILI null testes. We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs. These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage.

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Only thirteen microRNAs are conserved between D. melanogaster and the mouse; however, conditional loss of miRNA function through mutation of Dicer causes defects in proliferation of premeiotic germ cells in both species. This highlights the potentially important, but uncharacterized, role of miRNAs during early spermatogenesis. The goal of this study was to characterize on postnatal day 7, 10, and 14 the content and editing of murine testicular miRNAs, which predominantly arise from spermatogonia and spermatocytes, in contrast to prior descriptions of miRNAs in the adult mouse testis which largely reflects the content of spermatids. Previous studies have shown miRNAs to be abundant in the mouse testis by postnatal day 14; however, through Next Generation Sequencing of testes from a B6;129 background we found abundant earlier expression of miRNAs and describe shifts in the miRNA signature during this period. We detected robust expression of miRNAs encoded on the X chromosome in postnatal day 14 testes, consistent with prior studies showing their resistance to meiotic sex chromosome inactivation. Unexpectedly, we also found a similar positional enrichment for most miRNAs on chromosome 2 at postnatal day 14 and for those on chromosome 12 at postnatal day 7. We quantified in vivo developmental changes in three types of miRNA variation including 5′ heterogeneity, editing, and 3′ nucleotide addition. We identified eleven putative novel pubertal testis miRNAs whose developmental expression suggests a possible role in early male germ cell development. These studies provide a foundation for interpretation of miRNA changes associated with testicular pathology and identification of novel components of the miRNA editing machinery in the testis.