Male germ cells establish a unique heterochromatin domain, the XY-body, early in meiosis. How this domain is maintained through the end of meiosis and into post-meiotic germ cell differentiation is poorly understood. ADAD2 is a late meiotic male germ cell-specific RNA-binding protein, loss of which leads to post-meiotic germ cell defects. Analysis of ribosome association in Adad2 mouse mutants revealed defective translation of Mdc1, a key regulator of XY-body formation, late in meiosis. As a result, Adad2 mutants show normal establishment but failed maintenance of the XY-body. Observed XY-body defects are concurrent with abnormal autosomal heterochromatin and ultimately lead to severely perturbed post-meiotic germ cell heterochromatin and cell death. These findings highlight the requirement of ADAD2 for Mdc1 translation, the role of MDC1 in maintaining meiotic male germ cell heterochromatin and the importance of late meiotic heterochromatin for normal post-meiotic germ cell differentiation.

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Ruminants are major producers of meat and milk, thus managing their reproductive potential is a key element in cost-effective, safe, and efficient food production. Of particular concern, defects in male germ cells and female germ cells may lead to significantly reduced live births relative to fertilization. However, the underlying molecular drivers of these defects are unclear. Small noncoding RNAs, such as piRNAs and miRNAs, are known to be important regulators of germ-cell physiology in mouse (the best-studied mammalian model organism) and emerging evidence suggests that this is also the case in a range of ruminant species, in particular bovine. Similarities exist between mouse and bovids, especially in the case of meiotic and postmeiotic male germ cells. However, fundamental differences in small RNA abundance and metabolism between these species have been observed in the female germ cell, differences that likely have profound impacts on their physiology. Further, parentally derived small noncoding RNAs are known to influence early embryos and significant species-specific differences in germ-cell born small noncoding RNAs have been observed. These findings demonstrate the mouse to be an imperfect model for understanding germ-cell small noncoding RNA biology in ruminants and highlight the need to increase research efforts in this underappreciated aspect of animal reproduction.

ADAD1 is a testis-specific RNA binding protein expressed in post-meiotic spermatids whose loss leads to defective sperm morphology and male infertility. However, the drivers of the Adad1 phenotype remain unclear. Morphological and functional analysis of Adad1 mutant sperm demonstrated defective DNA compaction, abnormal head shaping, and reduced motility. RNA sequencing of Adad1 mutant testes revealed minimal transcriptome changes however ribosome association of many transcripts was reduced, suggesting ADAD1 may be required for their translational activation. Additionally, immunofluorescence of proteins encoded by select transcripts showed delayed protein accumulation. Further analyses demonstrated impaired subcellular localization of multiple proteins suggesting protein transport is also abnormal in Adad1 mutants. To clarify the mechanism giving rise to this, the manchette, a protein transport microtubule network, and the LINC complex, which connects the manchette to the nuclear lamin, were assessed across spermatid development. Proteins of both displayed delayed translation and/or localization in mutant spermatids implicating ADAD1 in their regulation, even in the absence of altered ribosome association. Finally, ADAD1’s impact on the nuclear pore complex (NPC), a regulator of both the manchette and the LINC complex, was examined. Reduced ribosome association of NPC encoding transcripts and reduced NPC protein abundance along with abnormal localization in Adad1 mutants confirmed ADAD1 is required for normal translation of the NPC in post-meiotic germ cells. Together, these studies lead to a model whereby ADAD1’s influence on nuclear transport leads to deregulation of the LINC complex and the manchette, ultimately generating the range of physiological defects observed in the Adad1 phenotype. Summary sentence: ADAD1 is a post-meiotic spermatid RNA binding protein that is required for normal translation of mRNAs important for post-meiotic differentiation and mRNAs associated with nuclear and intracellular transport.

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