Germline mutations in the Piwi gene and its human and mouse counterparts (Hiwi and Miwi, respectively) are directly responsible for azoospermia, according to data from infertile men and mouse models published in Cell.

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In most cells, DNA is packaged on a histone octamer; however, in sperm, DNA is packaged on protamines, which enable a greater degree of DNA condensation in order to enclose the DNA in the tiny sperm head. Histone-to-protamine exchange is complex and thought to involve global ubiquitination of H2A and H2B, triggered specifically at the spermiogenesis stage of spermatogenesis.

Piwi proteins are essential for spermatogenesis across species, enlisting germline-specific Piwi-interacting RNAs (piRNAs) and protecting the integrity of the genome in germ cells. The four human homologues Hiwi, Hili, Hiwi2, and Piwil3, are expressed in the human testis, but their functions have so far remained unknown.

Gou and colleagues hypothesized that mutations in the conserved destruction box (D-box) of the N-terminal domain of HIWI might have a role in human infertility. They began their investigations by screening for mutations in the D-box element of Hiwi in 413 patients with idiopathic azoospermia and 300 fertile control men by sequencing the D-box region. Heterozygous mutations were identified in three azoospermic men (R218A/L221A (Patient 1), L221G/N225H (Patient 2), and L221R (Patient 3)), all of which impaired Hiwi ubiquitination.

In order to better understand the role of these genes in azoospermia, Gou et al. created a conditional knock-in mouse model, based on the mutations identified in Patient 1, with R218A/L221A mutations in mouse Miwi. Crossing of these mice with a primordial germ cell (PGC)-specific TNAP-Cre transgenic mouse resulted in heterozygous germline-specific R218A/L221A knock-in mice (Miwi+/DB-Cre). All the male Miwi+/DB-Cre mice were infertile, whereas the females displayed normal fertility. Thus, the mouse model faithfully recapitulated the human disease phenotype in the azoospermic patient. Histopathological assessment of testes from Miwi+/DB-Cre mice showed that condensed spermatids at steps 14–16 were greatly reduced in the seminiferous tubules of Miwi+/DB-Cre testes and that testes of knock-in mice were 25% lighter than the testes of control mice.

Impaired histone-to-protamine exchange is a potential underlying mechanism

Sperm function studies indicated little movement and forward progression of sperm produced by Miwi+/DB-Cre mice, and that their ability to fertilize oocytes was substantially impaired. Most of the Miwi+/DB-Cre sperm showed abnormally curved back heads, with efficient staining of the nuclei with acidic aniline, which indicates poorly condensed chromatin. Transcriptome and piRNA level analysis in Miwi+/DB-Cre elongating spermatids uncovered only insignificant differences in knock-in and control mice, suggesting that the D-box mutations do not significantly affect gene expression. However, proteome analysis showed significant upregulation of levels of histones H2A, H2B, H3, H4, and their variants in Miwi+/DB-Cre sperm, and associated downregulation of protamines PRM1 and PRM2. Western blotting showed significant decreases in protamines PRM2 and PRM2, suggesting impaired histone-to-protamine exchange is a potential underlying mechanism to explain the functional abnormalities in knock-in sperm and their human counterparts.