PP1γ1 is unable to substitute for the mammal-specific PP1γ2 isoform to support male fertility and sperm function

In brief: Protein phosphatase 1 catalytic subunit gamma isoform 2 (PP1γ2) is a unique phosphatase expressed only in mammalian testes and sperm cells. The PP1γ2 isoform is indispensable for sperm motility and fertility and cannot be replaced by the PP1γ1 isoform for these functions. Abstract: The serine-threonine phosphatase has four paralogs - PP1α, PP1β, PP1γ1 and PP1γ2 - encoded by three genes, Ppp1ca, Ppp1cb and Ppp1cc. Protein phosphatase PP1γ2, one of two isoforms of the gene Ppp1cc, is expressed in spermatogenic cells in the testes and sperm, while PP1γ1 is found in somatic cells. The two PP1γ isoforms, formed by alternate splicing that occurs only in mammals, are identical except at their C-termini. Global or testis-specific knockout of Ppp1cc in mice results in male infertility due to disrupted spermiation and mid-to-late spermiogenesis. Transgenic expression of PP1γ2, driven by a testis-specific promoter in differentiating spermatogenic cells, rescues spermatogenesis and fertility in the Ppp1cc-null mice. Why PP1γ2 is essential and present only in mammalian sperm is a mystery. We have generated a knock-in mouse where the Ppp1cc gene is edited to express only PP1γ1. Spermatogenesis was normal in knock-in mice. Testis-expressed PP1γ1 in the knock-in mice and PP1γ2 in the wild-type mice were incorporated in equal amounts into sperm. Sperm bearing PP1γ1 have reduced flagellar beat amplitude and motility, and male mice were severely sub-fertile. Although in the wild-type mice, PP1γ2 is present in both the head and tail, in the knock-in mice, PP1γ1 is absent in sperm heads, leading to an altered intra-sperm protein phosphatase landscape. Phosphoproteomic analysis of sperm proteins suggested a plausible molecular basis for compromised PP1γ1 functions: it identified GSK3α, a known substrate of PP1, to be dysregulated in knock-in sperm. This study provides a preliminary explanation for the isoform-specific requirement of PP1γ2 for male fertility.