Analysis of Ppp1cc-Null Mice Suggests a Role for PP1gamma2 in Sperm Morphogenesis1

Abstract

Serine/threonine protein phosphatase 1 (PP1) consists of four ubiquitously expressed major isoforms, two of which, PP1gamma1 and PP1gamma2, are derived by alternative splicing of a single gene, Ppp1cc. PP1gamma2 is the most abundant isoform in the testis, and is a key regulator of sperm motility. Targeted disruption of the Ppp1cc gene causes male infertility in mice due to impaired spermiogenesis. This study was undertaken to determine the expression patterns of specific PP1 isoforms in testes of wild-type mice and to establish how the defects produced in Ppp1cc-null developing sperm are related to the loss of PP1gamma isoform expression. We observed that PP1gamma2 was prominently expressed in the cytoplasm of secondary spermatocytes and round spermatids as well as in elongating spermatids and testicular and epididymal spermatozoa, whereas its expression was weak or absent in spermatogonia, pachytene spermatocytes, and interstitial cells. In contrast, a high level of PP1gamma1 expression was observed in interstitial cells, whereas much weaker expression was observed in all stages of spermatogenesis. Another PP1 isoform, PP1alpha, was predominant in spermatogonia, pachytene spermatocytes, and interstitial cells. Examining the temporal expression of PP1 enzymes in testes revealed a striking postnatal increase in PP1gamma2 levels compared with other isoforms. Testicular sperm tails from Ppp1cc-null mice showed malformed mitochondrial sheaths and extra outer dense fibers in both the middle and principal pieces. These data suggest that in addition to its previously documented role in motility, PP1gamma2 is involved in sperm tail morphogenesis.

PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis

“…Defective mitochondrial sheath morphology has been observed previously in both the Ppp1cc and Tssk1/2 knockout mouse models (Chakrabarti et al 2007, Shang et al 2010. Comparative analysis of these structures via EM revealed key similarities between the two mutants.…”

“…Despite these differences, the time point of peak PPP1CC2 protein levels in the testis overlaps with that of TSSK1/2, and there are several key similarities between the knockout phenotypes. Both mutants exhibit a significant reduction in the number and motility of epididymal spermatozoa as well as prominent defects in the organization of the mitochondrial sheaths (Varmuza et al 1999, Chakrabarti et al 2007, Soler et al 2009, Shang et al 2010. Ppp1cc2 knock-in mice with low levels of transgene expression (O50% of heterozygous levels) are able to rescue the loss of post-meiotic germ cells found in Ppp1cc knockouts, but are still infertile and mitochondrial sheath abnormalities are still visible (Soler et al 2009, Sinha et al 2012.…”

“…Inside the seminiferous tubules, a widespread loss of germ cells is evident, most prominently in postmeiotic spermatids, leading to a breakdown of the spermatogenic cycle (Varmuza et al 1999, Forgione et al 2010). The few surviving germ cells feature a range of morphological abnormalities, including those involving meiosis, chromatin condensation, acrosome formation and mitochondrial organization (Varmuza et al 1999, Chakrabarti et al 2007, Forgione et al 2010. In the mouse testis, a number of different proteins have been shown to interact with PPP1CC2.…”

Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

“…The S/T phosphatases PPP1 and PPP2 are present in sperm cells, and were found to participate in the capacitation and acrosome reaction processes, and sperm motility (Furuya et al 1993, Ashizawa et al 2006, Chakrabarti et al 2007). In the mammalian phylum, four PPP1C isoforms were identified: PPP1CA (PP1ca), PPP1CB (PP1cb), PPP1CC1 (PP1cg1), and PPP1CC2 (PP1cg2), of which the latter two are alternative splice variants of the PPP1CC gene.…”

“…Current studies revealed PPP1CC2 as a testis-specific isoform that has been found to be required for flagellar and structural wholeness. In addition, it is also required for morphogenic processes during the spermiogenesis (Chakrabarti et al 2007).…”