Mammal specific protein phosphatase isoform, PPP1CC2, is essential for sperm function and male fertility
Kent State University / OhioLINK, 2012
Hochschulschrift
Zugriff:
Four isoforms of phospho-protein phosphatase 1 (PPP1), PPP1CA (α), PPP1CB (β), PPP1CC1 (γ1), PPP1CC2 (γ2) are derived from three genes in mammals (Ppp1ca, Ppp1cb, Ppp1cc). PPP1CC1 and PPP1CC2 are differentially spliced products of the Ppp1cc gene. All four isoforms are virtually identical (1-298 aa, approximately 90% identity) except at their extreme C-termini. While PPP1CC1, PPP1CA and PPP1CB are ubiquitous, PPP1CC2 is expressed largely in testis and is the sole PPP1 isoform present in spermatozoa. We have previously shown that sperm motility is inversely related to the activity of the protein phosphatase PPP1CC2. Furthermore, targeted disruption of Ppp1cc gene in mice, which eliminates both PPP1CC isoforms, results in male infertility due to lack of spermatogenesis suggesting an indispensible role for the proteins in spermatogenesis and sperm development. Mutant female mice are fertile and normal, presumably since PPP1CA or PPP1CB can substitute for the loss of PPP1CC isoforms. The goal of goal of this dissertation work was to identify the role of PPP1CC isoforms, PPP1CC1 and PPP1CC2 in spermatogenesis and male fertility. We wanted to determine if any one or both of the two PP1 isoforms are essential for spermatogenesis and sperm function. To this end we created transgenic mice lines eTg-G2 (γ2) and pTg-eG2 (γ2) expressing PPP1CC2 driven by either the endogenous promoter of Ppp1cc gene and by the testis specific Pgk2 promoter respectively. Based on in-silico and promoter assay studies a genomic fragment spanning 2.6 kb upstream of the transcription start site was identified as putative endogenous promoter and was thus subsequently used in these experiments. In addition, we also generated a third transgenic mouse line eTg-G1 (γ1) where PPP1CC1 transgene expression was driven by the same endogenous promoter fragment. Parallel to this, we used the Cre-Lox approach to generate germ cell conditional Ppp1cc gene knocdown mice to determine the somatic and germ cell requirement for PPP1CC1 and PPP1CC2.Our analysis of transgenic line eTg-G2 show that Ppp1cc gene promoter is regulated in testis manner and transgene expressed under its control show wild-type pattern of testis predominant PPP1CC2 expression. We have identified putative testis specific factors like Spz1, A-Myb which in combination with Sp1 might regulate the promoter in a testis manner. As expected Pgk2-driven expression of PPP1CC2 was also testis-specific. More importantly, we conclude from our study of the eTg-G2 and the pTg-G2 transgenic lines for the first time that expression of PPP1CC2 alone, via either promoter, is able not only to restore normal spermatogenesis, but the fertility of Ppp1cc null mice as well, provided that transgenic PPP1CC2 expression in testis reaches at least a lower threshold level equivalent to approximately 50% of its expression by a Ppp1cc +/- male. We conclude that the endogenous Ppp1cc promoter normally functions in the testes to maintain a sufficient level of PPP1CC2 expression for normal spermatogenesis to occur, and that production of spermatozoa capable of fertilization in vivo can take place in the complete absence of PPP1CC1 expression. We also conclude from our analysis of the germ cell conditional knockout mice that a reduction in testis levels of PPP1CC2 results in reduced in sperm numbers, increased proportion of abnormal sperm and consequently male infertility. It also demonstrated for the first time the dispensability of Ppp1cc gene in testicular sertoli cells and pre-meiotic germ cells. From the study of eTg-G1 transgenic lines we conclude that PPP1CC1 when forced to express in developing germ cells though able to partially restore spermatogenesis cannot restore fertility due to lack of normal sperm morphogenesis and motility. However further studies are in progress to confirm this observation.A potential implication of my dissertation work is that one of the causes of infertility in men could be low expression or activity levels of PPP1CC2 in testis and spermatozoa. Thus PPP1CC2 activity/levels could be used as clinical diagnostic marker to assess male fertility.
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Mammal specific protein phosphatase isoform, PPP1CC2, is essential for sperm function and male fertility
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Autor/in / Beteiligte Person: | Sinha, Nilam |
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Veröffentlichung: | Kent State University / OhioLINK, 2012 |
Medientyp: | Hochschulschrift |
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