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PP1gamma2 and PPP1R11 are parts of a multimeric complex in developing testicular germ cells in which their steady state levels are reciprocally related.

Cheng L, Pilder S, Nairn AC, Ramdas S, Vijayaraghavan S - PLoS ONE (2009)

Bottom Line: A biochemical approach was employed to learn how expression versus deficiency of PP1gamma2, the predominant PP1 isoform in male germ cells, affects spermatogenesis.Methods used in this study include column chromatography, western blot and northern blot analyses, GST pull-down assays, immunoprecipitation, non-denaturing gel electrophoresis, phosphatase enzyme assays, protein sequencing, and immunohistochemistry.However, in PP1alpha- spleen, where PP1gamma2 normally is not expressed, PPP1R11 levels remained unchanged.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Kent State University, Kent, Ohio, United States of America.

ABSTRACT
Mice lacking the protein phosphatase 1 gamma isoforms, PP1gamma1 and PP1gamma2, are male-sterile due to defective germ cell morphogenesis and apoptosis. However, this deficiency causes no obvious abnormality in other tissues. A biochemical approach was employed to learn how expression versus deficiency of PP1gamma2, the predominant PP1 isoform in male germ cells, affects spermatogenesis. Methods used in this study include column chromatography, western blot and northern blot analyses, GST pull-down assays, immunoprecipitation, non-denaturing gel electrophoresis, phosphatase enzyme assays, protein sequencing, and immunohistochemistry. We report for the first time that in wild-type testis, PP1gamma2 forms an inactive complex with actin, protein phosphatase 1 regulatory subunit 7 (PPP1R7), and protein phosphatase 1 regulatory subunit 11 (PPP1R11), the latter, a potent PP1 inhibitor. Interestingly, PPP1R11 protein, but not its mRNA level, falls significantly in PP1gamma- testis where mature sperm are virtually absent. Conversely, both mature sperm numbers and the PPP1R11 level increase substantially in PP1gamma- testis expressing transgenic PP1gamma2. PPP1R11 also appears to be ubiquitinated in PP1gamma- testis. The levels of PP1gamma2 and PPP1R11 were increased in phenotypically normal PP1alpha- testis. However, in PP1alpha- spleen, where PP1gamma2 normally is not expressed, PPP1R11 levels remained unchanged. Our data clearly show a direct reciprocal relationship between the levels of the protein phosphatase isoform PP1gamma2 and its regulator PPP1R11, and suggest that complex formation between these polypeptides in testis may prevent proteolysis of PPP1R11 and thus, germ cell apoptosis.

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Increasing steady state levels of PPP1R11, PP1γ2 and Sds22 in testis parallel the temporal progression of spermiogenesis.In each experiment, postnatal testis levels of PP1γ2, Sds22, and PPP1R11 were monitored by assessing equal concentrations of protein (25 µg) from extracts prepared from each age group. Proteins were separated by SDS-PAGE, and gels were assayed by western blotting with antibodies against PP1γ2, Sds22, and PPP1R11. Results indicating that postnatal expression of PP1γ2, Sds22, and PPP1R11 increased over the age range in which the initial round of spermiogenesis takes place were visualized by chemiluminescence. A duplicate blot was probed with an anti-actin antibody to demonstrate equal protein loading.
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pone-0004861-g007: Increasing steady state levels of PPP1R11, PP1γ2 and Sds22 in testis parallel the temporal progression of spermiogenesis.In each experiment, postnatal testis levels of PP1γ2, Sds22, and PPP1R11 were monitored by assessing equal concentrations of protein (25 µg) from extracts prepared from each age group. Proteins were separated by SDS-PAGE, and gels were assayed by western blotting with antibodies against PP1γ2, Sds22, and PPP1R11. Results indicating that postnatal expression of PP1γ2, Sds22, and PPP1R11 increased over the age range in which the initial round of spermiogenesis takes place were visualized by chemiluminescence. A duplicate blot was probed with an anti-actin antibody to demonstrate equal protein loading.

Mentions: To gain further insight into possible functional relationships between PPP1R11, Sds22, and PP1γ2 in developing male germ cells, the expression and localization patterns of the three proteins in testis were compared. Western blot analysis showed that significant amounts of Sds22 were present in the testis at days 8 and 18 post partum when little PP1γ2 or PPP1R11 was detectable. However, the steady state levels of PP1γ2, PPP1R11, and Sds22, all rose to their highest levels between days 30 and 45 (Fig. 7). This pattern of high level co-expression correlates closely with a time course over which spermatids differentiate into mature testicular spermatozoa. Consistent with this data, immunohistochemical analysis of testis sections confirmed that Sds22 was relatively more abundant in the cytoplasm of spermatocytes than either PP1γ2 or PPP1R11, with PP1γ2 more pronounced than PPP1R11 in these cells (Figs. 8A, 8B, & 8C). PPP1R11 fluorescence intensity in the cytoplasms of round and elongating spermatids and in spermatozoa confirmed that it is primarily a haploid germ cell-restricted protein (Fig. 8C).


PP1gamma2 and PPP1R11 are parts of a multimeric complex in developing testicular germ cells in which their steady state levels are reciprocally related.

Cheng L, Pilder S, Nairn AC, Ramdas S, Vijayaraghavan S - PLoS ONE (2009)

Increasing steady state levels of PPP1R11, PP1γ2 and Sds22 in testis parallel the temporal progression of spermiogenesis.In each experiment, postnatal testis levels of PP1γ2, Sds22, and PPP1R11 were monitored by assessing equal concentrations of protein (25 µg) from extracts prepared from each age group. Proteins were separated by SDS-PAGE, and gels were assayed by western blotting with antibodies against PP1γ2, Sds22, and PPP1R11. Results indicating that postnatal expression of PP1γ2, Sds22, and PPP1R11 increased over the age range in which the initial round of spermiogenesis takes place were visualized by chemiluminescence. A duplicate blot was probed with an anti-actin antibody to demonstrate equal protein loading.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654099&req=5

pone-0004861-g007: Increasing steady state levels of PPP1R11, PP1γ2 and Sds22 in testis parallel the temporal progression of spermiogenesis.In each experiment, postnatal testis levels of PP1γ2, Sds22, and PPP1R11 were monitored by assessing equal concentrations of protein (25 µg) from extracts prepared from each age group. Proteins were separated by SDS-PAGE, and gels were assayed by western blotting with antibodies against PP1γ2, Sds22, and PPP1R11. Results indicating that postnatal expression of PP1γ2, Sds22, and PPP1R11 increased over the age range in which the initial round of spermiogenesis takes place were visualized by chemiluminescence. A duplicate blot was probed with an anti-actin antibody to demonstrate equal protein loading.
Mentions: To gain further insight into possible functional relationships between PPP1R11, Sds22, and PP1γ2 in developing male germ cells, the expression and localization patterns of the three proteins in testis were compared. Western blot analysis showed that significant amounts of Sds22 were present in the testis at days 8 and 18 post partum when little PP1γ2 or PPP1R11 was detectable. However, the steady state levels of PP1γ2, PPP1R11, and Sds22, all rose to their highest levels between days 30 and 45 (Fig. 7). This pattern of high level co-expression correlates closely with a time course over which spermatids differentiate into mature testicular spermatozoa. Consistent with this data, immunohistochemical analysis of testis sections confirmed that Sds22 was relatively more abundant in the cytoplasm of spermatocytes than either PP1γ2 or PPP1R11, with PP1γ2 more pronounced than PPP1R11 in these cells (Figs. 8A, 8B, & 8C). PPP1R11 fluorescence intensity in the cytoplasms of round and elongating spermatids and in spermatozoa confirmed that it is primarily a haploid germ cell-restricted protein (Fig. 8C).

Bottom Line: A biochemical approach was employed to learn how expression versus deficiency of PP1gamma2, the predominant PP1 isoform in male germ cells, affects spermatogenesis.Methods used in this study include column chromatography, western blot and northern blot analyses, GST pull-down assays, immunoprecipitation, non-denaturing gel electrophoresis, phosphatase enzyme assays, protein sequencing, and immunohistochemistry.However, in PP1alpha- spleen, where PP1gamma2 normally is not expressed, PPP1R11 levels remained unchanged.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Kent State University, Kent, Ohio, United States of America.

ABSTRACT
Mice lacking the protein phosphatase 1 gamma isoforms, PP1gamma1 and PP1gamma2, are male-sterile due to defective germ cell morphogenesis and apoptosis. However, this deficiency causes no obvious abnormality in other tissues. A biochemical approach was employed to learn how expression versus deficiency of PP1gamma2, the predominant PP1 isoform in male germ cells, affects spermatogenesis. Methods used in this study include column chromatography, western blot and northern blot analyses, GST pull-down assays, immunoprecipitation, non-denaturing gel electrophoresis, phosphatase enzyme assays, protein sequencing, and immunohistochemistry. We report for the first time that in wild-type testis, PP1gamma2 forms an inactive complex with actin, protein phosphatase 1 regulatory subunit 7 (PPP1R7), and protein phosphatase 1 regulatory subunit 11 (PPP1R11), the latter, a potent PP1 inhibitor. Interestingly, PPP1R11 protein, but not its mRNA level, falls significantly in PP1gamma- testis where mature sperm are virtually absent. Conversely, both mature sperm numbers and the PPP1R11 level increase substantially in PP1gamma- testis expressing transgenic PP1gamma2. PPP1R11 also appears to be ubiquitinated in PP1gamma- testis. The levels of PP1gamma2 and PPP1R11 were increased in phenotypically normal PP1alpha- testis. However, in PP1alpha- spleen, where PP1gamma2 normally is not expressed, PPP1R11 levels remained unchanged. Our data clearly show a direct reciprocal relationship between the levels of the protein phosphatase isoform PP1gamma2 and its regulator PPP1R11, and suggest that complex formation between these polypeptides in testis may prevent proteolysis of PPP1R11 and thus, germ cell apoptosis.

Show MeSH
Related in: MedlinePlus