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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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Testicular phenotypes related to the expression of PP1γ2.A. Immunofluorescence of PP1γ- (KO), wild-type (WT), and PP1γ2-rescue testis sections probed with anti-PPP1R11 antibody. Negative (−) control = no primary antibody. B. Histological analysis of hematoxalin-stained testis sections from PP1γ- (Null) and PP1γ2-rescue (Rescue) mice demonstrating an antiapoptotic effect of PP1γ2. C. Western blot of testis protein extracts from PP1γ- (left lane), PP1γ2-rescue (center lane), and wild-type (right lane) animals probed with anti-Sds22, anti-PP1γ2, anti-PPP1R11, and anti-actin antibodies. D. Upper panel: PPP1R11 level in PP1γ- spleen is not significantly diminished compared to its level in wild-type spleen, but is significantly diminished in tissues normally expressing PP1γ2 (testis and brain); Lower panel: histogram of immunosignal ratios of PPP1R11 in PP1γ- vs wild-type tissues. E. Northern blot analysis showing that Ppp1r11 mRNA levels are comparable in PP1γ- and wild-type brain and testis, respectively. Actin mRNA levels are used as gel loading control. F. Left panel: PP1γ isoform levels are increased and PPP1R11 levels rise dramatically in PP1α- vs wild-type testis and brain, but PP1γ1 and PPP1R11 levels in PP1α- spleen are unchanged; Right panel: histogram of immunosignal ratios of PPP1R11 in PP1α- vs wild-type tissues.
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pone-0004861-g009: Testicular phenotypes related to the expression of PP1γ2.A. Immunofluorescence of PP1γ- (KO), wild-type (WT), and PP1γ2-rescue testis sections probed with anti-PPP1R11 antibody. Negative (−) control = no primary antibody. B. Histological analysis of hematoxalin-stained testis sections from PP1γ- (Null) and PP1γ2-rescue (Rescue) mice demonstrating an antiapoptotic effect of PP1γ2. C. Western blot of testis protein extracts from PP1γ- (left lane), PP1γ2-rescue (center lane), and wild-type (right lane) animals probed with anti-Sds22, anti-PP1γ2, anti-PPP1R11, and anti-actin antibodies. D. Upper panel: PPP1R11 level in PP1γ- spleen is not significantly diminished compared to its level in wild-type spleen, but is significantly diminished in tissues normally expressing PP1γ2 (testis and brain); Lower panel: histogram of immunosignal ratios of PPP1R11 in PP1γ- vs wild-type tissues. E. Northern blot analysis showing that Ppp1r11 mRNA levels are comparable in PP1γ- and wild-type brain and testis, respectively. Actin mRNA levels are used as gel loading control. F. Left panel: PP1γ isoform levels are increased and PPP1R11 levels rise dramatically in PP1α- vs wild-type testis and brain, but PP1γ1 and PPP1R11 levels in PP1α- spleen are unchanged; Right panel: histogram of immunosignal ratios of PPP1R11 in PP1α- vs wild-type tissues.

Mentions: Because PPP1R11 shows haploid germ cell-restricted expression, we performed an immunohistochemical evaluation of its expression pattern in PP1γ- testis where apoptosis is most pronounced in haploid germ cells [33]. As shown in Figure 9A, the localization of PPP1R11 appeared roughly similar in both PP1γ- and wild-type testis, though fluorescence intensity was significantly reduced in the former, and the depleted spermatids appeared to be disorganized in PP1γ- testis, possibly reflecting the apoptotic state of these cells. However, to determine the effect of the absence of PP1γ2 vs. PP1γ1 on the PP1γ- testicular phenotype, including effects on expression/stability of proteins with which PP1γ2 interacts, we produced PP1γ- mice expressing transgenic PP1γ2 specifically in the testis (PP1γ2-rescue mice; see Materials and Methods). Immunohistochemical staining of sections from PP1γ2-rescue testis suggested a significant recovery of PPP1R11 expression levels (Fig. 9A). Interestingly, a comparison of histological sections from the testis of PP1γ- and PP1γ2-rescue mice revealed that PP1γ2 expression in the mutant testis had an obvious anti-apoptotic effect (Fig. 9B).


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)

Testicular phenotypes related to the expression of PP1γ2.A. Immunofluorescence of PP1γ- (KO), wild-type (WT), and PP1γ2-rescue testis sections probed with anti-PPP1R11 antibody. Negative (−) control = no primary antibody. B. Histological analysis of hematoxalin-stained testis sections from PP1γ- (Null) and PP1γ2-rescue (Rescue) mice demonstrating an antiapoptotic effect of PP1γ2. C. Western blot of testis protein extracts from PP1γ- (left lane), PP1γ2-rescue (center lane), and wild-type (right lane) animals probed with anti-Sds22, anti-PP1γ2, anti-PPP1R11, and anti-actin antibodies. D. Upper panel: PPP1R11 level in PP1γ- spleen is not significantly diminished compared to its level in wild-type spleen, but is significantly diminished in tissues normally expressing PP1γ2 (testis and brain); Lower panel: histogram of immunosignal ratios of PPP1R11 in PP1γ- vs wild-type tissues. E. Northern blot analysis showing that Ppp1r11 mRNA levels are comparable in PP1γ- and wild-type brain and testis, respectively. Actin mRNA levels are used as gel loading control. F. Left panel: PP1γ isoform levels are increased and PPP1R11 levels rise dramatically in PP1α- vs wild-type testis and brain, but PP1γ1 and PPP1R11 levels in PP1α- spleen are unchanged; Right panel: histogram of immunosignal ratios of PPP1R11 in PP1α- vs wild-type tissues.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654099&req=5

pone-0004861-g009: Testicular phenotypes related to the expression of PP1γ2.A. Immunofluorescence of PP1γ- (KO), wild-type (WT), and PP1γ2-rescue testis sections probed with anti-PPP1R11 antibody. Negative (−) control = no primary antibody. B. Histological analysis of hematoxalin-stained testis sections from PP1γ- (Null) and PP1γ2-rescue (Rescue) mice demonstrating an antiapoptotic effect of PP1γ2. C. Western blot of testis protein extracts from PP1γ- (left lane), PP1γ2-rescue (center lane), and wild-type (right lane) animals probed with anti-Sds22, anti-PP1γ2, anti-PPP1R11, and anti-actin antibodies. D. Upper panel: PPP1R11 level in PP1γ- spleen is not significantly diminished compared to its level in wild-type spleen, but is significantly diminished in tissues normally expressing PP1γ2 (testis and brain); Lower panel: histogram of immunosignal ratios of PPP1R11 in PP1γ- vs wild-type tissues. E. Northern blot analysis showing that Ppp1r11 mRNA levels are comparable in PP1γ- and wild-type brain and testis, respectively. Actin mRNA levels are used as gel loading control. F. Left panel: PP1γ isoform levels are increased and PPP1R11 levels rise dramatically in PP1α- vs wild-type testis and brain, but PP1γ1 and PPP1R11 levels in PP1α- spleen are unchanged; Right panel: histogram of immunosignal ratios of PPP1R11 in PP1α- vs wild-type tissues.
Mentions: Because PPP1R11 shows haploid germ cell-restricted expression, we performed an immunohistochemical evaluation of its expression pattern in PP1γ- testis where apoptosis is most pronounced in haploid germ cells [33]. As shown in Figure 9A, the localization of PPP1R11 appeared roughly similar in both PP1γ- and wild-type testis, though fluorescence intensity was significantly reduced in the former, and the depleted spermatids appeared to be disorganized in PP1γ- testis, possibly reflecting the apoptotic state of these cells. However, to determine the effect of the absence of PP1γ2 vs. PP1γ1 on the PP1γ- testicular phenotype, including effects on expression/stability of proteins with which PP1γ2 interacts, we produced PP1γ- mice expressing transgenic PP1γ2 specifically in the testis (PP1γ2-rescue mice; see Materials and Methods). Immunohistochemical staining of sections from PP1γ2-rescue testis suggested a significant recovery of PPP1R11 expression levels (Fig. 9A). Interestingly, a comparison of histological sections from the testis of PP1γ- and PP1γ2-rescue mice revealed that PP1γ2 expression in the mutant testis had an obvious anti-apoptotic effect (Fig. 9B).

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