PP1γ2 and PPP1R11 Are Parts of a Multimeric Complex in Developing Testicular Germ Cells in which their Steady State Levels Are Reciprocally Related

Mice lacking the protein phosphatase 1 gamma isoforms, PP1γ1 and PP1γ2, 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 PP1γ2, 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, PP1γ2 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 PP1γ-null testis where mature sperm are virtually absent. Conversely, both mature sperm numbers and the PPP1R11 level increase substantially in PP1γ-null testis expressing transgenic PP1γ2. PPP1R11 also appears to be ubiquitinated in PP1γ-null testis. The levels of PP1γ2 and PPP1R11 were increased in phenotypically normal PP1α-null testis. However, in PP1α-null spleen, where PP1γ2 normally is not expressed, PPP1R11 levels remained unchanged. Our data clearly show a direct reciprocal relationship between the levels of the protein phosphatase isoform PP1γ2 and its regulator PPP1R11, and suggest that complex formation between these polypeptides in testis may prevent proteolysis of PPP1R11 and thus, germ cell apoptosis

Sterility and Gene Expression in Hybrid Males of Xenopus laevis and X. muelleri

BACKGROUND: Reproductive isolation is a defining characteristic of populations that represent unique biological species, yet we know very little about the gene expression basis for reproductive isolation. The advent of powerful molecular biology tools provides the ability to identify genes involved in reproductive isolation and focuses attention on the molecular mechanisms that separate biological species. Herein we quantify the sterility pattern of hybrid males in African Clawed Frogs (Xenopus) and apply microarray analysis of the expression pattern found in testes to identify genes that are misexpressed in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri). METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X. muelleri) were examined using a novel sperm assay that allowed quantification of live, dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm relative to the parental species. Hybrid spermatozoa were larger in size and accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene expression in testes was combined with a correction for sequence divergence derived from genomic hybridizations to identify candidate genes involved in the sterility phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of misexpression. There were only about 140 genes misexpressed in hybrids compared to X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri. CONCLUSIONS/SIGNIFICANCE: Our results provide an important correlation between phenotypic characteristics of sperm and gene expression in sterile hybrid males. The broad pattern of gene misexpression suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome in hybrids. These findings significantly contribute to growing evidence for allelic dominance in hybrids and have implications for the mechanism of species differentiation at the transcriptome level

PP1c2 and PPP1R11 Are Parts of a Multimeric Complex in Developing Testicular Germ Cells in which their Steady State Levels Are Reciprocally Related PP1c2 and PPP1R11 Are Parts of a Multimeric Complex in Developing Testicular Germ Cells in which their Stea

Abstract Mice lacking the protein phosphatase 1 gamma isoforms, PP1c1 and PP1c2, 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 PP1c2, 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, PP1c2 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 PP1c-null testis where mature sperm are virtually absent. Conversely, both mature sperm numbers and the PPP1R11 level increase substantially in PP1c-null testis expressing transgenic PP1c2. PPP1R11 also appears to be ubiquitinated in PP1c-null testis. The levels of PP1c2 and PPP1R11 were increased in phenotypically normal PP1a-null testis. However, in PP1a-null spleen, where PP1c2 normally is not expressed, PPP1R11 levels remained unchanged. Our data clearly show a direct reciprocal relationship between the levels of the protein phosphatase isoform PP1c2 and its regulator PPP1R11, and suggest that complex formation between these polypeptides in testis may prevent proteolysis of PPP1R11 and thus, germ cell apoptosis

A mouse chromosome 17 gene encodes a testes-specific transcript with unusual properties

We have characterized a novel mouse gene (D17Si11) on chromosome 17 that expresses a major transcript observed uniquely in the testes. The D17Si11 locus has been mapped to the central region of chromosome 17 between H-2 and C3. Sequence analysis demonstrates several unusual features of this locus and its transcript: first is the presence of complementary sets of alternating purine and pyrimidine residues within the 3' region of the transcript that could form double-stranded, hairpin-like secondary structures with properties similar to that of Z-DNA; second is the existence of a hypothetical, long open reading frame in the nucleotide strand that is complementary to the testes transcripts. This complementary strand open reading frame is three times the size of the longest potential open reading frame present in the transcript itself. Although a function for D17Si11 has yet to be determined, the gene is relatively nonpolymorphic in mice and appears conserved in mammals