The imprinted polycomb group gene Sfmbt2 is required for trophoblast maintenance and placenta development.

Imprinted genes play important roles in placenta development and function. Parthenogenetic embryos, deficient in paternally expressed imprinted genes, lack extra-embryonic tissues of the trophoblast lineage. Parthenogenetic trophoblast stem cells (TSCs) are extremely difficult to derive, suggesting that an imprinted gene(s) is necessary for TSC establishment or maintenance. In a candidate study, we were able to narrow the list to one known paternally expressed gene, Sfmbt2. We show that mouse embryos inheriting a paternal Sfmbt2 gene trap null allele have severely reduced placentae and die before E12.5 due to reduction of all trophoblast cell types. We infected early embryos with lentivirus vectors expressing anti-Sfmbt2 shRNAs and found that TSC derivation was significantly reduced. Together, these observations support the hypothesis that loss of SFMBT2 results in defects in maintenance of trophoblast cell types necessary for development of the extra-embryonic tissues, the placenta in particular.

What does genetics tell us about imprinting and the placenta connection?

Genomic imprinting is an epigenetic gene silencing phenomenon that is specific to eutherians in the vertebrate lineage. The acquisition of both placentation and genomic imprinting has spurred interest in the possible evolutionary link for many years. In this review we examine the genetic evidence and find that while many imprinted domains are anchored by genes required for proper placenta development in a parent of origin fashion, an equal number of imprinted genes have no apparent function that depends on imprinting. Examination of recent data from studies of molecular and genetic mechanisms points to a maternal control of the selection and maintenance of imprint marks, reinforcing the importance of the oocyte in the healthy development of the placenta and fetus.

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

The mouse protein phosphatase gene Ppp1cc is essential for male fertility, with mutants displaying a failure in spermatogenesis including a widespread loss of post-meiotic germ cells and abnormalities in the mitochondrial sheath. This phenotype is hypothesized to be responsible for the loss of the testis-specific isoform PPP1CC2. To identify PPP1CC2-interacting proteins with a function in spermatogenesis, we carried out GST pull-down assays in mouse testis lysates. Amongst the identified candidate interactors was the testis-specific protein kinase TSSK1, which is also essential for male fertility. Subsequent interaction experiments confirmed the capability of PPP1CC2 to form a complex with TSSK1 mediated by the direct interaction of each with the kinase substrate protein TSKS. Interaction between PPP1CC2 and TSKS is mediated through an RVxF docking motif on the TSKS surface. Phosphoproteomic analysis of the mouse testis identified a novel serine phosphorylation site within the TSKS RVxF motif that appears to negatively regulate binding to PPP1CC2. Immunohistochemical analysis of TSSK1 and TSKS in the Ppp1cc mutant testis showed reduced accumulation to distinct cytoplasmic foci and other abnormalities in their distribution consistent with the loss of germ cells and seminiferous tubule disorganization observed in the Ppp1cc mutant phenotype. A comparison of Ppp1cc and Tssk1/2 knockout phenotypes via electron microscopy revealed similar abnormalities in the morphology of the mitochondrial sheath. These data demonstrate a novel kinase/phosphatase complex in the testis that could play a critical role in the completion of spermatogenesis.

Tandem affinity purification in transgenic mouse embryonic stem cells identifies DDOST as a novel PPP1CC2 interacting protein.

Members of the PP1 family of protein phosphatases achieve functional diversity through numerous and varied protein-protein interactions. In mammals, there are four PP1 isoforms, the ubiquitously expressed PPP1CA, PPP1CB, and PPP1CC1, and the testis specific splice isoform PPP1CC2. When the mouse Ppp1cc gene is deleted, the only phenotypic consequence is a failure of spermatogenesis in homozygous males. To elucidate the function of the Ppp1cc gene, we sought to identify novel protein-protein interactions. To this end, we have created SBP-3XFLAG-PPP1CC1 and SBP-3XFLAG-PPP1CC2 knock-in mouse embryonic stem cell lines using a gene-trap-based system. Tandem affinity purification using our knock-in cell lines identified 11 significant protein-protein interactions, including nine known PP1 interacting proteins and two additional proteins (ATP5C1 and DDOST). Reciprocal in vitro sedimentation assays confirmed the interaction between PPP1CC2 and DDOST that may have physiological implications in spermatogenesis. Immunolocalization studies revealed that DDOST localized to the nuclear envelope in dissociated spermatogenic cells and persists throughout spermatogenesis. The knock-in system described in this paper can be applied in creating tandem affinity-tagged knock-in embryonic stem cell lines with any gene for which a compatible gene-trap line is available.

Recent acquisition of imprinting at the rodent Sfmbt2 locus correlates with insertion of a large block of miRNAs.

BACKGROUND: The proximal region of murine Chr 2 has long been known to harbour one or more imprinted genes from classic genetic studies involving reciprocal translocations. No imprinted gene had been identified from this region until our study demonstrated that the PcG gene Sfmbt2 is expressed from the paternally inherited allele in early embryos and extraembryonic tissues. Imprinted genes generally reside in clusters near elements termed Imprinting Control Regions (ICRs), suggesting that Sfmbt2 might represent an anchor for a new imprinted domain. RESULTS: We analyzed allelic expression of approximately 20 genes within a 3.9 Mb domain and found that Sfmbt2 and an overlapping non-coding antisense transcript are the only imprinted genes in this region. These transcripts represent a very narrow imprinted gene locus. We also demonstrate that rat Sfmbt2 is imprinted in extraembryonic tissues. An interesting feature of both mouse and rat Sfmbt2 genes is the presence of a large block of miRNAs in intron 10. Other mammals, including the bovine, lack this block of miRNAs. Consistent with this association, we show that human and bovine Sfmbt2 are biallelic. Other evidence indicates that pig Sfmbt2 is also not imprinted. Further strengthening the argument for recent evolution of Sfmbt2 is our demonstration that a more distant muroid rodent, Peromyscus also lacks imprinting and the block of miRNAs. CONCLUSIONS: These observations are consistent with the hypothesis that the block of miRNAs are driving imprinting at this locus. Our results are discussed in the context of ncRNAs at other imprinted loci. Accession numbers for Peromyscus cDNA and intron 10 genomic DNA are [Genbank:HQ416417 and Genbank:HQ416418], respectively.

Loss of protein phosphatase 1c{gamma} (PPP1CC) leads to impaired spermatogenesis associated with defects in chromatin condensation and acrosome development: an ultrastructural analysis.

Human male infertility affects approximately 5% of men, with one-third suffering from testicular failure, likely the result of an underlying genetic abnormality that disrupts spermatogenesis during development. Mouse models of male infertility such as the Ppp1cc knockout mouse display very similar phenotypes to humans with testicular failure. Male Ppp1cc mutant mice are sterile due to disruptions in spermatogenesis that begin during prepubertal testicular development, and continue into adulthood, often resulting in loss of germ cells to the point of Sertoli cell-only syndrome. The current study employs light and electron microscopy to identify new morphological abnormalities in Ppp1cc mutant seminiferous epithelium. This study reveals that germ cells become delayed in their development around stages VII and VIII of spermatogenesis. Loss of these cells likely results in the reduced numbers of elongating spermatids and spermatozoa previously observed in mutant animals. Interestingly, Ppp1cc mutants also display reduced numbers of spermatogonia compared with their wild-type counterparts. Using electron microscopy, we have shown that junction complexes in Ppp1cc mutants are ultrastructurally normal, and therefore do not contribute to the breakdown in tissue architecture seen in mutants. Electron microscopy revealed major acrosomal and chromatin condensation defects in Ppp1cc mutants. Our observations are discussed in the context of known molecular changes in Ppp1cc mutant testes.

Identification of potentially damaging amino acid substitutions leading to human male infertility.

There are a number of known genetic alterations found in men with nonobstructive azoospermia, or testicular failure, such as Y microdeletions and cytogenetic abnormalities. However, the etiology of nonobstructive azoospermia is unknown in the majority of men. The aim of this study was to investigate the possibility that unexplained cases of nonobstructive azoospermia are caused by nonsynonymous single-nucleotide polymorphisms (SNPs) in the coding regions of autosomal genes associated with sperm production and fertility. Using a candidate gene approach based on genetics of male infertility in mice, we resequenced nine autosomal genes from 78 infertile men displaying testicular failure using custom-made next-generation resequencing chips. Analysis of the data revealed several novel heterozygous nonsynonymous SNPs in four of nine sequenced genes in 14 of 78 infertile men. Eight SNPs in SBF1, three SNPs in LIMK2, two SNPs in LIPE, and one SNP in TBPL1 were identified. All of the novel mutations were in a heterozygous configuration, suggesting that they may be de novo mutations with dominant negative properties.

Unique trophoblast chromatin environment mediated by the PcG protein SFMBT2.

Stem/progenitor cells are maintained by a chromatin environment, mediated in part by Polycomb group (PcG) proteins, which depress differentiation. The trophoblast-specific PcG protein SFMBT2 is known to be required for maintenance of trophoblast progenitors. Rather than binding to trophoblast-specific genes repressed in TSC, SFMBT2 is concentrated at chromocentres and regions rich in repetitive elements, specifically LINE sequences and major satellites, suggesting that it is involved in higher-order organization of the trophoblast genome. It is also found enriched at a subset of ncRNAs. Comparison of ChIP-seq datasets for other chromatin proteins reveals several stereotypical distribution patterns, suggesting that SFMBT2 interacts with several different types of chromatin complexes specific to the trophoblast lineage.

The PcG gene Sfmbt2 is paternally expressed in extraembryonic tissues.

Genomic imprinting has dramatic effects on placental development, as has been clearly observed in interspecific hybrid, somatic cell nuclear transfer, and uniparental embryos. In fact, the earliest defects in uniparental embryos are evident first in the extraembryonic trophoblast. We performed a microarray comparison of gynogenetic and androgenetic mouse blastocysts, which are predisposed to placental pathologies, to identify imprinted genes. In addition to identifying a large number of known imprinted genes, we discovered that the Polycomb group (PcG) gene Sfmbt2 is imprinted. Sfmbt2 is expressed preferentially from the paternal allele in early embryos, and in later stage extraembryonic tissues. A CpG island spanning the transcriptional start site is differentially methylated on the maternal allele in e14.5 placenta. Sfmbt2 is located on proximal chromosome 2, in a region known to be imprinted, but for which no genes had been identified until now. This possibly identifies a new imprinted domain within the murine genome. We further demonstrate that murine SFMBT2 protein interacts with the transcription factor YY1, similar to the Drosophila PHO-RC.

Positive regulation of TRAF6-dependent innate immune responses by protein phosphatase PP1-γ.

Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events. Importantly, these activities were found to be critical for cellular innate responses to pathogen challenge and microbial clearance in both mouse macrophages and human monocyte lines. These data indicate that PP1-γ phosphatase activity regulates overall TRAF6 E3 ubiquitin ligase function and promotes NF-κB-mediated innate signaling responses.

The application of proteomic approaches to the study of mammalian spermatogenesis and sperm function.

Spermatogenesis is the process by which terminally differentiated sperm are produced from male germline stem cells. This complex developmental process requires the coordination of both somatic and germ cells through phases of proliferation, meiosis, and morphological differentiation, to produce the cell responsible for the delivery of the paternal genome. With infertility affecting ~ 15% of all couples, furthering our understanding of spermatogenesis and sperm function is vital for improving the diagnosis and treatment of male factor infertility. The emerging use of proteomic technologies has played an instrumental role in our understanding of spermatogenesis by providing information regarding the genes involved. This article reviews existing proteomic literature regarding spermatogenesis and sperm function, including the proteomic characterization of spermatogenic cell types, subcellular proteomics, post-translational modifications, interactomes, and clinical studies. Future directions in the application of proteomics to the study of spermatogenesis and sperm function are also discussed.

Imprinting and extraembryonic tissues-mom takes control.

Genomic imprinting is an epigenetic mechanism that silences one parental allele of a small subset of genes. Many imprinted genes exhibit this property only in extraembryonic tissues-placenta and yolk sac. This has led to the idea that imprinting in mammals coevolved with some aspect of placentation. Nevertheless, many studies of imprinting have ignored the extraembryonic tissues, the yolk sac and its precursor, the primitive endoderm, in particular. The primitive endoderm is involved in very early signaling events during a critical stage in development, gastrulation, during which body plan axes and head process neuroectoderm are established. Improper signaling from primitive endoderm as a result of abnormal expression of imprinted genes has the capacity to effect long-term defects in embryonic/fetal tissues that might hitherto have been overlooked. We discuss these gaps in the knowledge, propose a mechanism for genomic imprinting based on current data, and suggest a line of investigation that will expand our understanding of this unique regulatory mechanism and its impact on development.

Metakaryotic stem cell lineages in organogenesis of humans and other metazoans.

A non-eukaryotic, metakaryotic cell with large, open mouthed, bell shaped nuclei represents an important stem cell lineage in fetal/juvenile organogenesis in humans and rodents. each human bell shaped nucleus contains the diploid human DNA genome as tested by quantitative Feulgen DNA cytometry and fluorescent in situ hybridization with human pan-telomeric, pan-centromeric and chromosome specific probes. From weeks approximately 5-12 of human gestation the bell shaped nuclei are found in organ anlagen enclosed in sarcomeric tubular syncytia. Within syncytia bell shaped nuclear number increases binomially up to 16 or 32 nuclei; clusters of syncytia are regularly dispersed in organ anlagen. Syncytial bell shaped nuclei demonstrate two forms of symmetrical amitoses, facing or "kissing" bells and "stacking" bells resembling separation of two paper cups. Remarkably, DNA increase and nuclear fission occur coordinately. Importantly, syncytial bell shaped nuclei undergo asymmetrical amitoses creating organ specific ensembles of up to eight distinct closed nuclear forms, a characteristic required of a stem cell lineage. Closed nuclei emerging from bell shaped nuclei are eukaryotic as demonstrated by their subsequent increases by extra-syncytial mitoses populating the parenchyma of growing anlagen. From 9-14 weeks syncytia fragment forming single cells with bell shaped nuclei that continue to display both symmetrical and asymmetrical amitoses. These forms persist in the juvenile period and are specifically observed in bases of colonic crypts. Metakaryotic forms are found in organogenesis of humans, rats, mice and the plant Arabidopsis indicating an evolutionary origin prior to the divergence of plants and animals.

A testis specific isoform of endophilin B1, endophilin B1t, interacts specifically with protein phosphatase-1c gamma2 in mouse testis and is abnormally expressed in PP1c gamma null mice.

Male mice homozygous for a null mutation in the protein phosphatase-1c gamma (PP1c gamma) gene are infertile, displaying a severe impairment in spermatogenesis that is not compensated by the presence of PP1c alpha and PP1c beta in mutant testes. A lack of the PP1c gamma2 splice variant seems the most likely cause of the mutant phenotype, as it is the most heavily expressed PP1c gamma isoform in wild type testes. Yeast two-hybrid screening using PP1c gamma2 has identified several new binding partners, including endophilin B1t, a testis enriched isoform of endophilin B1a which differs from the somatic form by virtue of a carboxy terminal deletion spanning the last 10 amino acids. The testis isoform did not show an interaction with PP1c alpha, or with a truncated PP1c gamma2 mutant lacking the unique carboxy terminus. In contrast, somatic endophilin B1a did not interact with any of the PP1c isoforms. Sedimentation and co-immunoprecipitation experiments using native testis proteins verified binding of endophilin B1t to PP1c gamma2. Immunohistochemistry on wild type testis sections revealed a stage specific expression pattern for endophilin that appeared concentrated at discrete puncta throughout the seminiferous epithelium. Punctate endophilin expression in cells adjacent to the lumen was absent in PP1c gamma null mice. Phosphatase assays indicate that chimeric endophilin B1t is able to inhibit recombinant PP1c gamma2 activity toward phosphorylase a while having little effect on the activity of PP1c alpha. A potential role for endophilin B1t in mammalian spermatogenesis is discussed within the context of the PP1c gamma knockout testis phenotype.

Epigenetics and the renaissance of heresy.

Classic neo-Darwinian theory is predicated on the notion that all heritable phenotypic change is mediated by alterations of the DNA sequence in genomes. However, evidence is accumulating that stably heritable phenotypes can also have an epigenetic basis, lending support to the long-discarded notion of inheritance of acquired traits. As many of the examples of epigenetic inheritance are mediated by position effects, the possibility exists that chromosome rearrangements may be one of the driving forces behind evolutionary change by exerting position effect alterations in gene activity, an idea articulated by Richard Goldschmidt. The emerging evidence suggests that Goldschmidt's controversial hypothesis deserves a serious reevaluation.

Teratogenic effects of parthenogenetic cells from LTXBO mice, a strain which develops ovarian teratomas at high frequency.

LTXBO mice develop ovarian teratomas at high frequency. The phenotype of tumour tissues is unusual in that most contain trophoblast elements. Since the tumours are derived from parthenogenetically activated oocytes, they would not be expected to produce trophoblast. The developmental potential of parthenogenetic cells from these mice was tested in aggregation chimeras. No contribution to trophoblast tissues was observed. However, a high incidence of morphological abnormalities was seen, suggesting that the parthenogenetic cells exerted a teratogenic effect.

Increased recombination frequency showing evidence of loss of interference is associated with abnormal testicular histopathology.

Nondisjunction leading to aneuploid gametes has been linked genetically to both increases and decreases in recombination frequency on the aneuploid chromosome. In the present study, we present physical evidence of increased frequency of recombination nodules as measured by Mut-S-like homologue-1 (MLH1) foci on pachytene chromosomes from sterile male mice homozygous for a mutation in the protein phosphatase 1cgamma (PP1cgamma) gene. The pattern of elevated recombination frequency in PP1cgamma mutant spermatocytes is consistent with a loss of interference. Previous studies demonstrated: (1) spermiogenesis is impaired starting at step 8 with a severe reduction in elongating and condensed spermatids; (2) spermatids and sperm exhibit elevated rates of DNA fragmentation; and (3) haploid gametes exhibit elevated levels of aneuploidy. Morphometric analysis of developing testes revealed that the first wave of meiosis proceeds at a normal rate in mutant testes, a surprising result given that the PP1 inhibitor okadaic acid has been shown to accelerate progression of spermatocytes from pachytene to the first meiotic division (MI). Evidence of abnormal testicular histopathology is apparent at 3 weeks, before the appearance of haploid gametes, eliminating the possibility that the mutant phenotype is caused by the presence of abnormal spermatids, but coincident with the appearance of the first set of mid to late pachytene spermatocytes. These observations lead us to conclude that the PP1cgamma mutation causes a complex phenotype, including subtle adverse effects on meiosis, possibly mediated by defective signaling between germ cells and Sertoli cells.

Development to blastocyst is impaired when intracytoplasmic sperm injection is performed with abnormal sperm from infertile mice harboring a mutation in the protein phosphatase 1cgamma gene.

Idiopathic azoospermia, characterized by abnormal spermatogenesis, is commonly treated by performing intracytoplasmic sperm injection (ICSI) with sperm retrieved from testicular biopsies. However, no controlled experiments have been performed using an animal model to assess the efficacy or safety of the procedure. We have performed ICSI with testicular sperm obtained in a similar manner from testes of male mice homozygous for a null mutation in the protein phosphatase 1cgamma gene (PP1cgamma) or those of their wild-type littermates. PP1cgamma mutant testicular sperm are less resistant to sonication than are wild-type sperm and display a range of morphological abnormalities, similar to those reported for testicular sperm from idiopathic azoospermic men. PP1cgamma mutant sperm are unable to support development to the blastocyst stage, resulting in arrested development either before or just after compaction. A comparison of testicular and epididymal sperm from wild-type males revealed that the epididymal sperm caused embryos to fragment at an elevated rate. These results suggest that ICSI with any kind of testicular sperm carries an increased risk of embryo fragmentation and that abnormal testicular sperm has an added risk of embryo wastage at later preimplantation stages.

Identification of the spermatogenic zip protein Spz1 as a putative protein phosphatase-1 (PP1) regulatory protein that specifically binds the PP1cgamma2 splice variant in mouse testis.

The spermatogenic zip protein (Spz1) was originally isolated from a mouse testis library and identified as a novel member of the basic helix-loop-helix family of transcription factors. Here we identify Spz1 as a specific binding partner of the gamma2 catalytic subunit of protein phosphatase-1. Male mice homozygous for a null mutation in the protein phosphatase-1cgamma (PP1cgamma) gene are infertile and display a distinct impairment in spermiogenesis despite the continued presence of closely related PP1c isoforms. Yeast two-hybrid screening using the PP1cgamma2 splice variant has identified Spz1 as an interacting protein and possible mediator of the sterile PP1cgamma mutant phenotype. Spz1 was shown to interact specifically with PP1cgamma2 but did not show an interaction with PP1calpha or with a truncated version of PP1cgamma2 lacking 18 amino acids from the C terminus. Interaction between full-length Spz1 and PP1cgamma2 was verified by co-immunoprecipitation and co-localization experiments in COS-1 cells as well as gel-shift and sedimentation assays using whole testis lysates. Immunohistochemistry on wild type testis sections reveals a stage-specific expression pattern for Spz1 during spermatogenesis that appeared grossly abnormal in the testes of PP1cgamma mutant mice. Phosphatase assays using recombinant PP1c indicate that increasing concentrations of Spz1 are able to inhibit PP1cgamma2 activity while having little effect on the activity of PP1calpha. Furthermore, an interaction between PP1cgamma2 and Spz1 was shown to prevent binding of the latter to the consensus E-box promoter sequence. We propose that the interaction between Spz1 and PP1cgamma2 may be required for proper regulation of spermatogenesis and fertility in males.

Protein phosphatase 1cgamma is required in germ cells in murine testis.

The protein phosphatase 1cgamma (PP1cgamma) gene is required for spermatogenesis. Males homozygous for a null mutation are sterile, and display both germ cell and Sertoli cell defects. As these two cell types are physically and functionally intimately connected in the testis, the question arises as to whether the primary site of PP1cgamma action is in Sertoli cells, germ cells, or both. We generated chimeric males by embryo aggregation to test whether wild type Sertoli cells are capable of rescuing mutant germ cells. To distinguish between the desired XY-XY chimeras and uninformative XX-XY chimeras, we designed an adaptation of the single nucleotide primer extension (SNuPE) assay. None of the XY-XY chimeras sired pups derived from mutant germ cells, indicating that the protein is required in germ cells for production of functional sperm. Analysis of a chimeric testis revealed intermediate phenotypes when compared with PP1cgamma-/- testes, suggestive of cell nonautonomous effects. We conclude that PP1cgamma is required in a cell autonomous fashion in germ cells. There may be an additional cell nonautonomous role played by this gene in testes, possibly mediated by defective signaling between germ cells and Sertoli cells.