An evolutionarily conserved ribosome-rescue pathway maintains epidermal homeostasis.

Ribosome-associated mRNA quality control mechanisms ensure the fidelity of protein translation1,2. Although these mechanisms have been extensively studied in yeast, little is known about their role in mammalian tissues, despite emerging evidence that stem cell fate is controlled by translational mechanisms3,4. One evolutionarily conserved component of the quality control machinery, Dom34 (in higher eukaryotes known as Pelota (Pelo)), rescues stalled ribosomes 5 . Here we show that Pelo is required for mammalian epidermal homeostasis. Conditional deletion of Pelo in mouse epidermal stem cells that express Lrig1 results in hyperproliferation and abnormal differentiation of these cells. By contrast, deletion of Pelo in Lgr5-expressing stem cells has no effect and deletion in Lgr6-expressing stem cells induces only a mild phenotype. Loss of Pelo results in accumulation of short ribosome footprints and global upregulation of translation, rather than affecting the expression of specific genes. Translational inhibition by rapamycin-mediated downregulation of mTOR (mechanistic target of rapamycin kinase) rescues the epidermal phenotype. Our study reveals that the ribosome-rescue machinery is important for mammalian tissue homeostasis and that it has specific effects on different stem cell populations.

Heat shock protein A4 ablation leads to skeletal muscle myopathy associated with dysregulated autophagy and induced apoptosis.

BACKGROUND: Molecular chaperones assist protein folding, facilitate degradation of misfolded polypeptides, and thereby maintain protein homeostasis. Impaired chaperone activity leads to defective protein quality control that is implicated in multiple skeletal muscle diseases. The heat shock protein A4 (HSPA4) acts as a co-chaperone for HSP70. Previously, we showed that Hspa4 deletion causes impaired protein homeostasis in the heart. However, its functional role in skeletal muscle has not been explored. METHODS: We performed a comparative phenotypic and biochemical analyses of Hspa4 knockout (KO) mice with wild-type (WT) littermates. RESULTS: HSPA4 is markedly upregulated in regenerating WT muscle in vivo, and in differentiated myoblasts in vitro. Hspa4-KO mice are marked by growth retardation and increased variability in body weight, accompanied by 35% mortality rates during the peri-weaning period. The surviving Hspa4-KO mice experienced progressive skeletal muscle myopathy, characterized by increased number of muscle fibers with centralized nuclei, heterogeneous myofiber size distribution, inflammatory cell infiltrates and upregulation of embryonic and perinatal myosin heavy chain transcripts. Hspa4-KO muscles demonstrated an accumulation of autophagosome-associated proteins including microtubule associated protein1 light chain 3-II (LC3-II) and p62/sequestosome accompanied by increased number of TUNEL-positive nuclei. CONCLUSIONS: Our findings underscore the indispensable role of HSPA4 in maintenance of muscle integrity through contribution in skeletal muscle autophagy and apoptosis, which might provide a novel therapeutic strategy for skeletal muscle morbidities.

Ultra-structure of the sperm head-to-tail linkage complex in the absence of the spermatid-specific LINC component SPAG4.

Tight connection between sperm head and tail is crucial for the transport of the male genome and fertilization. The linkage complex, the sperm head-to-tail coupling apparatus (HTCA), originates from the centrosome and anchors to the nuclear membrane. In contrast to its ultra-structural organization, which is already well known for decades, its protein composition largely still awaits future deciphering. SUN-domain proteins are essential components of a complex that links the cytoskeleton to the peripheral nucleoskeleton, which is the nuclear lamina. Here, we studied the impact of the SUN protein SPAG4/SUN4 on the formation of the HTCA. SPAG4/SUN4 is specifically expressed in haploid male germ cells showing a polarized distribution towards the posterior pole in late spermatids that corresponds to the tail attachment site. SPAG4-deficient male mice are infertile with compromised manchette formation and malformed sperm heads. Nonetheless, sperm tails are present demonstrating dispensability of a proper manchette for their formation. Ultra-structural analyses revealed that the development of the sperm head-to-tail linkage complex in the absence of SPAG4 resembles that in the wild type. However, in SPAG4-deficient sperm, the attachment site is diminished with obvious lateral detachment of the HTCA from the nucleus. Our results thus indicate that SPAG4, albeit not essential for the formation of the HTCA per se, is, nevertheless, required for tightening the sperm head-to-tail anchorage by provoking the correct attachment of the lateral parts of the basal plate to the implantation fossa.

Pelota mediates gonocyte maturation and maintenance of spermatogonial stem cells in mouse testes.

Pelota (Pelo) is an evolutionarily conserved gene, and its deficiency in Drosophila affects both male and female fertility. In mice, genetic ablation of Pelo leads to embryonic lethality at the early implantation stage as a result of the impaired development of extra-embryonic endoderm (ExEn). To define the consequences of Pelo deletion on male germ cells, we temporally induced deletion of the gene at both embryonic and postnatal stages. Deletion of Pelo in adult mice resulted in a complete loss of whole-germ cell lineages after 45 days of deletion. The absence of newly emerging spermatogenic cycles in mutants confirmed that spermatogonial stem cells (SSCs) were unable to maintain spermatogenesis in the absence of PELO protein. However, germ cells beyond the undifferentiated SSC stage were capable of completing spermatogenesis and producing spermatozoa, even in the absence of PELO. Following the deletion of Pelo during embryonic development, we found that although PELO is dispensable for maintaining gonocytes, it is necessary for the transition of gonocytes to SSCs. Immunohistological and protein analyses revealed the attenuation of FOXO1 transcriptional activity, which induces the expression of many SSC self-renewal genes. The decreased transcriptional activity of FOXO1 in mutant testes was due to enhanced activity of the PI3K/AKT signaling pathway, which led to phosphorylation and cytoplasmic sequestration of FOXO1. These results suggest that PELO negatively regulates the PI3K/AKT pathway and that the enhanced activity of PI3K/AKT and subsequent FOXO1 inhibition are responsible for the impaired development of SSCs in mutant testes.

Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double-mutant mice.

Heat shock proteins HSPA4L and HSPA4 are closely related members of the HSP110 family and act as cochaperones. We generated Hspa4l(-/-)Hspa4(-/-) mice to investigate a functional complementarity between HSPA4L and HSPA4 during embryonic development. Hspa4l(-/-)Hspa4(-/-) embryos exhibited marked pulmonary hypoplasia and neonatal death. Compared with lungs of wild-type, Hspa4l(-/-), and Hspa4(-/-) embryos, Hspa4l(-/-)Hspa4(-/-) lungs were characterized by diminished saccular spaces and increased mesenchymal septa. Mesenchymal hypercellularity was determined to be due to an increased cell proliferation index and decreased cell death. A significant increase in expression levels of prosurvival protein B cell leukemia/lymphoma 2 may be the cause for inhibition of apoptotic process in lungs of Hspa4(-/-)Hspa4l(-/-) embryos. Accumulation of glycogen and diminished expression of surfactant protein B, prosurfactant protein C, and aquaporin 5 in saccular epithelium suggested impaired maturation of type II and type I pneumocytes in the Hspa4l(-/-)Hspa4(-/-) lungs. Further experiments showed a significant accumulation of ubiquitinated proteins in the lungs of Hspa4l(-/-)Hspa4(-/-) embryos, indicating an impaired chaperone activity. Our study demonstrates that HSPA4L and HSPA4 collaborate in embryonic lung maturation, which is necessary for adaptation to air breathing at birth.

Embryo implantation failure and other reproductive defects in Ube2q1-deficient female mice.

The ubiquitination process is indispensable for proteome regulation. Three classes of ubiquitin (Ub)-related proteins can be distinguished: E1, E2 and E3. Proteins from the E2 class are responsible for the transfer of Ubls from E1 to the target protein. For this activity, interaction with class E3 ligases is usually required. Ub-conjugating enzyme E2Q 1 (UBE2Q1) belongs to the E2 class of Ub-related enzymes and is demonstrated to be involved in the regulation of membrane B4GALT1 protein. Here, we demonstrate that human UBE2Q1 and mouse Ube2q1 are widely expressed and highly conserved genes. To elucidate the function of UBE2Q1 protein, we generated knockout mouse model. No overt phenotype was detected in UBE2Q1-deficient males, but in mutant females, pleiotropic reproductive defects were observed including altered oestrus cycle, abnormal sexual behaviour and reduced offspring care. Moreover, in the uterus of mutant females, significantly increased embryonic lethality and decreased implantation capacity of homozygous mutant embryos were noticed. We found that Ube2q1 is not expressed in the uterus of non-pregnant females but its expression is up-regulated during pregnancy. Taken together, Ube2q1 is involved in different aspects of female fertility.

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy.

BACKGROUND: In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. RESULTS: We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and ß-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, ß-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. CONCLUSIONS: Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted ß-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.

Targeted disruption of Hspa4 gene leads to cardiac hypertrophy and fibrosis.

Failure of molecular chaperones to direct the correct folding of newly synthesized proteins leads to the accumulation of misfolded proteins in cells. HSPA4 is a member of the heat shock protein 110 family (HSP110) that acts as a nucleotide exchange factor of HSP70 chaperones. We found that the expression of HSPA4 is upregulated in murine hearts subjected to pressure overload and in failing human hearts. To investigate the cardiac function of HSPA4, Hspa4 knockout (KO) mice were generated and exhibited cardiac hypertrophy and fibrosis. Hspa4 KO hearts were characterized by a significant increase in heart weight/body weight ratio, elevated expression of hypertrophic and fibrotic gene markers, and concentric hypertrophy with preserved contractile function. In response to pressure overload, cardiac hypertrophy and remodeling were further aggravated in the Hspa4 KO compared to wild type (WT) mice. Cardiac hypertrophy in Hspa4 KO hearts was associated with enhanced activation of gp130-STAT3, CaMKII, and calcineurin-NFAT signaling. Protein blot and immunofluorescent analyses showed a significant accumulation of polyubiquitinated proteins in cardiac cells of Hspa4 KO mice. These results suggest that the myocardial remodeling of Hspa4 KO mice is due to accumulation of misfolded proteins resulting from impaired chaperone activity. Further analyses revealed a significant increase in cross sectional area of cardiomyocytes, and in expression levels of hypertrophic markers in cultured neonatal Hspa4 KO cardiomyocytes suggesting that the hypertrophy of mutant mice was a result of primary defects in cardiomyocytes. Gene expression profile in hearts of 3.5-week-old mice revealed a differentially expressed gene sets related to ion channels, muscle-specific contractile proteins and stress response. Taken together, our in vivo data demonstrate that Hspa4 gene ablation results in cardiac hypertrophy and fibrosis, possibly, through its role in protein quality control mechanism.

Heat-shock protein HSPA4 is required for progression of spermatogenesis.

Heat-shock protein 110 (HSP110) family members act as nucleotide exchange factors (NEF) of mammalian and yeast HSP70 chaperones during the ATP hydrolysis cycle. In this study, we describe the expression pattern of murine HSPA4, a member of the HSP110 family, during testis development and the consequence of HSPA4 deficiency on male fertility. HSPA4 is ubiquitously expressed in all the examined tissues. During prenatal and postnatal development of gonad, HSPA4 is expressed in both somatic and germ cells; however, expression was much higher in germ cells of prenatal gonads. Analyses of Hspa4-deficient mice revealed that all homozygous mice on the hybrid C57BL/6J×129/Sv genetic background were apparently healthy. Although HSPA4 is expressed as early as E13.5 in male gonad, a lack of histological differences between Hspa4(-/-) and control littermates suggests that Hspa4 deficiency does not impair the gonocytes or their development to spermatogonia. Remarkably, an increased number of the Hspa4-deficient males displayed impaired fertility, whereas females were fertile. The total number of spermatozoa and their motility were drastically reduced in infertile Hspa4-deficient mice compared with wild-type littermates. The majority of pachytene spermatocytes in the juvenile Hspa4(-/-) mice failed to complete the first meiotic prophase and became apoptotic. Furthermore, down-regulation of transcription levels of genes known to be expressed in spermatocytes at late stages of prophase I and post-meiotic spermatids leads to suggest that the development of most spermatogenic cells is arrested at late stages of meiotic prophase I. These results provide evidence that HSPA4 is required for normal spermatogenesis.

Perturbed differentiation of murine embryonic stem cells upon Pelota deletion due to dysregulated FOXO1/ß-catenin signaling.

Differentiation of the embryonic stem cells (ESCs) is regulated by a variety of different signaling pathways. Genetic depletion of murine Pelota gene (Pelo) leads to early embryonic lethality. Here, we aimed at determining the embryonic stage and deciphering the dysregulated signaling pathways affected upon Pelo deletion. We found that development of PELO-null embryos is perturbed between the embryonic days E4.5 and E5.5, at which first differentiation process of ESCs takes place. Molecular analysis revealed enhanced activity of phosphoinositide 3-kinase-protein kinase B/ AKT (PI3K-PKB/AKT) signaling, but nuclear accumulation of forkhead box O1 (FOXO1), and upregulation of the pluripotency-related gene, Oct4, in mutant ESCs cultured under differentiation condition. Despite increased levels of nuclear ß-catenin in PELO-null ESCs as a result of decreased activity of glycogen synthase kinase-3ß, the activity of the canonical wingless (Wnt)/ß-catenin/T-cell factor (TCF) was significantly attenuated as judged by the promoter reporter assay, downregulated Wnt/ß-catenin target genes, and impaired cell proliferation. Interestingly, we demonstrated an increased binding of ß-catenin to FOXO1 in PELO-mutant ESCs cultured under differentiation condition that could explain, on one side, the nuclear accumulation of FOXO1 protein and hence persistent pluripotency of PELO-mutant ESCs, and on the other side, the dysregulated transcriptional activity of ß-catenin/TCF and therefore attenuated PELO-null ESC self-renewal. Taken together, our results strongly suggest that PELO deletion averts ESC differentiation through promoting FOXO1/ß-catenin binding with subsequent dysregulation of FOXO1 and canonical ß-catenin/TCF signaling pathways.

Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton.

BACKGROUND: Pelota (PELO) is an evolutionary conserved protein, which has been reported to be involved in the regulation of cell proliferation and stem cell self-renewal. Recent studies revealed the essential role of PELO in the No-Go mRNA decay, by which mRNA with translational stall are endonucleotically cleaved and degraded. Further, PELO-deficient mice die early during gastrulation due to defects in cell proliferation and/or differentiation. RESULTS: We show here that PELO is associated with actin microfilaments of mammalian cells. Overexpression of human PELO in Hep2G cells had prominent effect on cell growth, cytoskeleton organization and cell spreading. To find proteins interacting with PELO, full-length human PELO cDNA was used as a bait in a yeast two-hybrid screening assay. Partial sequences of HAX1, EIF3G and SRPX protein were identified as PELO-interacting partners from the screening. The interactions between PELO and HAX1, EIF3G and SRPX were confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Furthermore, the PELO interaction domain was mapped to residues 268-385 containing the c-terminal and acidic tail domain. By bimolecular fluorescence complementation assay (BiFC), we found that protein complexes resulting from the interactions between PELO and either HAX1, EIF3G or SRPX were mainly localized to cytoskeletal filaments. CONCLUSION: We could show that PELO is subcellularly localized at the actin cytoskeleton, interacts with HAX1, EIF3G and SRPX proteins and that this interaction occurs at the cytoskeleton. Binding of PELO to cytoskeleton-associated proteins may facilitate PELO to detect and degrade aberrant mRNAs, at which the ribosome is stalled during translation.

Hspa4l-deficient mice display increased incidence of male infertility and hydronephrosis development.

The Hspa4l gene, also known as Apg1 or Osp94, belongs to the HSP110 heat shock gene family, which includes three genes encoding highly conserved proteins. This study shows that Hspa4l is expressed ubiquitously and predominantly in the testis. The protein is highly expressed in spermatogenic cells, from late pachytene spermatocytes to postmeiotic spermatids. In the kidney, the protein is restricted to cortical segments of distal tubules. To study the physiological role of this gene in vivo, we generated mice deficient in Hspa4l by gene targeting. Hspa4l-deficient mice were born at expected ratios and appeared healthy. However, approximately 42% of Hspa4l(-/-) male mice suffered from fertility defects. Whereas the seminiferous tubules of Hspa4l(-/-) testes contained all stages of germ cells, the number of mature sperm in the epididymis and sperm motility were drastically reduced. The reduction of the sperm count was due to the elimination of a significant number of developing germ cells via apoptosis. No defects in fertility were observed in female mutants. In addition, 12% of null mutant mice developed hydronephrosis. Concentrations of plasma and urine electrolytes in Hspa4l(-/-) mice were similar to wild-type values, suggesting that the renal function was not impaired. However, Hspa4l(-/-) animals were preferentially susceptible to osmotic stress. These results provide evidence that Hspa4l is required for normal spermatogenesis and suggest that Hspa4l plays a role in osmotolerance.

Fas-associated factor (FAF1) is required for the early cleavage-stages of mouse embryo.

FAF1 was initially isolated as a Fas-associated factor and was subsequently found to interact with a subset of additional proteins that are involved in many cellular events including Fas-mediated apoptosis, heat shock signalling pathways and ubiquitin-dependent processes. Here, we describe that the 74-kDa FAF1 is ubiquitously expressed, while the expression of its post-translational-processed 49-kDa isoform is restricted to post-meiotic male germ cells. In ovary, FAF1 protein is localized predominantly in the cytoplasm of oocytes in all follicle stages. To determine the function of FAF1 in vivo, we analysed a mouse mutant line in which a gene trap vector was inserted in the Faf1 locus. The mutation disrupts the Faf1 and leads to lethality of the Faf1(GT/GT) embryos near the 2-cell stage. Analysis of FAF1 expression revealed that the protein is present in early preimplantation stages, while embryonic expression of Faf1 mRNA becomes appreciable at 4-cell stage. These results indicate that the death of Faf1(GT/GT) at the 2-cell stage may coincide with the depletion of maternal FAF1 in these embryos. Thus, our results indicate that the FAF1 gene product is necessary for early embryonic development.

Directed overexpression of insulin in Leydig cells causes a progressive loss of germ cells.

The primary goal of this study was to determine the 5'region of the Insl3 gene that specifically targets the expression of human insulin to Leydig cells, and to explore whether the testicular proinsulin is efficiently processed to insulin that is able to rescue the diabetes in different mouse models of diabetes. We show here that the sequence between nucleotides -690 and +4 of mouse Insl3 promoter is sufficient to direct the Leydig cell-specific expression of the human insulin transgene (Insl3-hIns). We also found that the 3'untranslated region (3'UTR) of Insl3 was effective in enhancing transgene expression of the insulin in vivo. Expression analysis revealed that the temporal expression pattern of the hIns transgene in Leydig cells of transgenic testes is roughly the same as that of the endogenous Insl3. Despite the Leydig cells translate human proinsulin and secrete a significant level of free C-peptide into the serum, the Leydig cell-derived insulin is not able to overcome the diabetes in different mouse models of diabetes, suggesting a lack of glucose sensing mechanisms in the Leydig cells. A consequence of overexpression of the human proinsulin in Leydig cells was the decrease of fertility of transgenic males at older ages. Germ cells in transgenic males were able to initiate and complete spermatogenesis. However, there was a progressive and age-dependent degeneration of the germ cells that lead to male infertility with increasing age.

Reduction of spermatogenesis but not fertility in Creb3l4-deficient mice.

Creb3l4 belongs to the CREB/ATF family of transcription factors that are involved in mediating transcription in response to intracellular signaling. This study shows that Creb3l4 is expressed at low levels in all organs and in different stages of embryogenesis but is present at very high levels in the testis, particularly in postmeiotic male germ cells. In contrast to CREB3L4 in the human prostate, of which specific expression was detected, Creb3l4 transcripts in the mouse prostate could be detected only by RT-PCR. To identify the physiological function of Creb3l4, the murine gene was inactivated by replacement with the gene encoding green fluorescent protein. Surprisingly, Creb3l4-deficient mice were born at expected ratios, were healthy, and displayed normal long-term survival rates. Despite a significant reduction in the number of spermatozoa in the epididymis of Creb3l4(-)(/)(-) mice, the breeding of mutant males with wild-type females was productive and the average litter size was not significantly altered in comparison to wild-type littermates. Further analyses revealed that the seminiferous tubules of Creb3l4(-)(/)(-) mice contained all of the developmental stages, though there was evidence for increased apoptosis of meiotic/postmeiotic germ cells. These results suggest that Creb3l4 plays a role in male germ cell development, but its loss is insufficient to completely compromise the production of spermatozoa.

Relaxin Family Member Insulin-Like Peptide 6 Ameliorates Cardiac Fibrosis and Prevents Cardiac Remodeling in Murine Heart Failure Models.

BACKGROUND: The insulin/insulin-like growth factor/relaxin family represents a group of structurally related but functionally diverse proteins. The family member relaxin-2 has been evaluated in clinical trials for its efficacy in the treatment of acute heart failure. In this study, we assessed the role of insulin-like peptide 6 (INSL6), another member of this protein family, in murine heart failure models using genetic loss-of-function and protein delivery methods. METHODS AND RESULTS: Insl6-deficient and wild-type (C57BL/6N) mice were administered angiotensin II or isoproterenol via continuous infusion with an osmotic pump or via intraperitoneal injection once a day, respectively, for 2 weeks. In both models, Insl6-knockout mice exhibited greater cardiac systolic dysfunction and left ventricular dilatation. Cardiac dysfunction in the Insl6-knockout mice was associated with more extensive cardiac fibrosis and greater expression of fibrosis-associated genes. The continuous infusion of chemically synthesized INSL6 significantly attenuated left ventricular systolic dysfunction and cardiac fibrosis induced by isoproterenol infusion. Gene expression profiling suggests liver X receptor/retinoid X receptor signaling is activated in the isoproterenol-challenged hearts treated with INSL6 protein. CONCLUSIONS: Endogenous Insl6 protein inhibits cardiac systolic dysfunction and cardiac fibrosis in angiotensin II- and isoproterenol-induced cardiac stress models. The administration of recombinant INSL6 protein could have utility for the treatment of heart failure and cardiac fibrosis.

Sox15 is required for skeletal muscle regeneration.

The Sox genes define a family of transcription factors that play a key role in the determination of cell fate during development. The preferential expression of the Sox15 in the myogenic precursor cells led us to suggest that the Sox15 is involved in the specification of myogenic cell lineages or in the regulation of the fusion of myoblasts to form myotubes during the development and regeneration of skeletal muscle. To identify the physiological function of Sox15 in mice, we disrupted the Sox15 by homologous recombination in mice. Sox15-deficient mice were born at expected ratios, were healthy and fertile, and displayed normal long-term survival rates. Histological analysis revealed the normal ultrastructure of myofibers and the presence of comparable amounts of satellite cells in the skeletal muscles of Sox15(-/-) animals compared to wild-type animals. These results exclude the role of Sox15 in the development of satellite cells. However, cultured Sox15(-/-) myoblasts displayed a marked delay in differentiation potential in vitro. Moreover, skeletal muscle regeneration in Sox15(-/-) mice was attenuated after application of a crush injury. These results suggest a requirement for Sox15 in the myogenic program. Expression analyses of the early myogenic regulated factors MyoD and Myf5 showed the downregulation of the MyoD and upregulation of the Myf5 in Sox15(-/-) myoblasts. These results show an increased proportion of the Myf5-positive cells and suggest a role for Sox15 in determining the early myogenic cell lineages during skeletal muscle development.

Disruption of the pelota gene causes early embryonic lethality and defects in cell cycle progression.

Mutations in either the Drosophila melanogaster pelota or pelo gene or the Saccharomyces cerevisiae homologous gene, DOM34, cause defects of spermatogenesis and oogenesis in Drosophila, and delay of growth and failure of sporulation in yeast. These phenotypes suggest that pelota is required for normal progression of the mitotic and meiotic cell cycle. To determine the role of the pelota in mouse development and progression of cell cycle, we have established a targeted disruption of the mouse PELO: Heterozygous animals are variable and fertile. Genotyping of the progeny of heterozygous intercrosses shows the absence of Pelo(-/-) pups and suggests an embryo-lethal phenotype. Histological analyses reveal that the homozygous Pelo deficient embryos fail to develop past day 7.5 of embryogenesis (E7.5). The failure of mitotic active inner cell mass of the Pelo(-/-) blastocysts to expand in growth after 4 days in culture and the survival of mitotic inactive trophoplast indicate that the lethality of Pelo-null embryos is due to defects in cell proliferation. Analysis of the cellular DNA content reveals the significant increase of aneuploid cells in Pelo(-/-) embryos at E7.5. Therefore, the percent increase of aneuploid cells at E7.5 may be directly responsible for the arrested development and suggests that Pelo is required for the maintenance of genomic stability.

Asthenozoospermia in mice with targeted deletion of the sperm mitochondrion-associated cysteine-rich protein (Smcp) gene.

The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp(-/-) female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcp-deficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp(-/-) mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.

Pelota Regulates Epidermal Differentiation by Modulating BMP and PI3K/AKT Signaling Pathways.

The depletion of evolutionarily conserved pelota protein causes impaired differentiation of embryonic and spermatogonial stem cells. In this study, we show that temporal deletion of pelota protein before epidermal barrier acquisition leads to neonatal lethality due to perturbations in permeability barrier formation. Further analysis indicated that this phenotype is a result of failed processing of profilaggrin into filaggrin monomers, which promotes the formation of a protective epidermal layer. Molecular analyses showed that pelota protein negatively regulates the activities of bone morphogenetic protein and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in the epidermis. To address whether elevated activities of bone morphogenetic protein and PI3K/AKT signaling pathways were the cause for the perturbed epidermal barrier in Pelo-deficient mice, we made use of organotypic cultures of skin explants from control and mutant embryos at embryonic day 15.5. Inhibition of PI3K/AKT signaling did not significantly affect the bone morphogenetic protein activity. However, inhibition of bone morphogenetic protein signaling caused a significant attenuation of PI3K/AKT activity in mutant skin and, more interestingly, the restoration of profilaggrin processing and normal epidermal barrier function. Therefore, increased activity of the PI3K/AKT signaling pathway in Pelo-deficient skin might conflict with the dephosphorylation of profilaggrin and thereby affect its proper processing into filaggrin monomers and ultimately the epidermal differentiation.