Unmethylated state of 5′ upstream CpG islands may be necessary but not sufficient for the testis-enriched expression of ZNF230/Znf230

The testis-enriched genes ZNF230/Znf230 are located on human chromosome 11p15/mouse chromosome 7 near conserved imprinting control regions. Typical CpG islands (CGIs) extend from the promoter to the first exon in each of these genes. To investigate the correlation between the methylation status of the above CGIs and the expression patterns of the two genes, we performed bisulfite genomic sequencing of genomic DNA from human and mouse tissues and cells. The results showed that the CGIs of ZNF230/Znf230 were completely unmethylated in all selected tissues and cells, regardless of the expression levels of the two genes. Further experiments using Znf230-second-exon-knockout mice to investigate the imprinting status of Znf230 showed that its expression was not affected by genomic imprinting. However, an in vitro methylation assay illustrated that the methylation of these CpG sites could repress the expression of the luciferase reporter gene. Furthermore, chromatin immunoprecipitation with anti-Specificity protein 1 (Sp1) antibody showed that Sp1 could bind to the CGIs in the ZNF230/Znf230 gene promoter. Thus, we propose that the unmethylated state of ZNF230/Znf230 CGIs may be a prerequisite for their expression but not sufficient for their abundant expression in the testis, and that Sp1 binding may be one factor involved in preserving the methylation-free state of ZNF230/Znf230 CGIs.     

The testis‐specific gene 1700102P08Rik is essential for male fertility

Male infertility is a rising problem around the world. Often the cause of male infertility is unclear, and this hampers diagnosis and treatment. Spermatogenesis is a complex process under sophisticated regulation by many testis‐specific genes. Here, we report the testis‐specific gene 1700102P08Rik is conserved in both the human and mouse and highly expressed in spermatocytes. To investigate the role of 1700102P08Rik in male fertility, knockout mice were generated by CRISPR‐Cas9. 1700102P08Rik knockout male mice were infertile with smaller testis and epididymis, but female knockout mice retained normal fertility. Spermatogenesis in the 1700102P08Rik knockout male mouse was arrested at the spermatocyte stage, and no sperm were found in the epididymis. The deletion of 1700102P08Rik causes apoptosis in the testis but did not affect the serum concentration of testosterone, luteinizing hormone, and follicle‐stimulating hormone or the synapsis and recombination of homologous chromosomes. We also found that 1700102P08Rik is downregulated in spermatocyte arrest in men. Together, these results indicate that the 1700102P08Rik gene is essential for spermatogenesis and its dysfunction leads to male infertility.     

PLIN1 deficiency affects testicular gene expression at the meiotic stage in the first wave of spermatogenesis

PLIN1, a lipid droplet associated protein, has been implicated in playing a key role in the regulation of lipolysis and lipid storage in adipocytes. PLIN1 is found to be highly expressed in Leydig cells of testis, suggesting a potential role in steroidogenesis and spermatogenesis. In this study, we showed that PLIN1 was expressed in testis and that its mRNA levels declined significantly with development. To investigate the role of PLIN1, we take advantage of PLIN1-null mice. We found that the number of seminiferous tubules containing round spermatids was significantly increased at P21 (postnatal day 21). Furthermore, microarray analysis showed that there were 538 differentially expressed genes between PLIN1-null and wild-type mice at P21. The up-regulated genes in knockout mice were enriched in spermatogenesis by Gene Ontology classification. Among them, Prm1 and Wbp2nl are important for spermatogenesis which were confirmed by real-time PCR. Unexpectedly, the levels of serum testosterone and serum 17β-estradiol as well as steroidogenic genes are not altered in the PLIN1-null mice. Compared to the wild-type mice, no significant difference of fertility was found in the PLIN1-null mice. Therefore, these findings indicated that PLIN1 disruption leads to the increase of round spermatid-containing seminiferous tubules at the meiotic stage of the first wave of spermatogenesis through regulating spermatogenic related genes.     

Rad54 is required for the normal development of male and female germ cells and contributes to the maintainance of their genome integrity after genotoxic stress

Rad54 is an important factor in the homologous recombination pathway of DNA double-strand break repair. However, Rad54 knockout (KO) mice do not exhibit overt phenotypes at adulthood, even when exposed to radiation. In this study, we show that in Rad54 KO mouse the germline is actually altered. Compared with the wild-type (WT) animals, these mice have less premeiotic germ cells. This germ cell loss is found as early as in E11.5 embryos, suggesting an early failure during mutant primordial germ cells development. Both testicular and ovarian KO germ cells exhibited high radiation sensitivity leading to a long-term gametogenesis defect at adulthood. The KO female germline was particularly affected displaying decreased litter size or sterility. Spermatogenesis recovery after irradiation was slower and incomplete in Rad54 KO mice compared with that of WT mice, suggesting that loss of germ stem cell precursors is not fully compensated along the successive rounds of spermatogenesis. Finally, spermatogenesis recovery after postnatal irradiation is in part regulated by glial-cell-line-derived neurotrophic factor (GDNF) in KO but not in irradiated WT mice, suggesting that Sertoli cell GDNF production is stimulated upon substantial germ cell loss only. Our findings suggest that Rad54 has a key function in maintaining genomic integrity of the developing germ cells.     

Contrasting effects of IGF binding protein-3 expression in mammary tumor cells and the tumor microenvironment

IGFBP-3 has both stimulatory and inhibitory effects on cancer progression. The growth of EO771 mammary carcinoma cells as syngeneic tumors in C57BL/6 mice is reduced in Igfbp3-null (BP3KO) mice, suggesting that systemic IGFBP-3 enhances tumor progression. In this study we assessed the growth of EO771 cells expressing human IGFBP-3 in BP3KO mice. Cells expressing hIGFBP-3 showed decreased proliferation in vitro and increased levels of IGF-1 receptor (IGF1R) protein but not mRNA, consistent with sequestration of endogenous IGF by IGFBP-3. The growth rate of these cells was restored by exposure to IGF-1 or analogues with reduced affinity for IGFBP-3 (long Arg³-IGF-1) or IGF1R (Leu²⁴-IGF-1). In EO771 cells implanted orthotopically into mice, hIGFBP-3 expression by the cells inhibited tumor establishment in BP3KO but not wild-type mice. For tumors that successfully established, final weight was not affected significantly by hIGFBP-3 expression. However, final tumor weight was inversely related to intratumoral T cell counts, and sera from BP3KO mice with tumors showed low-titer immunoreactivity against IGFBP-3. The contrasting effects on tumor establishment and progression of IGFBP-3 expressed by mammary carcinoma cells, compared to systemic stromal and circulating IGFBP-3, highlights the complexity of growth regulation by IGFBP-3 in mammary tumors.     

A Novel Testis-Enriched Gene Spata33 Is Expressed during Spermatogenesis

With an increasing incidence of male idiopathic infertility, identification of novel genes involved in spermatogenesis is an important aspect for the understanding of human testicular failure. In the present study, we have identified a novel gene Spata33, also called as 4732415M23Rik or C16orf55, which is conserved in mammalian species. Spata33 was predominantly expressed in the postpartum and adult mouse testes at mRNA and protein levels. Its expression was increased during the first wave of the spermatogenesis, indicating that Spata33 may be associated with the meiotic process. Further immunohistochemistry analysis revealed that Spata33 was mainly expressed in the spermatocytes, spermatogonia and round spermatids. Its expression was uniformly distributed in the nucleus and cytosol in these germ cells, which was further confirmed by Spata33-tagged with GFP staining in the GC-1 and TM4 cells. These results indicated that Spata33 was predominantly expressed in the mouse testis and associated with spermatogenesis. Identification and characterization of the novel testis-enriched gene Spata33 may provide a new route for understanding of spermatogenesis failure.     

Toll-like receptor 9 signaling has anti-inflammatory effects on the early phase of Helicobacter pylori-induced gastritis

Helicobacter pylori (H. pylori)-induced immune responses in the gastric mucosa are skewed toward T helper (Th) 1 phenotype, which is characterized by predominant production of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by helper T cells. Toll-like receptors (TLRs) play an essential role in mucosal defense against microbes through the recognition of bacterial molecules. Among the members of the TLR family, TLR9 recognizes bacterial unmethylated CpG DNA sites, and signal transduction of TLR9 induces production of a variety of cytokines, including type-I IFN (IFN-α/β). We investigated the expression and role of TLR9 in H. pylori-induced gastritis in mice. Expression of TLR9 mRNA in the gastric tissue increased after infection with H. pylori. TLR9 was mainly expressed in the macrophages, dendritic cells, and CD3⁺ cells in the gastric mucosa. Neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA were higher in TLR9 knockout (KO) mice than in wild-type mice at 2 and 4 months after H. pylori inoculation. These differences in inflammatory parameters between H. pylori-infected wild-type and TLR9 KO mice disappeared 6 months after H. pylori inoculation. Expression of interleukin-4 mRNA, typical Th2 cytokine, in the gastric tissue did not differ between H. pylori-infected wild-type and TLR9 KO mice. Expression level of IFN-α/β mRNA in the TLR9 KO mice was lower than that in wild-type mice by 4 months after inoculation. Administration of IFN-α reduced H. pylori infection-induced increase in neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA in TLR9 KO mice. Our findings suggest that TLR9 signaling plays important roles in the suppression of H. pylori-induced gastritis in the early phase via downregulation of Th1-type cytokines modulated by IFN-α.     

Expression and localization of the spermatogenesis-related gene, Znf230, in mouse testis and spermatozoa during postnatal development

Znf230, the mouse homologue of the human spermatogenesis-related gene, ZNF230, has been cloned by rapid amplification of cDNA ends (RACE). This gene is expressed predominantly in testis, but its expression in different testicular cells and spermatogenic stages has not been previously analyzed in detail. In the present study, the cellular localization of the Znf230 protein in mouse testis and epididymal spermatozoa was determined by RT-PCR, immunoblotting, immunohistochemistry and immunofluorescence. It is primarily expressed in the nuclei of spermatogonia and subsequently in the acrosome system and the entire tail of developing spermatids and spermatozoa. The results indicate that Znf230 may play an important role in mouse spermatogenesis, including spermatogenic cell proliferation and sperm maturation, as well as motility and fertilization.     

Excessive Astrocyte-Derived Neurotrophin-3 Contributes to the Abnormal Neuronal Dendritic Development in a Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS) is a form of inherited mental retardation in humans that results from expansion of a CGG repeat in the Fmr1 gene. Recent studies suggest a role of astrocytes in neuronal development. However, the mechanisms involved in the regulation process of astrocytes from FXS remain unclear. In this study, we found that astrocytes derived from a Fragile X model, the Fmr1 knockout (KO) mouse which lacks FMRP expression, inhibited the proper elaboration of dendritic processes of neurons in vitro. Furthermore, astrocytic conditioned medium (ACM) from KO astrocytes inhibited proper dendritic growth of both wild-type (WT) and KO neurons. Inducing expression of FMRP by transfection of FMRP vectors in KO astrocytes restored dendritic morphology and levels of synaptic proteins. Further experiments revealed elevated levels of the neurotrophin-3 (NT-3) in KO ACM and the prefrontal cortex of Fmr1 KO mice. However, the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) were normal. FMRP has multiple RNA–binding motifs and is involved in translational regulation. RNA–binding protein immunoprecipitation (RIP) showed the NT-3 mRNA interacted with FMRP in WT astrocytes. Addition of high concentrations of exogenous NT-3 to culture medium reduced the dendrites of neurons and synaptic protein levels, whereas these measures were ameliorated by neutralizing antibody to NT-3 or knockdown of NT-3 expression in KO astrocytes through short hairpin RNAs (shRNAs). Prefrontal cortex microinjection of WT astrocytes or NT-3 shRNA infected KO astrocytes rescued the deficit of trace fear memory in KO mice, concomitantly decreased the NT-3 levels in the prefrontal cortex. This study indicates that excessive NT-3 from astrocytes contributes to the abnormal neuronal dendritic development and that astrocytes could be a potential therapeutic target for FXS.     

Fat/Vessel-derived Secretory Protein (Favine)/CCDC3 Is Involved in Lipid Accumulation

We previously identified a novel gene encoding Favine/CCDC3 (NCBI protein entry {"type":"entrez-protein","attrs":{"text":"NP_083080","term_id":"58037351","term_text":"NP_083080"}}NP_083080), a possible secretory factor, the mRNA of which is highly expressed in adipose tissue and the aorta. The Favine mRNA levels are increased in the course of differentiation of rat primary adipocytes and are more elevated in the adipose tissue of genetically obese and diet-induced obese mice than in lean mice. However, its biological function has not yet been elucidated until now. Here, we tested the hypothesis that Favine is involved in lipid metabolism in adipocytes. We found that overexpression of Favine promoted 3T3-L1 adipocyte differentiation. To further investigate the function of Favine in vivo, we generated Favine knock-out (KO) mice. Favine KO mice exhibited a lean phenotype as they aged. The weights of white adipose tissue and liver were less, and adipocyte size was smaller in Favine KO mice compared with wild-type littermates (WT). Expression levels of lipogenic genes, such as fatty-acid synthase (FAS), acetyl-CoA carboxylase α (ACC1), and diacylglycerol O-acyltransferase-2 (Dgat2), were decreased in adipose tissue of Favine KO mice. In 1-year-old mice, Favine deficiency decreased the number of inflammatory cells in white adipose tissue and diminished hepatic steatosis. In vitro, deficiency of Favine attenuated differentiation of primary adipocytes. Taken together, these data demonstrate that Favine has adipogenic and lipogenic effects on adipocytes.     

Developmental retardation in neonates of aldehyde reductase (AKR1A)-deficient mice is associated with low ascorbic acid and high corticosterone levels

Aldehyde reductase encoded by the Akr1a gene catalyzes the NADPH-dependent reduction of a variety of aldehyde compounds, and it plays a role in the biosynthesis of ascorbic acid (AsA) by converting D-glucuronate to L-gulonate. Although supplementing drinking water with AsA (1.5 mg/mL) ameliorates the fertility of Akr1a ^−/− (KO) female mice, litter sizes in the KO mice are typically smaller than those for Akr1a ^+/+ (WT) mice, and about one-third of the neonates have a reduced stature. Half of the neonates in the smallest, developmentally retarded group died before weaning, and the remaining half (less than 6 g in weight) also barely grew to adulthood. While no difference was found in the number of fetuses between the KO and WT mice at 14.5-embryonic days, the sizes of the KO fetuses had already diverged. Among the organs of these retarded KO neonates at 30 d, the spleen and thymus were characteristically small. While an examination of spleen cells showed the normal proportion of immune cells, apoptotic cell death was increased in the thymus, which would lead to thymic atrophy in the retarded KO neonates. Plasma AsA levels were lower in the small neonates despite the fact that their mothers had received sufficient AsA supplementation, and the corticosterone levels were inversely higher compared to wild-type mice. Thus, insufficient AsA contents together with a defect in corticosterone metabolism might be the cause of the retarded growth of the AKR1A-deficient mice embryos and neonates.     

Absence of Inhibin Alpha and Retinoblastoma Protein Leads to Early Sertoli Cell Dysfunction

Sertoli cells, the support cells of mammalian spermatogenesis, are regulated by a number of nuclear factors and express retinoblastoma (RB) tumor suppressor protein. We hypothesized that RB is an important mediator of Sertoli cell tumorigenesis in inhibin α knockout (Inha KO) mice. In our previous mouse studies, we found that conditional knockout (cKO) of Rb in Sertoli cells caused progressive Sertoli cell dysfunction. Initially, loss of RB had no gross effect on Sertoli cell function as the mice were fertile with normal testis weights at 6 weeks of age, but by 10–14 weeks of age, mutant mice demonstrated severe Sertoli cell dysfunction and infertility. Although double knockout (dKO) of Rb and Inha did not result in exacerbation of the tumorigenic phenotype of Inha-null mice, we found that the dKO mice demonstrate an acceleration of Sertoli cell dysfunction compared to Rb cKO mice. Specifically, in contrast to Rb cKO mice, Inha/Rb dKO mice showed signs of Sertoli cell dysfunction as early as 4 weeks of age. These results demonstrate that RB is not essential for Sertoli cell tumorigenesis in Inha KO mice but that loss of Inha accelerates the infertility phenotype of Rb cKO mice.     

Deficiency of myotubularin-related protein 14 influences body weight,metabolism, and inflammation in an age-dependent manner

Background

Myotubularin-related protein 14 (MTMR14) is a novel phosphoinositide phosphatase with roles in the maintenance of normal muscle performance, autophagy, and aging in mice. Our initial pilot study demonstrated that MTMR14 knock out (KO) mice gain weight earlier than their wild-type (WT) littermates, which suggests that this gene may also be involved in metabolism regulation.

Results

The present study evaluated the role of MTMR14 in the development of aging-associated obesity. We found that aged MTMR14 KO mice fed a normal chow diet exhibited increased serum triglyceride, total cholesterol, and glucose levels compared to age-matched WT controls. Lipid accumulation was also increased in aged KO mice. Several inflammatory cytokines and adipokines were dramatically dysregulated in the metabolic tissues of aged MTMR14 KO mice compared to control mice. Circulating inflammatory cytokines were significantly elevated and plasma adipokine levels were abnormally regulated in aged MTMR14 KO mice. These data suggest that MTMR14 deficiency caused a late-onset inflammation and metabolic dysfunction. Further study demonstrated that this exacerbated metabolic dysfunction and inflammation may be regulated by the phosphoinositide 3 kinase/protein kinase B and extracellular signal-regulated protein kinase signaling pathways.

Conclusions

Our current research suggests that MTMR14 deletion induces overweight and adult obesity accompanied by chronic inflammation in an age-dependent manner.