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The piwi-like 2 (piwil2) gene is widely expressed in tumors and protects cells from apoptosis induced by a variety of stress stimuli. However, the role of Piwil2 in Fas-mediated apoptosis remains unknown. Here, we present evidence that Piwil2 inhibits Fas-mediated apoptosis. By a bacterial two-hybrid screening, we identify a new Piwil2-interacting partner, keratin 8 (K8), a major intermediate filament protein protecting the cell from Fas-mediated apoptosis. Our results show that Piwil2 binds to K8 and p38 through its PIWI domain and forms a Piwil2/K8/P38 triple protein-protein complex. Thus, Piwil2 increases the phosphorylation level of K8 Ser-73 and then inhibits ubiquitin-mediated degradation of K8. As a result, the knockdown of Piwil2 increases the Fas protein level at the membrane. In addition to our previous finding that Piwil2 inhibits the expression of p53 through the Src/STAT3 pathway, here we demonstrate that Piwil2 represses p53 phosphorylation through p38. Our present study indicates that Piwil2 plays a role in Fas-mediated apoptosis for the first time and also can affect p53 phosphorylation in tumor cells, revealing a novel mechanism of Piwil2 in apoptosis, and supports that Piwil2 plays an active role in tumorigenesis.

The mouse testis-enriched Znf230 gene, which encodes a type of RING finger protein, is present primarily in the nuclei of spermatogonia, the acrosome and the tail of spermatozoa. To investigate the role of Znf230 in spermatogenesis, we generated Znf230-deficient mice by disrupting Znf230 exon-5 and exon-6 using homologous recombination. The homozygous Znf230-knockout (KO) mice did not exhibit Znf230 mRNA expression and Znf230 protein production. Znf230 KO mice exhibited no obvious impairment in body growth or fertility. Male Znf230 KO mice had integral reproductive systems and mature sperm that were regular in number and shape. The developmental stages of male germ cells of Znf230 KO mice were also normal. We further examined variations in the transcriptomes of testicular tissue between Znf230 KO and wild-type mice through microarray analysis. The results showed that the mRNA level of one unclassified transcript 4921513I08Rik was increased and that the mRNA levels of three other transcripts, i.e., 4930448A20Rik, 4931431B13Rik and potassium channel tetramerisation domain containing 14(Kctd14), were reduced more than two-fold in Znf230 KO mice compared with wild-type mice. Using our current examination techniques, these findings suggested that Znf230 deficiency in mice may not affect growth, fertility or spermatogenesis.

Tissue-specific gene expression is regulated by epigenetic modification involving trans-acting factors. Here, we identified that the human MAGEB16 gene and its mouse homolog, Mageb16, are only expressed in the testis. To investigate the mechanism governing their expression, the promoter methylation status of these genes was examined in different samples. Two CpG islands (CGIs) in the 5' upstream region of MAGEB16 were highly demethylated in human testes, whereas they were methylated in cells without MAGEB16 expression. Similarly, the CGI in Mageb16 was hypomethylated in mouse testes but hypermethylated in other tissues and cells without Mageb16 expression. Additionally, the expression of these genes could be activated by treatment with the demethylation agent 5'-aza-2'-deoxycytidine (5'-aza-CdR). Luciferase assays revealed that both gene promoter activities were inhibited by methylation of the CGI regions. Therefore, we propose that the testis-specific expression of MAGEB16 and Mageb16 is regulated by the methylation status of their promoter regions. [BMB Reports 2014; 47(2): 86-91]

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.

Testis gene Znf230 may play a role in mammalian spermatogenesis according to previous reports. Deleting 5 0 important exons to block the formation of protein was a routine way in gene-knockout experiments. To investigate the physiological function of Znf230 gene, the mutant mice with disrupted exon 2 of Znf230 were generated in this study. Results showed that, mutant Znf230 mice were fertile and showed normal body, genitourinary organs, testes weights, and spermatid number but the litter size of the offspring reduced with unclear reasons. Hematoxylin and eosin staining showed that the testicular tissue of mutant mice was intact. Reverse transcriptase polymerase chain reaction analysis showed that two novel mutant transcripts appeared in the mutant mice: the short one including exon-1 and exon-3 to -6, the long one unexpectedly containing a partial sequence from the pPNT vector acting as a new exon 2. Bioinformatic analysis of the long transcript revealed that it might code a 24-kDa N-terminal mutant protein with the same 182 amino acids as that of the wild-type Znf230 in the C-terminus, indicating that the potential functional region of C3HC4-type RING finger was intact in mutant protein. Western blot and immunohistochemistry analyses also implied that this Nterminal mutation of Znf230 might not disrupt the possible role that wild-type Znf230 played in spermatogenesis. In summary, a potential exon structure in the targeting vector sequence involved in the expression of targeting Znf230 gene and disturbed the strategy of this gene-targeting experiment.

Transfection of plasmid vectors coexpressing short hairpin RNA (shRNA) for focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) significantly inhibited protein level of FAK and EGFR. Knockdown of FAK and EGFR expression significantly inhibited cell proliferation and induced cell apoptosis of A549 lung cancer cells in vitro. In A549 subcutaneous xenograft model, mice treated for 3 weeks with plasmid that coexpresses FAK and EGFR shRNA had significantly smaller tumors than those in control mice (P

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.

Piwi (P-element-induced wimpy testis) proteins have been shown to play important roles in maintenance of germ line stem cells, germ cell proliferation and differentiation, and control of Piwi-interacting RNAs (PiRNAs). PiRNAs comprise a broad class of small noncoding RNAs that function as an endogenous defense system against transposable elements. Fibroblast growth factor (Fgf) signals, mediated partly by no tail gene (ntl), are responsible for patterning embryo and mesoderm formation. To understand the function of Piwi proteins, we used zebrafish as a model system. In zebrafish, piwi-like 2 gene (piwil2) is also required for germ cell differentiation and meiosis. Here we report that piwil2 knockdown is able to inhibit the expression of fibroblast growth factor 8a (fgf8a). In contrast, injection with piwil2 mRNA enhances fgf8a expression. Knockdown of piwil2 reduces the inductive effect of fgf8a on dorsalized phenotype, in which embryos extend to an oval shape at the end of epiboly stage. Coinjection with fgf8a and piwil2 mRNAs led to more seriously dorsalized phenotype than coinjection with fgf8a mRNA and piwil2-cMO. In addition, knockdown of piwil2 inhibits the inductive effect of fgf8a on ntl, whereas overexpression of piwil2 enhances the inductive effect of fgf8a on ntl. We also demonstrate that piwil2 positively regulates ntl expression at bud stage, while piwil2 negatively regulates ntl expression at 24 hours post-fertilization. Thus, the functional consequences of piwil2 expression vary during early development of zebrafish embryo. Taken together, we suggest that zebrafish piwil2 is a mediator of Fgf signals in gastrula period.

Using the method of isolation of specific nucleic acids associated with proteins (SNAAP), we have identified 10 candidate target mRNA substrates bound by mT-STAR (mouse T-STAR protein) from testis extract. Among them, our study focused on Fabp9, a gene that is essential for male gametogenesis, and showed that mT-STAR could directly bind to Fabp9 mRNAs. The binding sites are in a short sequence of the coding region and 3' untranslated region of Fabp9 mRNA. These suggest that mT-STAR can regulate the metabolism and expression of Fabp9. In conclusion, identification of mT-STAR-bound mRNA substrates might help to illustrate the potential spectrum of the process and provide valuable insight into the biological function of this RNA-binding protein in spermatogenesis.

Genetic variations of the human RETN gene are associated with metabolic phenotypes, including obesity, insulin sensitivity, diabetes, and coronary heart disease (CHD). There are few studies of two gene variants, -394CG and -420CG, in Chinese population. This study investigated the distribution of RETN gene, single nucleotide polymorphisms (SNPs), in Chinese Han population and the association of the polymorphisms with type 2 diabetes mellitus (T2DM) and CHD. In a population-based, case-control genetic association study, a total of 961 subjects were recruited from the community, including 318 T2DM patients, 273 CHD patients, and 370 unrelated healthy control individuals. Serum lipid levels were detected. Two SNPs of RETN gene, -394CG and -420CG, were genotyped by PCR-RFLP. Unknown Polymorphisms were screened with the technique of denaturing high performance liquid chromatography (DHPLC). The frequencies of RETN -394G allele in T2DM group, CHD group, and control group were 0.3066, 0.3555, and 0.3481, respectively, which are met with the Hardy-Weinberg equilibrium. There is a significant difference of the comparison of sex in T2DM group of RETN gene SNP-394CG (P0.05). Compared with controls, there was no significant difference in the distribution of genotypes and allele frequencies of -394CG polymorphic site in T2DM patients and CHD patients, respectively. No direct association was found between the -394CG polymorphism and T2DM or CHD. The frequencies of RETN -420G allele in T2DM group, CHD group, and control group were 0.4009, 0.3725, and 0.3859, respectively, which are met with the Hardy-Weinberg equilibrium. The frequencies of RETN -420G allele in T2DM groups and control groups of Chinese population are significantly different from those in European population (0.40 vs. 0.27, 0.39 vs. 0.26) (P0.01). Compared with controls, there was no significant difference in distribution of genotypes and allele frequencies of -420CG polymorphic site in T2DM patients and CHD patients, respectively. No direct association was found between the -420CG polymorphism and T2DM or CHD. In addition, we found new potential SNP +593GC in exon 3 of RETN gene using DHPLC. The RETN gene exhibits sex and ethnic differences. +593GC of RETN gene might be a new potential SNP in exon 3 of RETN gene. Association between SNP -394CG and -420CG of RETN gene with T2DM and CHD in Chinese needs more exploration.

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.

The authors investigated the possible association of −4522C/T variation of adiponectin gene with coronary heart disease (CHD) and type 2 diabetes mellitus (T2DM). Genotyping of SNP −4522C/T in 304 patients with CHD, 389 patients with T2DM, and 405 age and sex-matched healthy control subjects was carried out by means of PCR-RFLP approach. No significant difference in the genotype or allele frequencies was found, either between patients with CHD and control subjects, or between patients with T2DM and control subjects. However, in the subgroup analysis, an association of the T/T genotype and T allele with type 2 diabetes combined with obesity (BMI ≥ 25 kg/m 2 ) was found ( P = 0.014 and P = 0.034, respectively). Also the homeostasis model assessment of insulin resistance (HOMA-IR) in T2DM patients with T/T genotype was significantly higher than that in T2DM patients carrying C allele ( P = 0.0069). The authors' findings for the first time demonstrated that SNP −4522 in the adiponectin gene was associated with T2DM that combined with obesity and higher insulin resistance index in patients with T2DM. This indicated that the variation might associate with an increased susceptibility to type 2 diabetic obesity and insulin resistance. But −4522C/T polymorphism did not contribute to the susceptibility of CHD.

Coronary atherosclerotic heart disease (CAD) is a multifactorial disorder resulting from numerous gene-gene and gene-environment interactions. Lecithin:cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport and the metabolism of high-density lipoprotein (HDL), is thought to be a candidate gene related to dyslipidemia and CAD. Variations in the LCAT gene were investigated in 190 CAD patients and 209 age- and gender-matched controls by denaturing high-performance liquid chromatography, and confirmed by sequencing and RFLP assay. In CAD patients, a novel single-nucleotide polymorphism (P143L) in exon 4 of the LCAT gene was discovered in nine males and two females (frequency of 5.79%), which was found in none of 209 controls. The genotype and allele distribution of P143L is significantly (P

Spondyloepiphyseal Dysplasia Tarda (SEDT; MIM 313400) is a rare genetically heterogeneous disorder of vertebral and epiphyseal growth resulting in disproportionally short-trunked short stature, barrel-shaped chest, and dysplasia of the large joints. It is caused by the mutations of SEDL gene. The distinctive radiological signs and the X-linked mode of inheritance make it easy to diagnose. Here a four-generation Chinese SEDT family has been analyzed and the disease-causing mutation has been found. After polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis and DNA sequencing, a previously unreported deletion of T in exon 5 of SEDL gene (i.e. 293delT) was observed and seven individuals in the family carried the mutation. It results in frameshift and a putative truncated protein with the 97 N-terminal amino acids, and 9 changed amino acids. Therefore, loss of function of the gene could be predicted. However, this mutation has not been detected in 50 age and sex matched unrelated controls.

Autosomal dominant polycystic kidney disease (ADPKD) is a common and severe renal disease. Mutations of PKD1 and PKD2 genes are responsible for approximately 85% and 15% of ADPKD cases, respectively. In the present study, PKD1 and PKD2 genes were analyzed in a large Chinese family with ADPKD using denaturing high-performance liquid chromatography and DNA sequencing. A novel mutation, c.3623-3624insGTGT in exon 15 of the PKD1 gene, was identified in all nine affected family members, but not in any unaffected consanguineous relatives or 100 unrelated controls. These findings suggest that the unique 4 bp insertion, c.3623-3624insGTGT, in the PKD1 gene might be the pathogenic mutation responsible for the disease in this family.

The zinc finger gene family represents one of the largest in the mammalian genome, with several of these genes reported to be involved in spermatogenesis. A newly discovered gene has been identified that is expressed abundantly in the testicular tissue of fertile men as determined by mRNA differential display. The gene encodes a C3HC4-type zinc finger protein motif (ring finger motif) consistent with a role in pre-meiotic or post-meiotic sperm development. The gene was named ZNF230 and mapped to the short arm of chromosome 11 (11p15). ZNF230 has two transcripts, of 1kb and 4.4kb in length. The shorter 1kb transcript was only detected in testicular tissue whereas the longer 4.4kb transcript was not detected in testis but was found in several other tissues. The lack of detectable ZNF230 expression in azoospermic patients by reverse transcriptase-mediated PCR analysis is interpreted to mean that this gene is involved in maintaining normal human male fertility.

As members of the same gene family, tumor suppressor genes P16/CDKN2/INK4A and P15/INK4B have a high degree of structural and functional homology with both P16 and P15 proteins involved directly in the regulation of cell cycles. However, the status of P16 and P15 genes in primary maxillofacial squamous cell carcinomas (MSCC) has not been reported. Studies on abnormalities of these genes including homozygous deletion, methylation of the 5 ′ CpG islands, and mutations were carried out in 65 primary MSCC with polymerase chain reaction (PCR), methylation-specific PCR (MSP), PCR-SSCP (single-strand conformation polymorphism), and DNA sequencing techniques. Of the 65 tumors, 22 (34%) were methylated; 7 (11%) displayed point mutations. The total frequency of alteration of the P16 gene was 43% (28/65). The methylation rate of P15 was 12% (8/65). No homozygous deletion was found in either the P16 gene or P15 gene. In all MSCC samples, almost half (49%) harbored an alteration of the P16 or P15 gene. The P16 gene was altered more frequently than P15 , and therefore is inactivated by methylation or mutation in a significant proportion of MSCC. The P15 gene appeared to play a lesser role in tumorigenesis of these tumors.

RNF114 [RING (really interesting new gene) finger protein 114] has been shown to be a novel psoriasis susceptibility gene, with a putative role in the regulation of immune responses, though the underlying mechanism was not fully identified. In the present study, to investigate whether RNF114 is involved in T cell activation, a series of fluorescence activated cell sortings (FACS) were performed. The analysis confirmed that RNF114 over-expression had a promotion effect on T cell activation with an average 43.97% increment and the upregulatory roles showed a dose-dependent effect with 18.44% increment. Interestingly, the two C2H2 domains were shown to play important but opposite roles in T cell activation. The deficiency of upstream C2H2 domain increased the efficiency of T cell activation by 12.81%, while the downstream C2H2 domain alone promoted it with an average level 25.12% higher than intact RNF114 protein. Combined with tandem affinity purification (TAP) and mass spectrometry, our investigations found 23 RNF114-interacting proteins that have distinct physiological roles in transcription, translation, DNA repair and signaling pathways. These findings, including recognition of RNF114 as a positive regulatory protein and identification of its interacting proteins, widen the understanding for investigating functions of RNF114 involved in T cell activation.

AZFc deletions cause a significant phenotypic heterogeneity with respect to spermatogenesis; however, the reason for this is poorly understood. Recently, testis-specific protein Y-encoded 1 (TSPY1) copy number variation (CNV) was determined to be a potential genetic modifier of spermatogenesis. We performed a large-scale cohort study to investigate the effect of TSPY1 CNV on spermatogenesis and to elucidate the possible contribution of TSPY1 genetic variation to the phenotypic expression of AZFc deletions. Haplogrouping of the Y-chromosome and quantification of the TSPY1 copy number were performed in 2272 Han Chinese males with different spermatogenic statuses (704 males with the b2/b4 or gr/gr deletion and 1568 non-AZFc-deleted males). Our data revealed that the TSPY1 copy number distributions were significantly different among non-AZFc-deleted males with different spermatogenic phenotypes. Lower sperm production and an elevated risk of spermatogenic failure were observed in males with fewer than 21 TSPY1 copies and in those with more than 55 copies relative to men with 21-35 copies. Similar results were observed in males with the gr/gr deletion. These findings indicate that TSPY1 CNV affects an individual's susceptibility to spermatogenic failure by modulating the efficiency of spermatogenesis and strongly suggest that there is a significant quantity effect of the TSPY1 copy number on the phenotypic expression of the gr/gr deletion. To our knowledge, this CNV is the first independent genetic factor that has been clearly observed to influence the spermatogenic status of gr/gr deletion carriers. A combined genetic analysis of the TSPY1 copy number and the gr/gr deletion could inform the clinical counselling of infertile couples.

Interleukin-15 (IL15) is a potential immunotherapeutic treatment for cancer. Caspy2 is an active zebra caspase for inducing apoptosis and immune response in murine tumors. In this study, we aim to evaluate the potential of gene therapy using IL15 and Caspy2 against the murine tumors. Plasmid expressing both Caspy2 and IL15 genes was constructed, encapsulated in DOTAP/cholesterol cationic liposome and injected intratumorally into the mice bearing CT26, B16-F10 and 4T1 carcinoma. We found that coexpression of IL15 and Caspy2 could significant inhibit tumor growth and prolong survival of the mice bearing CT26 or B16F10 tumor. A significant reduction in spontaneous lung metastasis was observed in the 4T1 tumor model. In CT26 model, the mice treated with IL15 and Caspy2 acquired a long-time protective immunity against the parental tumor cell rechallenge. Cytotoxic T lymphocytes and terminal deoxynucleotidyltransferase-mediated nick end labelling assays showed that the combination of capsy2 and IL15 could enhance both the apoptosis and immune response induction, which may account for its extraordinary antitumor effect. Furthermore, we showed that the observed tumor suppression by IL15 and Caspy2 concurred with the Caspy2-mediated downregulation of IL10 and upregulation of interferon-γ and tumor necrosis factor-α. Our results therefore suggested that the combination regimen might be a novel and effective strategy for cancer treatment.

Fertilization promoting peptid (FPP) is essential for capacitation and acrosome reaction. The mouse t-complex protein 11 (Tcp11) gene, which encodes the receptor of FPP, plays an important role in fertilization. We had identified three alternative splicing products of its human homologous gene, TCP11, nominated as TCP11a, TCP11b and TCP11c. Their testis-specific expression had been noted, suggesting that TCP11 may play an important role in spermatogenesis and sperm function. In order to explore the function of TCP11, we investigated its expression, subcellular location and binding protein in the sperm. RT-PCR assay shows that all isoforms of TCP11 are present in both human testis and sperm. However, we could only detect the expression of 56-kDa protein, representing TCP11a and TCP11c, but not TCP11b, by western blot analysis. Furthermore, the expression level of 56-kDa TCP11 protein was lower by about threefold in sperm samples containing over 15% of coiled sperms than the level in sperm samples with normal morphology. The coiled sperm, which shows a coiling or bending back of the tail on itself, is associated with infertility. In addition, several TCP11a-binding proteins were isolated using full-length TCP11a as bait. Among them, we focused on outer dense fiber 1 (ODF1), a component of sperm tail outer dense fibers, because outer dense fibers contribute to the distinct morphology and the function of sperm tail. Co-immunoprecipitation assays of sperm cell extracts confirmed that TCP11 protein interacted with ODF1. These results suggest that TCP11 may be responsible for the sperm tail morphology and motility.

Cancer/testis (CT) antigens exhibit highly tissue-restricted expression and are considered promising targets for cancer vaccines. Here we identified a novel CT gene ZNF645 which restrictively expresses in normal human testes and lung cancer patients (68.3%). To investigate the promoter methylation status of ZNF645, we carried out bisulfite genomic sequencing and found that the CpG island in its promoter was heavily methylated in normal lung tissues without the expression of ZNF645, whereas there was high demethylation in normal human testes and lung carcinoma tissues with its expression. Also ZNF645 could be remarkably activated in A549 and HEK293T cells treated by DNA demethylation agent 5`-aza-2`-deoxycytidine. And the dual luciferase assay revealed that the promoter activity of the ZNF645 was inhibited by methylation of the CpG island region. Therefore, we proposed that ZNF645 is a CT gene and activated in human testis and lung cancers by demethylation of its promoter region.

The ZNF313 gene has the highest transcription level in fertile male testes and may be related to human spermatogenesis. The deletion-mutated plasmids of ZNF313 promoter were constructed and transfected into HEK293 cells. The result showed that the fragment from nt -157 to +8 has a basal transcriptional activity. A functional Sp1 binding site was identified by site-directed mutation test and mithramycin A treatment. A 447-bp based at +233 to -213 exhibits a characteristic CpG island, which overlaps with the promoter region. Our work suggests that ZNF313 is controlled at the transcriptional level, and a common mechanism controlling the basal transcription of ZNF313 gene exists.

TSSK6 is a member of the testis-specific serine/threonine kinase family. Male Tssk6 knockout mice are infertile owing to spermatogenic impairment, including sperm count reduction, a decrease in motile sperm number and motility rates, and an increase in the number of sperms with abnormal morphology. We investigated the possible association between variations of the TSSK6 gene and spermatogenic impairment in humans. Mutation screening of TSSK6 was carried out in 519 patients with azoospermia (n = 273) or severe oligozoospermia (n = 246) and in 359 controls with normozoospermia by denaturing high-performance liquid chromatography and DNA sequencing. The frequencies of alleles and genotypes of gene polymorphism were compared between patients and controls. A novel triallelic polymorphism in TSSK6, c.822+126T>G/C, was identified. The frequencies of genotype TT and allele T were increased dramatically in infertile patients compared with controls, whereas genotype TG, allele G and allele C frequencies were significantly higher in controls than in patients. Further study revealed that the allele C frequency of controls was remarkably higher than that of patients with oligospermia. Our findings, for the first time, suggested an association of c.822+126T>G/C in TSSK6 with spermatogenic impairment in humans in which allele T may be a risk factor for male infertility, while alleles C and G may decrease susceptibility to male infertility.

Spermatogenesis is a complex process subject to strict controls at both levels of transcription and translation. It has been proposed that DAZL protein binds to RNA in the cytoplasm of germ cells and controls spermatogenesis. In male mice, loss of Dazl results in numerous defects throughout the mitotic and meiotic process of germ cell development. Tex19.1 also plays an important role during spermatogenesis and Tex19.1(-/-) knockout males exhibit impaired spermatogenesis. Mouse DAZL protein can bind to 3'UTR of mTex19.1 mRNAs and may repress mTex19.1 expression at the translational level. These have been confirmed by both electrophoretic mobility shift assay and translation assay in Zebrafish embryo detecting the luciferase activity. Taken together these data suggest that mDazl may regulate mTex19.1 expression through binding to 3'UTR of mTex19.1 mRNAs in germ cells.

Rbmy gene encodes a RNA-binding protein and its expression is limited to the nuclei of germ cells. Previous studies indicate that RBMY may function in pre-mRNA processing during spermatogenesis, although its precise target mRNAs remain unclear. By using specific nucleic acids associated with proteins and immunoprecipitation techniques, we have identified 12 potential target mRNAs bound by mouse RBMY protein from testis. We detect that both mRbmy-1 and mRbmy-2 transcripts co-exist in mouse testis and they differ mainly in the 5'UTR. Importantly, our result shows that mRBMY protein can bind to one of its own transcripts, mRbmy-2, suggesting that mRBMY may affect alternative splicing or regulate the expression of its own gene. Using electrophoretic mobility shift assay, we demonstrated that mRBMY protein can bind to the testis and sperm-specific spa17 mRNA and that the binding domain contains rich oligo(A), suggesting that mRBMY protein may have high affinity to oligo(A) rich sequences. In conclusion, the identification of RBMY target mRNAs will be helpful to further explore the biological function of RBMY in spermatogenesis.

Udgivelsesdato: 2009-May The ZNF230 gene is a recently cloned gene which is transcribed only in fertile male testes and may be related to human spermatogenesis. To characterize the multiple stage-specific transcription elements necessary for ZNF230 expression, we cloned ZNF230 promoter and constructed chimeric luciferase reporter Plasmids. Overexpression and site-directed mutation test were used to characterize the cis-element. The results showed ZNF230 gene promoter to be GC rich and not contain a TATA box. Deletion analysis of the 5'-flanking region of ZNF230 in HEK293 cells indicated that the sequence encompassing from nt -131 to +152 has a basal transcriptional activity. Site-directed mutation test and mithramycin A treatment demonstrated that the ZNF230 promoter contained a functional Sp1 site. Overexpression of the Sox5 protein activated the promoter activity. A 312-bp fragment surrounding the transcription start site exhibits a characteristic CpG island which overlaps with the promoter region. We also provided evidence that both the human and mouse znf230 promoter consist of Sp1 binding site and GC-rich sequences, suggesting Sp1 is required for the transcription of human and mouse ZNF230 genes. In conclusion, these findings suggest that ZNF230 is tightly controlled at transcriptional level and a common mechanism controls the basal transcription of ZNF230 gene.

The Dazl gene encodes a germ-cell-specific RNA-binding protein which is essential for spermatogenesis. It has been proposed that this protein (DAZL) binds to RNA in the cytoplasm of germ cells and controls spermatogenesis. Using the specific nucleic acids associated with proteins (SNAAP) technique, we identified 17 target mRNAs bound by mDAZL. Among these transcripts, we focused on TSSK2, which encodes a testis-specific serine/threonine kinase. To date, five TSSK family members have been cloned, and all are exclusively expressed in the testis. We demonstrated that in addition to the TSSK1 3'UTR , the 3'UTRs of TSSKs 2 and 4 were bound by human and mouse DAZL, and that human DAZL (hDAZL) bound to the 3'UTR of human TSSK5 (hTSSK5). Our results suggest that the Dazl gene may play different roles in human and mouse spermatogenesis by regulating different members of the downstream gene family. Udgivelsesdato: 2008

Autosomal recessive polycystic kidney disease (ARPKD) is one of the important genetic disorders in pediatric practice. Mutation of the polycystic kidney and hepatic disease gene 1 (PKHD1) was identified as the cause of ARPKD. The gene encodes a 67-exon transcript for a large protein of 4074 amino acids termed fibrocystin, but its function remains unknown. The neoplastic-like in cystic epithelial proliferation and the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis overactivity are known as the most important characteristics of ARPKD. Since the misregulation of Ca(2+) signaling may lead to aberrant structure and function of the collecting ducts in kidney of rat with ARPKD, present study aimed to investigate the further mechanisms of abnormal proliferation of cystic cells by inhibition of PKHD1 expression. For this, a stable PKHD1-silenced HEK-293T cell line was established. Then cell proliferation rates, intracellular Ca(2+) concentration and extracellular signal-regulated kinase 1/2 (ERK1/2) activity were assessed after treatment with EGF, a calcium channel blocker and agonist, verapamil and Bay K8644. It was found that PKHD1-silenced HEK-293T cell lines were hyperproliferative to EGF stimulation. Also PKHD1-silencing lowered the intracellular Ca(2+) and caused EGF-induced ERK1/2 overactivation in the cells. An increase of intracellular Ca(2+) in PKHD1-silenced cells repressed the EGF-dependent ERK1/2 activation and the hyperproliferative response to EGF stimulation. Thus, inhibition of PKHD1 can cause EGF-induced excessive proliferation through decreasing intracellular Ca(2+) resulting in EGF-induced ERK1/2 activation. Our results suggest that the loss of fibrocystin may lead to abnormal proliferation in kidney epithelial cells and cyst formation in ARPKD by modulation of intracellular Ca(2+).

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Recent studies indicate that miRNAs are mechanistically involved in the development of mammalian spermatogenesis. However, little work has been done to compare the miRNA expression patterns between immature and mature mouse testes. Here, we employed a miRNA microarray to detect 892 miRNAs in order to evaluate the expression patterns of miRNA. The expression of 19 miRNAs was significantly different between immature and mature individuals. Fourteen miRNAs were significantly upregulated and five miRNAs were downregulated in immature mice and this result was further confirmed by a quantitative real-time RT-PCR assay. Many target genes involved in spermatogenesis are predicted by MiRscan performing miRNA target scanning. Our data indicated specific miRNAs expression in immature mouse testis and suggested that miRNAs have a role in regulating spermatogenesis.

Chromosomal abnormality and Y chromosome microdeletion are regarded as two frequent genetic causes associated with spermatogenic failure in Caucasian population. To investigate the distribution of the two genetic defects in Chinese patients with azoospermia or severe oligozoospermia, karyotype analysis by G-banding was carried out in 358 idiopathic infertile men, including 256 patients with azoospermia and 102 patients with severe oligozoospermia, and screening of AZF region microdeletion of Y chromosome by multiplex PCR was performed in those patients without detectable chromosomal abnormality and 100 fertile controls. Of 358 patients, 39(10.9%) were found to have chromosomal abnormalities in which Klinefelter's syndrome (47, XXY) was the most common chromosomal aberration. The incidence of sex chromosomal abnormality in patients with azoospermia was significantly higher than that in patients with severe oligozoospermia (12.1% vs 1%). Among the rest of the 319 patients with normal karyotype, 46 (14.4%) were found to have microdeletions in AZF region. The prevalence rates of AZF microdeletion was 15% and 13.1% in patients with azoospermia and severe oligozoospermia respectively. The microdeletion in AZFc was the most frequent deletion and all the microdeletions in AZFa were found in azoospermic patients. No microdeletion in AZF region was detected in fertile controls. In conclusion, chromosomal abnormality and AZF region microdeletion of Y chromosome might account for about 25% of Chinese infertile patients with azoospermia or severe oligozoospermia, suggesting the two abnormalities are important genetic etiology of spermatogenic failure in Chinese population and it is essential to screen them during diagnosis of male infertility before in vitro assisted fertilization by introcytoplasmic sperm injection.

Amyloid beta protein (Abeta) is the principal component of neuritic plaques in Alzheimer's disease (AD). Abeta is derived from beta amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-site APP cleaving enzyme 1 (BACE1) has been identified as the major beta-secretase. BACE2 is the homolog of BACE1. The BACE2 gene is on chromosome 21 and has been implicated in the pathogenesis of AD. However, the function of BACE2 in Abeta generation is controversial. Some studies have shown that BACE2 cleaved APP at the beta-site whereas other studies showed it cleaved around the alpha-secretase site. To elucidate the involvement of BACE2 in AD pathogenesis, we compared BACE2 and BACE1 gene regulation and their functions in Abeta generation. We cloned and functionally characterized the human BACE2 promoter. The BACE2 gene is controlled by a TATA-less promoter. Though Sp1 can regulate both BACE1 and BACE2 genes, comparative sequence analysis and transcription factor prediction showed little similarity between the two promoters. BACE1 increased APP cleavage at the beta-site and Abeta production whereas BACE2 did not. Overexpression of BACE2 significantly increased sAPP levels in conditioned media but markedly reduced Abeta production. Knockdown of BACE2 resulted in increased APP C83. Our data indicate that despite being homologous in amino acid sequence, BACE2 and BACE1 have distinct functions and transcriptional regulation. BACE2 is not a beta-secretase, but processes APP within the Abeta domain at a site downstream of the alpha-secretase cleavage site. Our data argue against BACE2 being involved in the formation of neuritic plaques in AD.

The common polymorphism at codon 311 (C311S) of paraoxonase 2 gene (PON2) was investigated in 165 patients with sporadic late-onset Alzheimer's disease (LOAD) and 174 controls in Chinese. The PON2*C allele frequency was significantly increased in the patients as compared with controls. However, no significant difference was observed after stratification of apolipoprotein E (ApoE) epsilon4 allele. These results suggested that the PON2 polymorphism might be a risk factor for LOAD independent of ApoE epsilon4 status in Chinese.

Complete cDNA of mouse gene znf230 was cloned by rapid amplification of cDNA ends (RACE). This cDNA is 982 base pairs (bp) in length and encodes a 230 amino acids (aa) protein with a ring finger domain at its C-terminus. Ninety-one and 98% nucleotide (nt) and aa sequence identity are observed with its human homolog. Revealed by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR), this cDNA is only detected in testicular tissue, whereas the longer transcripts of 2.4 and 4.4 kb are ubiquitously expressed. The expression of znf230 in testis is developmentally regulated and first detected at day 6 postnatal (pn). It reaches adult level between day 14 and 21 pn during which round spermatids appear in seminiferous tubule. The protein of znf230 exhibits DNA binding activity and its ring finger domain may function as an activator module in transcription. Therefore, it is postulated that znf230 may function as a testis specific transcription factor during mouse spermatogenesis.

Hepatic lipase (HL) is a lipolytic enzyme in- volved in the metabolism of plasma lipoproteins, especially HDLs. Association of the polymorphisms in the promoter region of the LIPC gene to post-heparin plasma HL activity and the plasma HDL-C concentration has been investigated thoroughly, but to date little is known about this in the Chi- nese. In the present study, we analyzed the polymorphisms in the promoter region of LIPC gene in Chinese patients with coronary artery disease (CAD) using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. As the result, a novel single nucleotide poly- morphism � 586T-to-C was identified and no linkage of this variant with other polymorphisms in the promoter was found. Compared with the nonsymptomatic control sub- jects, excess of carriers of the � 586T/C substitution were detected in the CAD patients (43% vs. 31%, � 2 � 4.597, degree of freedom � 2, P � 0.032).The -586C allele carri- ers in the CAD patients had a significantly higher HDL-C level than the noncarriers (1.13 � 0.24 mmol/l vs. 0.91 � 0.14 mmol/l, P � 0.05). To test the functionality of this substitution, luciferase-reporter assays was performed in HepG2 cells. Promoter activity of the � 586C construct was decreased 2-fold than the � 586T construct. Our studies suggest that a T-to-C substitution at � 586 of the LIPC promoter is associated with a lowered HL activity and that this variation may contribute to the increased plasma HDL-C concentration in the Chinese. —Su, Z., S. Zhang, D. W. Nebert, L. Zhang, D. Huang, Y. Hou, L. Liao, and C. Xiao. A novel allele in the promoter of the HL is associated with increased concentration of HDL-C and decreased pro- moter activity. J. Lipid Res. 2002. 43: 1595-1601.

A human testis-specific gene was isolated by subtractive hybridization between the cDNA pools of adult and fetal testes, followed by rapid amplification of cDNA ends (RACE). This gene sequence is highly homologous to a large portion of the mouse Tcp11 gene which is important in sperm function because it encodes the receptor for fertilization-promoting peptide (FPP). The gene was mapped to human chromosome band 6p21 by fluorescence in-situ hybridization. The 9 exon gene spans a 22.8 kp genomic DNA sequence. The mature processed message encodes a 441 amino acid protein that is highly homologous to the mouse 566 amino acid protein after the first 142 amino acids. Results of Northern blot and RT-PCR analyses of RNA extracted from human tissues revealed that the gene is only expressed in fertile adult testes, but not in azoospermic testes, fetal testes nor in other human tissues. Taken together, our results along with the mouse Tcp11 function suggest that TCP11 gene is important in sperm function and fertility.

A novel human KRAB ( Kr uppel a ssociated b ox) type zinc finger protein encoding gene, ZNF463, was obtained by mRNA differential display and RACE. It consists of 1904 nucleotides and encodes a protein of 463 amino acids with an amino-terminal KRAB domain and 12 carboxy-terminal C2H2 zinc finger units. The gene is mapped to chromosome 19q13.3∼4 by FISH. As from Northern blot analysis ZNF463 is only expressed in testis, RT-PCR indicates that ZNF463 is expressed more highly in normal fertile adults than in fetus and azoospermic patients suggesting that it may play a role in human spermatogenesis.

Studies on mutations of BRCA1 gene in Oriental populations, especially in Chinese are sparse. To evaluate the contribution of BRCA1 mutations to sporadic breast cancer in Chinese, mutations in exon 11 from nucleotide positions 3533 to 3682(U14680), a highly variable region of BRCA1 gene, were screened by single-strand conformation polymorphism (SSCP) and DNA sequencing in 329 genomic DNAs from 95 Chinese women with sporadic breast cancer, 29 with benign breast disease and 50 controls. As results, 6 single nucleotide changes were found, and 5 of them (3545A>G, 3551G>T, 3607C>T, 3646T>A and 3661T>C) were newly discovered. The 3545A>G and 3607C>T were germline variations, and the other 3 were somatic variations. In addition, four new haplotypes were defined. Since these newly discovered nucleotide changes were only detected in patients, our results suggest that BRCA1 may also play a role in the development of sporadic breast cancer in Chinese populations. © 2002 Wiley-Liss, Inc.

4 widely used cohesived end-producing restriction enndonucleases (REs), EcoR I, Pst I, Sal I and Bam HI were tested in CHO cells for their aberration-inducing effects. It was demonstrated that all these REs significantly increased the frequencies of aberrant cells, the aberration frequencies per cell and the aberration frequencies per chromosome. The effects of REs on chromosomal aberrations are similar to ionizing radiation, but more minutes and interchange fiqures are observed. Polyploid cells are more susceptible to RE treatment, an interesting finding which may be explained by the mechanisms leading to the formation of polyploid cells.

Chromosomes with various degrees of centromere spreading and completely separated chromatids, as well as metaphasic chromosomes, were observed simultaneously in the same Burkitt's lymphoma cell. In another study, a pair of prematurely and completely separated chromatids was found in a metaphase of nasopharyngeal carcinoma cell. Based on these and other observations, the possible significance of centromere spreading and out-of-phase sister chromatid separation in the origin of chromosome nondisjunction in tumor cells is suggested and discussed.