Sizhong Zhang

Male infertility is one of the major diseases that affect human health and social life, and is influenced by many genetic and environmental factors. Epigenetic modification on DNA strands in response to environmental factors plays an important role in the process of spermatogenesis. Abnormalities of epigenetic regulation may affect both the quantity and quality of sperm production and result in disorders of male reproduction. We hereby review recent progress made in research on epigenetic regulation including DNA methylation, histone modification and non-coding RNA related with male infertility.

To investigate the temporal and spatial features of mouse Rnf148 gene expression and the function of RING finger domain of Rnf148 protein. The whole RNA was extracted from different tissues of adult mice, embryo in four developmental stages, and testes of postnatal mice respectively. RT-PCR and Northern blotting analysis were used to investigate the expression of Rnf148 gene in the above tissues. The in vitro expression vector for GST-Rnf148 fused protein was constructed, which encompassing the entire RING domain of Rnf148 protein. GST-Rnf148 fused protein was expressed in Escherichia coli. BL21(DE3) cells and purified with glutathione-sepharose 4B. In vitro ubiquitination assay was performed to analyze whether GST-Rnf148 fused protein possess the function of E3 ubiquitin ligase. The Mice Rnf148 mRNA expression was only observed in testis, and Northern blotting confirmed that there was only one 1.2 kb mRNA band present in mice testis. Rnf148 mRNA started to appear in the testis of day 21 mice, and then increased dramatically and reached to the highest level in day 25, and continued to express thereafter. GST-Rnf148 fused protein was induced and purified, in vitro ubiquitination reaction showed that the recombinant protein has E3 ubiquitin ligase activity. Rnf148 gene is specifically expressed in mice testis.

Testis gene Znf230 may play a role in mammalian spermatogenesis according to previous reports. Deleting 5′ 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 N-terminal 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.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disease with an incidence of 1 in 400 to 1000. The disease is genetically heterogeneous, with two genes identified: PKD1 (16p13.3) and PKD2 (4q21). Molecular diagnosis of the disease in at-risk individuals is complicated due to the structural complexity of PKD1 gene and the high diversity of the mutations. This study is the first systematic ADPKD mutation analysis of both PKD1 and PKD2 genes in Chinese patients using denaturing high-performance liquid chromatography (DHPLC). Methods: Both PKD1 and PKD2 genes were mutation screened in each proband from 65 families using DHPLC followed by DNA sequencing. Novel variations found in the probands were checked in their family members available and 100 unrelated normal controls. Then the pathogenic potential of the variations of unknown significance was examined by evolutionary comparison, effects of amino acid substitutions on protein structure, and effects of splice site alterations using online mutation prediction resources. Results: A total of 92 variations were identified, including 27 reported previously. Definitely pathogenic mutations (ten frameshift, ten nonsense, two splicing defects and one duplication) were identified in 28 families, and probably pathogenic mutations were found in an additional six families, giving a total detection level of 52.3% (34/65). About 69% (20/29) of the mutations are first reported with a recurrent mutation rate of 31%. Conclusions: Mutation study of PKD1 and PKD2 genes in Chinese Hans with ADPKD may contribute to a better understanding of the genetic diversity between different ethnic groups and enrich the mutation database. Besides, evaluating the pathogenic potential of novel variations should also facilitate the clinical diagnosis and genetic counseling of the disease.

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.

AJA publishes new findings in basic and clinical (including modern, traditional and epidemiological) research on Andrology.

Tssk2, a member of the testis‐specific serine/threonine kinase (TSSK) family, is expressed predominantly in the testis and is crucial for the formation and function of sperm cells in the mouse. Targeted deletion of Tssk1 and Tssk2 in male chimeric mice caused infertility because of haploinsufficiency of the genes. Therefore, it is reasonable to postulate that mutations in the human homolog TSSK2 gene may also play a role in impaired spermatogenesis in humans. To explore the possible association between mutations in the TSSK2 gene and idiopathic infertility in humans, mutation screening of the gene in 494 patients with azoospermia or severe oligozoospermia and 357 fertile controls was performed using denaturing high‐performance liquid chromatography and DNA sequencing. As a result, 4 single‐nucleotide transitions were identified, including c.80A>G (rs3747052), c.774C>T (rs1052756), c.839C>T (rs1052763), and c.1026G>A (rs1052773). Among them, significant differences in polymorphism frequencies were observed of c.80A>G (rs3747052) and c.774C>T (rs1052756) between the patients and controls; the allele G of c.80A>G (rs3747052) and allele T of c.774C>T (rs1052756) seem to be risk factors for the development of spermatogenic impairment, suggesting that the TSSK2 gene may be associated with male idiopathic infertility in humans.

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. [BMB reports 2010; 43(6): 400-406]

Summary: Recent studies have revealed that AZF deletion in Y chromosome is the most common known molecular genetic cause of spermatogenetic failure leading to male infertility. Characteristics of AZFa, AZFb and AZFc deletions and their association with spermatogenic impairment have been reported in a large number of populations. However, the distributions of those larger deletions resulted from P5/proximal‐P1, P5/distal‐P1 and P4/distal‐P1 recombinations are still unclear as the literature on their frequencies is limited, and the contribution of these deletions to spermatogenetic failure remain to be confirmed by population studies. In this study, we investigated such massive deletions in 387 idiopathic azoospermic, 269 oligozoospermic patients and 315 men with normal spermatogenesis using 21 AZFb/c specific sequence‐tagged sites. As a result, nine uninterrupted massive deletions were observed exclusively in men with azoospermia caused by either Sertoli cell only (SCO) or maturation arrest (MA). Prevalence of the deletion was 2.33%, in which five deletions arose from non‐allelic homologous recombination between the palindromes P5 and P1 and two between P4 and P1. In other two deletions, novel proximal breakpoints in the interval region between P4 and P3 were observed. Our findings strongly support that the massive deletions in the AZFb or AZFb+c regions are important genetic causes of SCO and/or MA resulting in azoospermia and, besides the P5/proximal‐P1, P5/distal‐P1 and P4/distal‐P1 deletions there may be other massive deletions in these regions resulting in severe spermatogenic impairment.