Histone H3 methylation orchestrates transcriptional program in mouse spermatogenic cell line.

Changes in histone modifications always correlate with altered transcriptional activities of genes. Recent studies have shown that the mutation of certain lysine residues to methionine in the histone variant H3.3 can act as a valuable tool to reduce specific H3 methylation levels. In our study, we used the mouse spermatogenic cell line GC-2 as a model to generate cells stably expressing H3.3 K4, H3.3 K9, H3.3 K27, and H3.3 K36M. The expression of these H3.3 K-to-M mutants influenced the expression of different subsets of genes, and a total of 891 differentially expressed genes were identified through global gene expression profiling. Moreover, the H3.3 K-to-M transgenes, especially H3.3 K36M, impacted the expression of endogenous retrovirus ERVK. This study gives a global view of how different H3 modifications regulate transcriptomes in spermatogenic cell lines, and identifies potential targets of H3 modifications in male germ line.

[Differentially expressed long non-coding RNAs in spermatozoa with different fertilizing abilities].

OBJECTIVE: To search for conception capacity-related long non-coding RNAs (lncRNA) and explore their possible roles in fertilization. METHODS: We obtained 10 semen samples, 5 of high and the other 5 of low fertilizing ability, extracted large RNAs, established a cDNA library, and performed RNA sequencing with the HiSeq 2000 sequencing system. Using the bioinformatics method, we assembled and predicted lncRNAs, screened differentially expressed genes between the two groups by NOIseq, analyzed the lncRNAs with the box plot and volcano plot, and determined their expression patterns by hierarchical cluster analysis. We examined the functional classification of differentially expressed lncRNAs by pathway and gene ontology (GO) enrichment and predicted those of some lncRNAs by lncRNA-mRNA interaction analysis and intersection analysis with up- and down-stream cis-acting elements. RESULTS: A total of 147 1615 lncRNAs were identified in all the semen samples, including 463 596 novel ones and 8 019 known ones, with 4 052 differentially expressed lncRNAs, 985 upregulated and the other 3 067 downregulated. Box plot and volcano plot filtering analyses showed statistically significant differences in the expressions of the lncRNAs between the two groups, and so did hierarchical cluster analysis. GO functional annotations manifested the involvement of the differentially expressed lncRNAs in the metabolic process, biological regulation, membrane and organelle formation, and protein-nucleotide binding. Pathway analysis showed that the differentially expressed lncRNAs were related to transport and catabolism, cell motility, signaling molecular interactions, signaling transduction, and signaling pathways in the development and immune systems. The functions of the 5 lncRNAs predicted were shown to be associated with sperm motility, acrosomal reaction and signal transduction during fertilization. CONCLUSIONS: Differentially expressed lncRNAs may play an important role in fertilization and become biomarkers for the assessment of sperm quality.

Cellular Mechanism Underlying Hydrogen Sulfide Mediated Epithelial K+ Secretion in Rat Epididymis.

As a novel gasotransmitter, hydrogen sulfide (H2S) elicits various physiological actions including smooth muscle relaxation and promotion of transepithelial ion transport. However, the pro-secretory function of H2S in the male reproductive system remains largely unclear. The aim of this study is to elucidate the possible roles of H2S in modulating rat epididymal intraluminal ionic microenvironment essential for sperm storage. The results revealed that endogenous H2S-generating enzymes cystathionine ß-synthetase (CBS) and cystathionine γ-lyase (CSE) were both expressed in rat epididymis. CBS located predominantly in epithelial cells whilst CSE expressed primarily in smooth muscle cells. The relative expression level of CBS and CSE escalated from caput to cauda regions of epididymis, which was paralleled to the progressively increasing production of endogenous H2S. The effect of H2S on epididymal epithelial ion transportation was investigated using short-circuit current (I SC), measurement of intracellular ion concentration and in vivo rat epididymal microperfusion. Our data showed that H2S induced transepithelial K+ secretion via adenosine triphosphate-sensitive K+ (KATP) channel and large conductance Ca2+-activated K+ (BKCa) channel. Transient receptor potential vanilloid 4 (TRPV4) channel-mediated Ca2+ influx was implicated in the activation of BKCa channel. In vivo studies further demonstrated that H2S promoted K+ secretion in rat epididymal epithelium. Inhibition of endogenous H2S synthesis caused a significant decrease in K+ concentration of cauda epididymal intraluminal fluid. Moreover, our data demonstrated that high extracellular K+ concentration actively depressed the motility of cauda epididymal sperm in a pH-independent manner. Collectively, the present study demonstrated that H2S was vital to the formation of high K+ concentration in epididymal intraluminal fluid by promoting the transepithelial K+ secretion, which might contribute to the maintenance of the cauda epididymal sperm in quiescent dormant state before ejaculation.

The reference values for semen parameters of 1213 fertile men in Guangdong Province in China.

Semen samples were collected from 1213 fertile men whose partners had a time-to-pregnancy (TTP) ≤12 months in Guangdong Province in Southern China, and semen parameters including semen volume, sperm concentration, total counts, motility, and morphology were evaluated according to the World Health Organization (WHO) 2010 guideline. All semen parameters analyzed were normal in ~62.2% of the total samples, whereas ~37.8% showed at least one of the semen parameters below normal threshold values. The fifth centiles (with 95% confidence intervals) were 1.3 (1.2-1.5) ml for semen volume, 20 × 10 6 (18×10 6 -20×10 6 ) ml-1 for sperm concentration, 40 × 10 6 (38×10 6 -44×10 6 ) per ejaculate for total sperm counts, 48% (47%-53%) for vitality, 39% (36%-43%) for total motility, 25% (23%-27%) for sperm progressive motility, 5.0% (4%-5%) for normal morphology. The pH values ranged from 7.2 to 8.0 with the mean ± standard deviation at 7.32 ± 0.17. No effects of age and body mass index were found on semen parameters. Occupation, smoking and alcohol abuse, varicocele appeared to decrease semen quality. Sperm concentration, but not sperm morphology, is positively correlated with TTP, whereas vitality is negatively correlated with TTP. Our study provides the latest reference values for the semen parameters of Chinese fertile men in Guangdong Province, which are close to those described in the new WHO guidelines (5 th Edition).

The intervention effect of Rosiglitozone in ovarian fibrosis of PCOS rats.

OBJECTIVE: To explore the Intervention effect of Rosiglitozone in ovarian fibrosis of PCOS rats. METHODS: 60 female SD rats were randomly divided into 3 groups: control group, model group and treatment group. The model and treatment groups were established by subcutaneous injection of DHEA, while the treatment group was given RGZ. The serum hormone values, pathohistology of ovarian structure of rats, ovarian ultrastructure and the expressions of TGF-ß(1) and CTGF were detected. RESULTS: The PCOS model was established successfully. The expression intensity of TGF-ß(1) and CTGF in Oocytes of the PCOS groups was 9.545±2.954 and 9.665±2.400, respectively and was significantly higher than that of the control group 6.636±2.264 and 7.036±2.133; after treatment with rosiglitazone, the expression was significantly decreased 6.980±2.421 and 6.642±2.721 as compared with that of the model group (P<0.05, P<0.001). The values in serum of the PCOS groups were 3.749±2.054 and 0.265±0.129, and 1.914±1.801 and 0.096±0.088 in the control group which had statistically significant difference (P<0.05, P<0.001). After treatment with rosiglitazone, the values were 2.3100±1.825 and 0.112±0.187 and were significantly different with those of the model group (P<0.05, P<0.001). CONCLUSION: TGF-ß(1) and CTGF play an important role in the development of ovary fibrosis in PCOS. However, RGZ may postpone the development of fibrosis by decreasing the levels of TGF-ß(1) and CTGF.

Enhancement of immune responses to hepatitis B vaccine in immunodepressed mice by CpG oligodeoxynucleotide.

AIM: To investigate enhancement of immune response to hepatitis B vaccine in immunosuppressed mice by CpG oligodeoxynucleotide (ODN). METHODS: Immunodepressed C57BL/6 mice caused by cytoxan were given an injection of either hepatitis B vaccine alone or hepatitis B vaccine and CpG ODN into the left tibialis anterior muscle, then the mice were given another shot after 2 weeks using the same formulation. Blood was collected at 5 weeks after immunization and anti-hepatitis B surface antigen IgG and IL-12 levels were measured by ELISA. Spleens of immunized mice were observed under microscope. RESULTS: There was a two-fold increase in anti-hepatitis B surface antigen IgG level when hepatitis B vaccine was mixed with CpG ODN to immunize immunodepressed mice. There was a significant increase in IL-12 level when hepatitis B vaccine was mixed with CpG ODN. Under optical microscope, there were fewer lymphocytes in immunodepressed mice than in normal control group. The proliferation level of spleen lymphocytes in CpG ODN group was elevated compared with that of normal control group and nuclei of lymphocytes became larger. CONCLUSION: CpG ODN can enhance the immune response to hepatitis B vaccine in immunodepressed mice.