Characterization of the protein expression and localization of hnRNP family members during murine spermatogenesis / 亚洲男科学杂志(英文版)

Mammalian testis exhibits remarkably high transcriptome complexity, and spermatogenesis undergoes two periods of transcriptional cessation. These make the RNA-binding proteins (RBPs) the utmost importance during male germ cell development. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RBPs implicated in many steps of RNA processing; however, their roles in spermatogenesis are largely unknown. Here, we investigated the expression pattern of 12 hnRNP family members in mouse testes and found that most detected members are highly expressed in the testis. Furthermore, we found that most of the detected hnRNP proteins (hnRNPD, hnRNPK, hnRNPQ, hnRNPU, and hnRNPUL1) display the highest signals in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells, whereas hnRNPE1 exclusively concentrates in the manchette of elongating spermatids. The expression of these hnRNP proteins showed both similarities and specificity, suggesting their diverse roles in spermatogenesis.

Hyperoside protects mouse spermatocytes GC-2 cells from oxidative damage by activating the Keap1/Nrf2/HO-1 pathway / 南方医科大学学报

OBJECTIVE@#To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (H2O2)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the Keap1/Nrf2/HO-1 pathway in this protective mechanism.@*METHODS@#GC-2 cells were treated with 2.5 mmol/L azaacetylcysteine (NAC), 50, 100, and 200 μmol/L hyperoside, or the culture medium for 48 h before exposure to H2O2 (150 μmol/L) for 2 h. CCK-8 assay was used to detect the changes in cell viability, and cell apoptosis was analyzed using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity and malondialdehyde (MDA) in the culture medium. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of nuclear factor erythroid 2-related factor2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1); the nuclear translocation of Nrf2 was detected using immunofluorescence assay.@*RESULTS@#Exposure to H2O2 significantly lowered the proliferation rate, reduced the activities of SOD, GSH and CAT, and obviously increased MDA content, cell apoptosis rate, and the expressions of Keap1 and Nrf2 mRNA and Keap1 protein in GC-2 cells (P < 0.05 or 0.01). Treatment of the cells prior to H2O2 exposure with either NAC or 200 μmol/L hyperoside significantly increased the cell proliferation rate, enhanced the activities of SOD, GSH-PX and CAT, and lowered MDA content and cell apoptosis rate (P < 0.05). Treatment with 200 μmol/L hyperoside significantly decreased the mRNA and protein expressions of Keap1 and increased the expressions of HO-1 mRNA and the protein expressions of Nrf2 and HO-1 (P < 0.05 or 0.01). Hyperoside also caused obvious nuclear translocation of Nrf2 in the cells (P < 0.05).@*CONCLUSION@#Hyperoside protects GC-2 cells against H2O2- induced oxidative damage possibly by activation of the Keap1/Nrf2/HO-1 signaling pathway.

Chronology of meiosis & synaptonemal complex abnormalities in normal & abnormal spermatogenesis.

Background & objectives: The study was taken up to define criteria of normality for meiosis by assessing the frequency of meiotic prophase cell types, the frequency of pachytene substage in normal and abnormal spermatogenesis and to determine what synaptonemal complex. Methods: A quantitative and qualitative analysis of the first meiotic prophase was performed in 10 patients presenting with non-obstructive infertility and 10 controls, using dual colour immunocytochemistry with SCP3 and BRCA1 which visualise axial elements and synaptonemal complexes (SC). The respective frequencies of the leptotene, zygotene and pachytene stages as well as the frequencies of the four substages of pachytene were evaluated. The frequencies of the main types of meiotic abnormalities at pachytene were also assessed. Results: The frequencies of leptotene and zygotene stages were significantly higher in patients (7.95 and 9.75%) than in controls (2.30 and 1.45%), whereas the frequency of pachytene was significantly higher in controls than in patients (96.25 vs. 75.30%). Detailed analysis of the sex chromosomes revealed that the controls showed a presence of late pachytene substages (P3 + P4 = 64.40%), whereas the patients showed a early pachytene substages (P1 + P2 = 63.40%). From these results, a new index was defined to evaluate spermatogenesis: the Pachytene Index, or PI (PI = P1 + P2 / P1 + P2 + P3 + P4). The same abnormalities (asynapsis, fragmented SC, dotted SC, thin SC) were observed in controls and in patients, but with different frequencies. The most frequent abnormality was fragmented SC, with a significant difference between patients and controls (15.28 vs. 9.74%). There was a significant difference between patients and controls for the frequency of asynapsed nuclei (7.97 vs. 2.95%) while the difference in other abnormalities were not significant. Interpretation & conclusion: The accumulation of early primary spermatocytes is an indication that progression of meiosis is defective in spermatogenesis failures. The value of the PI less than 0.50 indicates that the kinetic of meiosis is normal at pachytene. There is no normal spermatogenesis when the frequency of one or several SC abnormalities is significantly higher than in controls and/ or when the PI is more than 0.50.

The Hsp60C gene in the 25F cytogenetic region in Drosophila melanogaster is essential for tracheal development and fertility.

Earlier studies have shown that of the four genes (Hsp60A, Hsp60B, Hsp60C, Hsp60D genes) predicted to encode the conserved Hsp60 family chaperones in Drosophila melanogaster, the Hsp60A gene (at the 10A polytene region) is expressed in all cell types of the organism and is essential from early embryonic stages, while the Hsp60B gene (at 21D region) is expressed only in testis, being essential for sperm individualization. In the present study, we characterized the Hsp60C gene (at 25F region), which shows high sequence homology with the other three Hsp60 genes of D. melanogaster. In situ hybridization of Hsp60C-specific riboprobe shows that expression of this gene begins in late embryonic stages (stage 14 onwards), particularly in the developing tracheal system and salivary glands; during larval and adult stages, it is widely expressed in many cell types but much more strongly in tracheae and in developing and differentiating germ cells. A P-insertion mutant (Hsp60C(1)) allele with the P transposon inserted at -251 position of the Hsp60C gene promoter was generated. This early larval recessive lethal mutation significantly reduces levels of Hsp60C transcripts in developing tracheae and this is associated with a variety of defects in the tracheal system, including lack of liquid clearance. About 10% of the homozygotes survive as weak, shortlived and completely sterile adults. Testes of the surviving mutant males are significantly smaller, with fewer spermatocytes, most of which do not develop beyond the round spermatid stage. In situ and Northern hybridizations show significantly reduced levels of the Hsp60C transcripts in Hsp60C(1) homozygous adult males. The absence of early meiotic stages in the Hsp60C(1) homozygous testes contrasts with the effect of testis-specific Hsp60B (21D) gene, whose mutation affects individualization of sperm bundles later in spermiogenesis. In view of the specific effects in tracheal development and in early stages of spermatogenesis, it is likely that, besides its functions as a chaperone, Hsp60C may have signalling functions and may also be involved in cation transport across the developing tracheal epithelial cells.