CFTR is essential for sperm fertilizing capacity and is correlated with sperm quality in humans.

BACKGROUND: Our previous studies have demonstrated the cystic fibrosis transmembrane conductance regulator (CFTR) is important for capacitation and male fertility in mouse and guinea pig spermatozoa. However, the exact function of CFTR on human sperm fertilizing capacity, and correlation with sperm quality has not been established. The present study may shed light on some unexplained male infertility, and on a possible new method for diagnosis of male infertility and strategy for male contraception. METHODS: To assess the effect of CFTR on human sperm fertilizing capacity, we examined sperm capacitation and the acrosome reaction using chlortetracycline staining, analyzed sperm hyperactivation by computer-assisted semen analysis (CASA), measured intracellular cAMP levels using ElA and evaluated sperm penetration of zona-free hamster eggs assay in fertile men. The percentage of spermatozoa expressing CFTR from fertile, healthy and infertile men (mainly teratospermic, asthenoteratospermic, asthenospermic and oligospermic) was conducted by indirect immunofluorescence staining. RESULTS: Progesterone significantly facilitated human sperm capacitation and ZP3 triggered the acrosome reaction, both were significantly inhibited by CFTR inhibitor-172 (CFTRinh-172; 10 nM-1 microM) in a dose-dependent manner. The presence of 100 nM CFTRinh-172 markedly depressed intracellular cAMP levels, sperm hyperactivation and sperm penetration of zona-free hamster eggs. In addition, the percentage of spermatozoa expressing CFTR in the fertile men was significantly higher than healthy and infertile men categories (P < 0.01). CONCLUSIONS: CFTR is essential for human sperm fertilizing capacity and the impairment of CFTR expression in spermatozoa is correlated with a reduction of sperm quality. These results suggest that defective expression of CFTR in human sperm may lead to the reduction of sperm fertilizing capacity.

[Expression of protein kinase C alpha in Chinese hamster ovary cells].

OBJECTIVE: To study the expression of protein kinase C (PKC) alpha subtype in Chinese hamster ovary (CHO) cells. METHODS: The PKC alpha primer pairs were designed based on the GenBank sequence of PKC alpha of a species with the highest homology to Chinese hamster identified using EMBL Data Library Clustalw tool. The sequence coding for PKC alpha, amplified from the CHO cells using RT-PCR, was ligated to the pGEM-T plasmid vector, and the recombinant vector was transformed into E.coli DH5alpha with the positive colones selected by blue/white screening. Restriction enzyme digestion, gel electrophoresis analysis, followed by sequencing of the digestion products were performed for identification of the recombinant. Western blotting was used to analyze the PKC alpha expression in the CHO cells. RESULTS: The presence of PKC alpha mRNA was detected in the CHO cells by RT-PCR. Western blotting also identified PKC alpha expression in the cells. CONCLUSIONS: PKC alpha expression has been identified in the CHO cells, which may facilitate further structural and functional study of PKC alpha and investigation of its role in the intracellular signal transduction pathways.