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

Human Reproduction, Vol.25, No.2 pp. 317–327, 2010 Advanced Access publication on November 18, 2009 doi:10.1093/humrep/dep406 ORIGINAL ARTICLE Andrology CFTR is essential for sperm fertilizing capacity and is correlated with sperm quality in humans Chu-Yan Li 1,3†, Ling-Ying Jiang 2†, Wen-Ying Chen1†, Kun Li1, Hui-Qiang Sheng4, Ya Ni1, Jian-Xin Lu 3, Wan-Xiang Xu 5, Song-Ying Zhang 2,6, and Qi-Xian Shi1,6 1 Unit of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013, China 2Assisted Reproductive Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310016, China 3Department of Laboratory Medicine, Wenzhou Medical College, Wenzhou, Zhejiang 325035, China 4The Sperm Bank of Zhejiang Province, Hangzhou, Zhejiang 310012, China 5Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China 6 Correspondence address. Tel: +86-571-86006028; Fax: +86-571-86044817; E-mail: (S-Y.Z)/Tel: þ86-57188215476; Fax: þ86-571-88075447; E-mail: (Q.-X.S.) 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 mM) 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. Key words: CFTR / human sperm / fertilizing capacity / sperm quality Introduction Cystic fibrosis transmembrane conductance regulator (CFTR) is known to act as a cAMP-activated Cl2 channel, mutations of which are responsible for cystic fibrosis (CF), a common hereditary disease characterized by defective Cl2 and HCO 3 transport with clinical manifestations of progressive lung disease, pancreatic insufficiency and infertility in both sexes (Quinton, 1990, 1999; Rowe et al., 2005; Chan et al., 2006). Almost 97% CF male patients are infertile due to congenital bilateral absence of the vas deferens (CBAVD) with resultant obstructive azoospermia (Wong, 1998; Cuppens and Cassiman, 2004). Other causes of azoospermia include isolated anomalies of the seminal vesicles, congenital unilateral absence of the vas deferens (Meschede et al., 1997) and Young’s syndrome (Le Lannou et al., 1995). The anatomical defect may be traced back to the embryonic stage, indicating a possible requirement of CFTR or CFTR-mediated anion secretion for normal development of the male genital tract. The absence of sperm in the ejaculatory duct of CF males has led us to consider possible involvement of CFTR in † CY, Li, LY, Jiang and WY, Chen, contributed equally to this work. & The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: 318 normal human sperm function. Moreover when spermatozoa from CF patients with CBAVD are used for intracytoplasmic sperm injection, fertilization rates are not reduced, suggesting a specific defect in zona pellucida penetration or membrane fusion capacity in these spermatozoa (Silber et al., 1994). Meanwhile, it is unclear whether CFTR mutations cause a reduction of sperm quality. Some investigators have provided evidence for this (van der Ven et al., 1996; Jakubiczka et al., 1999; Dohle et al., 2002), although others have argued against it (Jakubiczka et al., 1999; Mak et al., 2000; Stuhrmann and Dork, 2000; Ravnik-Glavac et al., 2001). Testicular biopsies of some CF men have revealed a decreased number of mature spermatids in the seminiferous epithelium of the testes, and many of the testicular spermatozoa are malformed. Importantly, van der Ven et al. (1996) reported that 17.5% healthy men with infertility due to the reduction of sperm quality and 14.3% men with azoospermia have at least one mutation in the CFTR gene. These observations strongly suggest the involvement of CFTR in spermatogenesis, although Mak et al. (2000) did not detect the CFTR mutations in infertile men with primary testicular failure. To date, the literature suggests that CFTR gene mutations in healthy men may cause a reduction of sperm quality, and this prompted us to hypothesize that CFTR plays an important role in human sperm fertilizing capacity and is correlated with sperm quality. It has been reported that CFTR is expressed in germ cells at specific stages of spermatogenesis (Trezise et al., 1993) and that functional CFTR protein is present in the sertoli cells (Boockfor et al., 1998), as well as in the epithelial cell of the epididymis in rats (Leung et al., 1996; Wong, 1998; Gong et al., 2000, 2001). Our previous studies demonstrated that CFTR is involved in the secretion and transport of HCO 3 in the epithelium of mouse uterine endometrium, defects of which lead to a reduction of sperm capacitation and fertilizing capacity (Wang et al., 2003). We have also demonstrated, using CF heterozygote mouse spermatozoa, that impaired CFTR function may result in the reduction of sperm fertilizing capacity (Xu et al., 2007). Hernandez-Gonzalez et al. (2007) showed that CFTR is involved in mouse sperm capacitation by regulating Naþ channel activity (ENaC). Recently, Chen et al. (2009) reported that a functional interaction between CFTR and SLC26A3 (a Cl =HCO 3 exchanger) lead to sperm capacitation, and the inhibition of the anion e (...truncated)