Chen Xi Zhou

An epididymis-specific |[beta]|-defensin is important for the initiation of sperm maturation

Although the role of the epididymis, a male accessory sex organ, in sperm maturation has been established for nearly four decades, the maturation process itself has not been linked to a specific molecule of epididymal origin. Here we show that Bin1b, a rat epididymis-specific β-defensin with antimicrobial activity, can bind to the sperm head in different regions of the epididymis with varied binding patterns. In addition, Bin1b-expressing cells, either of epididymal origin or from a Bin1b-transfected cell line, can induce progressive sperm motility in immotile immature sperm. This induction of motility is mediated by the Bin1b-induced uptake of Ca2+, a mechanism that has a less prominent role in maintaining motility in mature sperm. In vivo antisense experiments show that suppressed expression of Bin1b results in reduced binding of Bin1b to caput sperm and in considerable attenuation of sperm motility and progressive movement. Thus, β-defensin is important for the acquisition of sperm motility and the initiation of sperm maturation.

Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl− and HCO3− transport. Although >95% of all CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. Here we report that CFTR is detected in both human and mouse sperm. CFTR inhibitor or antibody significantly reduces the sperm capacitation, and the associated HCO3−-dependent events, including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower compared with that of the wild-type. These results suggest that CFTR in sperm may be involved in the transport of HCO3− important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD.

Involvement of CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in a wide variety of epithelial cells, mutations of which are responsible for the hallmark defective chloride secretion observed in cystic fibrosis (CF). Although CFTR has been implicated in bicarbonate secretion, its ability to directly mediate bicarbonate secretion of any physiological significance has not been shown. We demonstrate here that endometrial epithelial cells possess a CFTR-mediated bicarbonate transport mechanism. Co-culture of sperm with endometrial cells treated with antisense oligonucleotide against CFTR, or with bicarbonate secretion-defective CF epithelial cells, resulted in lower sperm capacitation and egg-fertilizing ability. These results are consistent with a critical role of CFTR in controlling uterine bicarbonate secretion and the fertilizing capacity of sperm, providing a link between defective CFTR and lower female fertility in CF.

Critical role of CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl- channel expressed in a wide variety of epithelial cells, mutations of which are responsible for hallmark defective Cl- and HCO3- secretion seen in cystic fibrosis (CF). However, the physiological role of CFTR in reproductive tracts is far from understood although infertility has been observed in CF patients of both sexes. Previously we have demonstrated the expression of CFTR in the female reproductive tract and the involvement of CFTR in mediating anion secretion by the endometrium. Our recent results show that endometrial epithelial cells possess a cAMP-activated HCO3- transport mechanism, which could be impaired with channel blockers known to block CFTR or antisense against CFTR. Co-culture of sperm with CFTR antisense-treated endometrial cells or HCO3- secretion-defective CF epithelial cells resulted in reduced sperm capacitation and egg-fertilizing ability. Addition of HCO3- to the culture media and transfection of wild-type CFTR into CF cells rescued the fertilizing capacity of sperm. Immunostaining and Western blot revealed that CFTR is expressed in rodent sperm and intracellular measurement of pH during sperm capacitation indicated that the entry of HCO3- into sperm could be inhibited by CFTR inhibitor. These results are consistent with a critical role of CFTR in controlling uterine HCO3- secretion and sperm fertilizing capacity, suggesting that CFTR may be a potential target for post-meiotic regulation of fertility.

Differential expression and localization of CFTR and ENaC in mouse endometrium during pre-implantation

Interaction between the cystic fibrosis transmembrane conductance regulator (CFTR), a CAMP‐activated Cl− channel, and epithelial Na+ channel (ENaC) has been proposed as the major mechanism regulating uterine fluid absorption and secretion. Differential expression of these ion channels may give rise to dynamic changes in the fluid environment affecting various reproductive events in the female reproductive tract. This study investigated the expression and localization of CFTR and ENaC during the pre‐implantation period. Semi‐quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry were used to study the expression and localization of CFTR and ENaC in uteri collected from mature superovulated female mice. RT‐PCR showed maximal ENaC and CFTR expression on day 3 after mating. Maximal immunoreactivity was also observed for both ENaC and CFTR on day 3 after mating. However, ENaC was immunolocalized to the apical membrane of both luminal and glandular epithelia, while CFTR was predominantly found in the stromal cells rather than the epithelial cells. Differential expression and localization of CFTR and ENaC provide a molecular mechanism by which maximal fluid absorption can be achieved immediately prior to implantation, to ensure the immobilization of the blastocyst necessary for implantation.

A sperm GPI-anchored protein elicits sperm-cumulus cross-talk leading to the acrosome reaction.

Abstract.The acrosome reaction has long been thought to be induced by the zona pellucida. Here we report the identification and function of a novel human sperm glycosylphosphatidylinositol (GPI)-anchored membrane protein, NYD-SP8. The release of the protein during sperm-egg interaction and its binding to the cumulus, the first layer of egg investment, elicits cross-talk between the gametes and produces calcium dependant release of progesterone, which lead to the acrosome reaction. An in vivo mouse model of NYD-SP8 immunization is also established showing a reduced fertility rate. Thus, contrary to accepted dogma, our study demonstrates for the first time that, prior to reaching the zona pellucida, sperm may release a surface protein that acts on the cumulus cells leading to the acrosome reaction, which may be important for determining the outcome of fertilization.

The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms.

Nitric oxide (NO), which is produced from l‐arginine by three isoforms of NO synthase (NOS), has been implicated in reproductive functions. However, the specific role of NOS isoforms in gamete function and fertilization is not clear. Three types of NOS knockout mice were super ovulated and fertilized in vitro and in vivo. The sperm count and motility, in vivo and in vitro fertilization rate as indicated by two‐cell embryos and blastocyst rate were examined. The sperm count and motility from all three knockout mice were not significantly different from that of the wild type. Inducible NOS (iNOS) knockout mice were found to have the largest number of two‐cell embryos/mouse collected after fertilization in vivo (P < 0.01), but the rate of blastocyst formation from two‐cell embryos in vitro was similar for all three knockouts. The rate of in vitro fertilization using either iNOS‐deficient sperm or oocytes, but not those deficient in the other two NOS isoforms, was significantly elevated when compared to that in the wild type (P < 0.001). While all three types of NOS do not seem to play a significant role in pre‐ejaculated sperm function, iNOS may play an inhibitory role in sperm and oocyte functions affecting the process of fertilization and early embryo development.