Louis Chukwuemeka Ajonuma

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.

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.

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.

CFTR is required for cellular entry and internalization of Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular Gram‐negative pathogen affecting over 600 million people worldwide with 92 million new cases occurring globally each year. C. trachomatis enter the cells and replicate to infect different tissues/organs, giving rise to a spectrum of pathological conditions; however, the exact mechanism or receptor(s) for their entry is not well understood. Here we report that CFTR (cystic fibrosis transmembrane conductance regulator), an apical epithelial anion channel, is required for cellular entry and internalization of C. trachomatis. Human epithelial cell lines expressing functional CFTR internalized more C. trachomatis than the cells expressing mutant Δ508 CFTR. The in vitro cellular uptake of C. trachomatis can be blocked by CFTR inhibitors or antibody, and the in vivo cellular uptake of C. trachomatis in CFTR mutant (CFTR−/−) mice was significantly less compared with that in the wild‐type. Direct interaction between CFTR and C. trachomatis LPS (lipopolysaccharide) is demonstrated by their immune‐co‐localization and co‐immunoprecipitation. Despite an increase in CFTR expression observed upon C. trachomatis LPS challenge, a reduction in its ion channel activity is observed, consistent with the notion that CFTR functions as a receptor for cellular entry and internationization of C. trachomatis, with compromised ion‐channel function. These findings, for the first time, demonstrate that CFTR functions as a cell‐surface receptor for epithelial cell entry, and internalization of C. trachomatis and these findings may lead to the development of new treatment strategies to curtail the spread of chlamydial infections.

Involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in the pathogenesis of hydrosalpinx induced by Chlamydia trachomatis infection

Background:  Genital Chlamydia (C) trachomatis infection has been recognized as the single most common cause of pelvic inflammatory disease leading to severe tubal damage, ectopic pregnancy, infertility and hydrosalpinx. However, the mechanism underlying the formation of hydrosalpinx induced by C. trachomatis infection remains largely unknown. We performed this study to determine the involvement of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐activated chloride channel that regulates epithelial electrolyte and fluid secretion, in hydrosalpinx fluid formation.

Ultrastructural characterization of whole hydrosalpinx from infertile Chinese women

Hydrosalpinx (HSP) has been shown to be detrimental to the outcome of assisted reproduction, but little is known of its pathology. This prospective study examined and detailed ultrastructural characterization of HSP of infertile women presenting for assisted reproductive treatments. Both light and electron microscopies were used to characterize HSP. Hematoxylin and eosin staining of HSP showed areas without epithelial cell lining or with abnormalities such as flattening of the epithelial layer and exfoliation of epithelial cells with occasional normal columnar epithelial lining. HSP muscle fibers were atrophic and occasionally replaced by fibrous tissues, or separated by areas of severe edema. Inflammatory cells could be found in hydrosalpinx fluid (HF) in the lumen in areas with flattened to no epithelial cells, without epithelial lining, as well as in dilated blood vessels and/or lymph vessels. Scanning electron microscopy of the epithelial surface revealed epithelial denudation‐severe loss of both cilia and microvilli and stomata exuding globular bodies on eroded ampulla surfaces. Severe chronic inflammation and damage to the epithelial lining and musculature of Fallopian tubes and the presence of inflammatory cells provides an explanation for HF formation, and thus for the detrimental effects of HF on reproductive processes and IVF outcome.

Involvement of cystic fibrosis transmembrane conductance regulator in infection-induced edema

Abnormal fluid accumulation in tissues, including the life‐threatening cerebral and pulmonary edema, is a severe consequence of bacteria infection. Chlamydia (C.) trachomatis is an obligate intracellular gram‐negative human pathogen responsible for a spectrum of diseases, causing tissue fluid accumulation and edema in various organs. However, the underlying mechanism for tissue fluid secretion induced by C. trachomatis and most of other infectious pathogens is not known. Here, we report that in mice C. trachomatis infection models, the expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel, is up regulated together with increased cytokine release and tissue fluid accumulation that can be reversed by treatment with antibiotic specific for C. trachomatis and CFTR channel blocker. However, C. trachomatis infection cannot induce tissue edema in CFTR tm1Unc mutant mice. Administration of exogenous IL‐1β to mice mimics the C. trachomatis infection‐induced CFTR upregulation, enhanced CFTR channel activity and fluid accumulation, further confirming the involvement of CFTR in infection‐induced tissue fluid secretion.

Differential effects of Matrigel and its components on functional activity of CFTR and ENaC in mouse endometrial epithelial cells.

Our previous studies have observed an effect of Matrigel, a solubilized basement membrane preparation extracted from the Engelbreth—Holm—Swarm (EHS) mouse sarcoma, on the expression of ion channels in mouse endometrial epithelia; namely the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐dependent Cl−channel, and the epithelial sodium channel (ENaC). The present study further investigated the effects of Matrigel and its individual components on the functional expression of CFTR and ENaC using the short‐circuit current (Isc) technique. The results showed that different components of Matrigel, namely growth factors, laminin and collagen, had differential effects on the functional activity of the two ion channels in murine endometrial epithelium. The information obtained may be useful for designing future in vitro culture models to investigate the functional roles of these ion channels in the endometrium.

Hydrosalpinx fluid and in vitro mouse embryo development.

To the Editors: It is with interest that we read the article entitled ”Effects of human hydrosalpinx fluid on in uitvo mouse embryogenesis”.” Many recent reports, including the meta-analyses of Zeyneloglu et al. and Camus et ~ l . ~ , ~ ) have confirmed that in the presence of a hydrosalpinx, implantation and pregnancy rates after in-uitro fertilization and embryo transfer (IVF/ET) are significantly impaired. However, some studies have failed to demonstrate any adverse effect.“’) In addition, while an adverse effect of hydrosalpinx fluid (HF) has not been reported in human embryo or in human cytotrophoblast cell culture?] this has been documented in mouse embryo development It has been suggested that the negative effect of HF on IVF/ET outcome may be due, at least in part, to the composition of HF. However, despite the similarity between HF, human tuba1 fluid (HTF) and serum, some variations do exist and they variably effect iiz vitro embryo development and clinical outcome. Saito et al.” analysed only two HF samples from their small sample size of 6, and of the 2 that were analysed, no mention was made of the values of pyruvate, lactate, bicarbonate and chloride, which are important components of a good embryo culture medium and necessary for early embryo development. It has been reported that pyruvate and lactate are preferred energy substrates to glucose in early embryo development. Ng et ~ 7 1 . ~ ~ 1 demonstrated that the low osmolality, low lactate and low protein levels present in HF caused a decrease in sperm motility and survival. We have also observed poor mouse embryo development in HF with low osmolality, low lactate and low protein levels (Ajonuma et al. unpublished observation). Strandell et investigated the effects of HF on human embryo development and implantation in vitro, and found that in 2 samples with extremely low osmolality, poor embryo development resulted. Murray et 01. reported an inhibitory effect of HF on mouse embryo development, which was significantly reduced when lactate was supplemented in culture. The morphology and the rate of embryo cleavage, which are pertinent to the quality of the blastocysts, are important parameters and deserve attention. Furthermore, the small sample size described by Saito et nl . may not be sufficient to show a significant difference. As part of an examination of the effect of HF on sperm motility, movement characteristics and survival rates, Ng et al.”’ demonstrated that HF was toxic to human spermatozoa using 15 HF samples in 45 different concentrations. However, in an earlier small trial of 3 samples, Schats et al.’”) found no toxic effect of HF on sperm survival. Saito et al . stated that their results demonstrated that early embryos develop normally in the microenvironment of HF. However, it would have been more appropriate for them to use pronuclear stage (one-cell) embryos in their study. Table 1, a summary of all previously published studies on mouse embryo culture in HF, shows that in all studies where pronuclear stage mouse embryos were used, embryo development was impaired at low HF concentrations and two-cell stage embryo development was only affected at very high HF concentrations. Thus, it is tempting to conclude that most HF milieu have an adverse influence on mouse embryo development from the pro-nuclear to two-cell stage, but not thereafter, particularly at low concentrations. Saito et nl.I) and Mukherjee et al.’) used the same ICR mice embryos (Table 1) but obtained different results, which may have been due to differences in embryo stage at the beginning of the experiment, a finding that is consistent with other studies. The adverse effect of HF on mouse but not on human embryos may be related to inter-species differences with respect to optimal culture condi-