Characterisation of renal chloride channel, CLCN5, mutations in hypercalciuric nephrolithiasis (kidney stones) disorders

Mutations of the renal-specific chloride channel (CLCN5) gene, which is located on chromosome Xp11.22, are associated with hypercalciuric nephrolithiasis (kidney stones) in the Northern European and Japanese populations. CLCN5 encodes a 746 amino acid channel (CLC-5) that has approximately 12 transmembrane domains, and heterologous expression of wild-type CLC-5 in Xenopus oocytes has yielded outwardly rectifying chloride currents that were markedly reduced or abolished by these mutations. In order to assess further the structural and functional relationships of this recently cloned chloride channel, additional CLCN5 mutations have been identified in five unrelated families with this disorder. Three of these mutations were missense (G57V, G512R and E527D), one was a nonsense (R648Stop) and one was an insertion (30:H insertion). In addition, two of the mutations (30:H insertion and E527D) were demonstrated to be de novo, and the G57V and E527D mutations were identified in families of Afro-American and Indian origin, respectively. The G57V and 30:H insertion mutations represent the first CLCN5 mutations to be identified in the N-terminus region, and the R648Stop mutation, which has been observed previously in an unrelated family, suggests that this codon may be particularly prone to mutations. Heterologous expression of the mutations resulted in a marked reduction or abolition of the chloride currents, thereby establishing their functional importance. These results help to elucidate further the structure-function relationships of this renal chloride channel.      

Greater numbers of human spermatozoa associate with endosalpingeal cells derived from the isthmus compared with those from the ampulla

A simple co-culture bioassay system was used to investigate whether or not the anatomical origin affected the ability of epithelial cells from the human uterine (Fallopian) tube to 'bind' spermatozoa. This study was also used to identify some of the factors which may be involved in the regulation of sperm-epithelial interactions in vitro by comparing different tissue culture models and assessing the effect of oestradiol concentration. Epithelial explants harvested from different regions of human uterine tubes were co-incubated with a known concentration of motile donor spermatozoa. All results were adjusted to reflect a standard sperm concentration of 5 x 10(6)/ml. More spermatozoa associated per field of isthmic compared to ampullary epithelium [isthmus 9.5 +/- 0.9, ampulla 7.1 +/- 0.7 (mean +/- SEM); n = 36, P < 0.05, ANOVA] and cells from post-menopausal patients had an apparently reduced ability to bind spermatozoa [isthmus 5.5 +/- 2.0, ampulla 4.3 +/- 1.4 (mean +/- SEM); n = 4]. Neither menstrual cycle stage nor addition of mid-cycle concentrations of 17beta-oestradiol (750 pmol/l) affected the number of spermatozoa which bound to epithelium from either tubal region. In addition, the number of spermatozoa which bound per field of polarized explants was greater (P < 0.05) than that bound to dissociated primary and passaged epithelial cell monolayers. This report is the first to provide evidence suggestive of a role for sperm- epithelial binding in the formation of an isthmic sperm reservoir in the human uterine tube. Results also indicate that oestrogen is not involved in the regulation of these interactions, and that cell polarity is an important factor for such associations in vitro.