Expression and location of taurine transporters and channels in the epididymis of infertile c-ros receptor tyrosine kinase-deficient and fertile heterozygous mice.

Defective sperm volume regulation causes infertility in c-ros knockout (KO) mice lacking the initial segment of the epididymis (IS). As taurine is in high concentration in the male tract and acts as an organic osmolyte in somatic cells, the taurine transporter (TauT), taurine content and taurine channel phospholemman (PLM) were examined in the epididymides of these and fertile heterozygous (+/-) mice. TauT and PLM genes were demonstrated by RT-PCR in the caput of +/- and c-ros knockout males. Northern blots revealed one transcript of TauT (8 kb) in all regions with different expression between segments (cauda > corpus > IS > caput) and no differences in expression between genotypes. Western blotting revealed three translation products of TauT in all epididymal regions (107, 69, 49 kDa) with higher expression of the 69 kDa and 49 isoforms in the -/- than +/- caput. Immunohistochemical staining revealed staining of principal cells and stronger staining of apical and clear cells in all epididymal regions. The expression of the PLM transcript (0.75 kb: cauda = corpus > caput > IS) was upregulated in the proximal caput and cauda of KO mice. Tissue taurine was higher in the cauda >corpus>IS congruent with caput in fertile males and significantly higher in the proximal caput of the KO male. By contrast, taurine concentrations in cauda epididymidal fluid and content per 10(6) sperm did not differ between genotypes. TauT and PLM may be involved in taurine regulation in the normal epididymis and the proximal accumulation of taurine in the infertile males.

Expression and cellular localization of contraception-associated protein.

Treatment of male rats with ornidazole results in reversible infertility, which is associated with the detection of the contraception-associated protein 1 (CAP1) in epididymal fluid. The protein, which is present in sperm but not detectable in epididymal fluid of fertile rats, seems to be shed from sperm during ornidazole administration. Cloning and characterization of the gene revealed a high degree of similarity between CAP1 and DJ-1 (Wagenfeld et al, 1998b) a protein that was recently found in humans and which has been classified as a novel oncogene. Reverse transcription of total ribonucleic acid (RNA) from various species indicated that a gene similar to CAP1 was also expressed in the testes of hamsters, mice, cynomolgus monkeys, and humans. Detection of RNA expression in rats at the cellular level by in situ hybridization revealed a stage-specific CAP1 expression in the cytoplasm of pachytene spermatocytes (stages IX-XIII), secondary spermatocytes (stage XIV), and round spermatids (stages I-VII). Immunolocalization of CAP1 in rat testis showed a strong staining of elongating spermatids (stages VI-VIII), indicating a translational delay of CAP1 expression. The location of CAP1 on sperm depended on the method of fixation used, with CAP1 being exhibited on the equatorial segment of the sperm head and cytoplasmic droplets. Flow cytometric analysis of sperm from ornidazole-fed rats revealed a significant decline (of 22%-24%) in the amount of sperm surface CAP1 compared with controls, which is associated with an altered location on the sperm head. These observations support a putative role of the protein in the fertilization process.

The cause of infertility of male c-ros tyrosine kinase receptor knockout mice.

Male homozygous transgenic c-ros knockout mice are sterile by natural mating, lack a part of their epididymis, and the epididymal sperm exhibit tail angulation in vivo and in vitro. To ascertain if this abnormal tail form caused the infertility, the number and nature of sperm in the tract of females mated to knockout and wild-type mice were determined. Percentage motility and numbers of sperm in the uterus 1 h after mating were similar between genotypes. The majority of the uterine sperm from the wild-type males had straight flagella, whereas 46-86% of knockout sperm were bent at the cytoplasmic droplet even when motile. Motile knockout sperm showed a 54 and 37% reduction in the straightline and curvilinear velocities compared with straight wild-type sperm. Sequential flushings of the oviduct 4 h after mating with the wild-type males contained sperm: 591 +/- 119 free, 371 +/- 70 loosely, and 122 +/- 47 tightly bound to the epithelium, but no knockout sperm were recovered from the oviduct or observed within the uterotubal junction in tissue sections. The infertility of c-ros knockout male mice can be explained by the sperm's inability to enter the oviduct, as a result of their bent tails forming the entangled sperm mass and their compromised flagellar vigor within the uterus.

Molecular cloning and expression of rat contraception associated protein 1 (CAP1), a protein putatively involved in fertilization.

Epididymal fluid from rats rendered infertile by oral administration of ornidazole contains a protein CAP1 (contraception-associated protein 1) that is absent from epididymal fluid, but present on epididymal sperm, from fertile vehicle-treated rats. The gene for CAP1 has been isolated from a rat testis cDNA library and its expression investigated in different tissues. The deduced protein sequence of CAP1 contains 189 amino-acid residues and database searches revealed a high degree of homology (83-95%) with human and mouse DJ-1 at the nucleotide and amino acid levels. Northern blot hybridisation from different rat tissues indicated that CAP1 is encoded by a 1.6-kilobase RNA transcript and seems to be ubiquitiously expressed in the rat with a high level of expression in the testis.

The appearance of basal cells in the developing murine epididymis and their temporal expression of macrophage antigens.

This work demonstrates similarities between epididymal basal cells and macrophages in the mouse. Light microscopic studies of the postnatal development of the murine epididymis showed that basal cells were not present before days 12, 14 and 16 in the cauda, caput and corpus epididymis, respectively. An increase in cell number per unit length of tubule perimeter was demonstrated in all segments between days 20 and 27, when testicular fluid and spermatozoa start entering the epididymis. In the adult, there were more basal cells per unit perimeter in the cauda than caput or corpus epididymis. Conspicuous and consistent expression by basal cells of antigens detected by antibodies against tissue-fixed macrophages (F4/80) and mature macrophages (Mac-1) occurred only after they became established within the epithelium. Basal cells in the cauda epididymis did not display either antigen in the adult, although they persisted in the caput region. Such developmental patterns are compatible with the hypothesis that basal cells play a role in immune defence against sperm autoantigens.

Shedding of a rat epididymal sperm protein associated with infertility induced by ornidazole and alpha-chlorohydrin.

The protein composition of epididymal fluid and sperm extracts of rats treated with the nitroimidazole compound ornidazole was investigated by two-dimensional gel electrophoresis. Epididymal luminal fluid from the corpus and cauda regions of male animals rendered infertile by ornidazole treatment contained a prominent protein (contraception-associated protein 1, CAP1) with a molecular mass of approximately 25 kDa and an isoelectric point (pI) of 5.8; it was not found in fluids, but was present in sperm, from fertile vehicle-fed rats. Infrared matrix-assisted laser desorption/ionization mass spectrometry indicated that the molecular mass of CAP1 was 20420+/-120 daltons. Analysis of 17 amino acids demonstrated 49% homology to a diuretic hormone from an insect (Acheta domesticus). Densitometric quantitation of CAP1 on silver-stained gels indicated its presence in greater amounts in cauda than in corpus fluid from treated animals, whereas fluid from the rete testis lacked CAP1. In vitro incubations of tissue from the caput, corpus, and cauda epididymidal regions with [35S]methionine gave no hint that CAP1 was a secretion product of the epididymal epithelium. The absence of CAP1 from luminal fluid obtained from the sperm-depleted corpus epididymidis of efferent duct-ligated ornidazole-fed rats suggested a spermatozoal origin. CAP1 was present in spermatozoa from the caput epididymidis but not from the rete testis in control animals. Less CAP1 was present in detergent extracts of cauda sperm from ornidazole-treated rats than in sperm from control animals, suggesting a contraceptive-related displacement of protein from sperm to fluid. The association of ornidazole- and alpha-chlorohydrin-induced infertility with the presence of CAP1 in epididymal fluid, probably originating from spermatozoa, suggests a critical role for this protein in fertilization.

Interaction of the human epididymal protein CD52 (HE5) with epididymal spermatozoa from men and cynomolgus monkeys.

A monoclonal antibody (CAMPATH-1 G) against the human lymphocyte surface protein CD52, which is similar to the epididymal secretion HE5, was used to ascertain the presence of this protein on maturing primate spermatozoa by flow cytometry. The percentage of human viable spermatozoa stained specifically with this antibody increased from sperm in spermatocoeles (0.5%), to the efferent ducts (3.8%), corpus (47.2%), and cauda (85.7%) epididymidis. Positive cells revealed staining mainly over the whole tail and postacrosomal region of the sperm head. Spermatozoa (approximately 10%) from both the efferent ducts and corpus epididymidis took up additional antigen when incubated with human distal cauda epididymidal plasma as a source of CD52, and 12-22% of human testicular sperm (from spermatocoeles) took up CD52 from human seminal plasma. In the cynomolgus monkey, nonspecific binding of control IgG was greater than that in human males and net CD52 staining was measurable only on approximately 30% of corpus sperm where it was mainly on the principal piece. Neither caput nor cauda sperm took up human CD52 upon incubation with human seminal plasma, but an additional 27% of corpus sperm expressed CD52. Such uptake of CD52 was drastically reduced, or did not occur, when seminal plasma had been fractionated by filtration through 0.1 microns filters (filtrate II) or 300,000 Da cutoff filters (filtrate III), respectively. Western blots revealed that CD52 contents were much reduced in filtrate II and nondetectable in filtrate III of seminal plasma. Similar reduction of CD52 in the filtrate of cauda epididymidal plasma indicates the association of this epididymal secretion with large molecular factors and suggests their involvement as carriers in the in vivo transfer of the secretion onto the epididymal sperm surface. The in vitro uptake of CD52 by some but not all immature sperm and the detection by Western blotting of much less CD52 in the corpus than the cauda luminal plasma suggest that the acquisition of this epididymal secretion by spermatozoa depends on their maturation status as well as the availability of the protein in the epididymal lumen.

Epithelial monolayers from human epididymal and efferent duct tubules; testosterone metabolism and effects of culture conditions on cell height and confluence.

Corpus epididymal and efferent duct epithelial cells on permeable supports formed confluent monolayers that resisted hydrodynamic equilibrium and created electrical resistance. Monolayers were formed sooner and were of better quality when fetal bovine serum (FBS), rather than bovine serum albumin (BSA), was present in glucose-free, rather than glucose-containing, media. Testosterone was converted to androstenedione by both cell types and conversion of both steroids to 5 alpha-reduced metabolites was higher in cells from the corpus epididymidis than from efferent ducts. Addition of heat-treated human spermatocoele fluid (similar to rete testis fluid) to the apical aspects of the cells increased cell heights when they were initially low, but some cytoplasmic damage was observed. New serum-free media (especially those designed for keratinocytes and mammary epithelial cells) could maintain cultured cells at heights found in situ.