The SV40 large T-antigen has been widely used to convert various cell types to a transformed phenotype, and also to induce progressive tumours in transgenic animals. The objectives of this review are to compare and discuss three different approaches to generate epididymal epithelial cell lines using the large T-antigen. In the first approach, retroviral transfection of primary cultures was used to immortalize canine epididymal cells in vitro; the other two approaches used transgenic mice expressing the large T-antigen. In one of these in vivo approaches, a construct consisting of the coding sequence of a temperature sensitive (ts) SV40 large T-antigen was inserted in a mouse genome. When the cells are exposed to the permissive temperature of 33 °C, functional expression of the large T-antigen occurs and cells start to proliferate. In the second in vivo approach a tissue-specific promoter, the 5kb GPX5 promoter, was used to direct expression of the large T-antigen to the epididymal duct epithelium.

The adjuvanticity of two gamma inulin/liposomes/Vitamin E combinations was evaluated in the mouse, in contraceptive vaccines with sperm protein extracts or a synthetic HE2 peptide ("Human Epididymis gene product"; residues 15-28) as antigen. The HE2 peptide was not conjugated to a protein carrier, to ensure that the antibodies elicited were specific against the HE2 peptide. The adjuvant combinations were designed to increase adjuvanticity, as their components have complementary mechanisms, and their performance was compared to Freund's adjuvant. Antibody production against native sperm structures was determined in sera by ELISA immunoassay and immunohistology. Toxicity of adjuvants was determined by histopathological study and treated mice were monitored for signs of pain or distress. Our results show that the gamma inulin (1-2 microm particle size)/liposomes/Vitamin E combination, with sperm protein extracts, is better than Freund's adjuvant because it elicits good antibody titres without any toxicity. When the synthetic HE2 peptide is used as antigen, the gamma inulin (1-2 microm particle size)/liposomes/Vitamin E combination is less effective than Freund's adjuvant; nevertheless, the anti-HE2 antibodies elicited are highly specific and recognize native structures in sperm.

Human Epididymis‐specific protein 6 [HE6 (GPR64)] is a highly conserved, tissue‐specific seven‐transmembrane receptor of the human epididymis. The rodent counterparts were cloned and 5′‐inverse PCR employed to confirm that the cDNA sequences were full length. Downstream from the highly conserved signal peptide‐coding sequence, the 5′‐regions contained at least six mini‐exons of less than 50 nucleotides. Multiple splice variants involving these mini‐exons were cloned in the human, the majority of which was also found in rodents. Northern blot analysis showed that the tissue distribution of the mRNA was very similar in human and rodents. The human HE6 gene was assigned to the X chromosome in a region, which is syntenic to the mouse. The HE6 sequence predicted a two‐subunit receptor of the LNB‐TM7 subfamily. A membrane preparation and protein solubilization method was adopted to identify the endogenous epididymal proteins. Two sets of peptides were chosen for antibody production, assuming that protein scission had occurred within the conserved GPS‐motif. Western blot analysis revealed abundant two‐subunit proteins in human and rodents, comprising an approximately 180 kDa hydrophilic ectosubunit and a <40 kDa hydrophobic endosubunit. Deglycosylation experiments showed that the large ectosubunits were highly glycosylated, the carbohydrate side chains dramatically increasing the apparent molecular mass. Immunohistochemical studies revealed that both subunits were associated with apical membranes of efferent ductule and proximal epididymal duct epithelia. Mol. Reprod. Dev. 64: 13–26, 2003. © 2003 Wiley‐Liss, Inc.

The surface of mammalian spermatozoa is covered by a dense coating of carbohydrate-rich molecules forming a 20-60 nm thick glycocalyx. The majority of sugar residues are attached to proteins which are either integrated within the sperm membrane, or are more or less loosely associated with it. It is estimated that there may be several hundred different glycoproteins comprising the glycocalyx, some of which are synthesized within the testis. Others, however, are produced by the epithelia of the efferent ducts, epididymis and possibly other accessory glands, and become associated with the spermatozoa post-testicularly during transit through, and storage in, the male tract. The acquisition of the mature glycocalyx is associated with the attainment of full sperm fertilizing ability. Until its complete molecular structure is elucidated, the complex function of the glycocalyx remains obscure, though it may be related to membrane maturation and immunoprotection in the female tract, as well as to sperm-zona binding and fertilization.

Summary Human post-testicular proteins were cloned by subtractive screening of epididymal cDNA libraries, employing testis as the primary negative control. This method identified six human epididymal cDNAs, named HE1-HE6, which are derived from abundant epididymal mRNAs. With the exception of HE5, which turned out to be identical to the lymphocyte surface antigen CD52, they represented completely novel human gene products. To date, there is little information on their function and the mechanism of their deposition on the sperm surface. Unlike the sperm coating antigens, CD52 binds firmly to the sperm membrane via its GPI anchor during epididymal passage. Its synthesis is carefully regulated by the epididymal epithelium. From the results of both in vivo and in vitro studies it was concluded that androgen and temperature are principal factors synergistically modulating epididymal CD52 expression. The human counterparts of two well-known major rodent epididymal proteins, secretory epididymal glutathione peroxidase (sGPX) and acidic epididymal glycoprotein (AEG = Protein DE), were not cloned by the subtractive screening approach, but by RT-PCR amplification.

Advances in understanding the role of the epididymis in human sperm maturation and fertility are dependent upon the availability of appropriate model systems in which to examine regulatory mechanisms and functions in a controllable fashion. Of a number of mammalian species studied by us and others, we suggest that the dog epididymis offers an excellent compromise in terms of the similarity of the specific genes expressed to those in the human, their mode of regulation, and the availability of tissues suitable for cell culture studies. We have developed a set of important tools in the form of epididymis-specific canine cDNA clones and cell culture methods, with which to examine the functioning of the epididymal epithelial cells in vitro, as well as the expression of specific genes in vivo, under normal and pathological conditions (e.g. cryptorchidism). Using this model system, we have elaborated the regional patterns of gene expression in vivo for several of the major secretory protein genes of the canine epididymis, and we have examined the importance of androgens and temperature on gene expression in vitro.

A novel gene product, HE6, showing homology to the seven transmembrane-domain (Tm7) receptor superfamily, has been cloned by differential screening from a human epididymal cDNA library. The cDNA clone represented an abundant approximately 5-kb mRNA, comprising 0.01% of the cDNA library. Northern blot analysis including various human tissues revealed an epididymis-specific expression. In situ transcript hybridization localized the mRNA within the epithelial cells lining the epididymal duct. Southern blot analysis, employing a fragment encoding part of the amino-terminal extracellular domain as a probe, identified an autosomal single-copy gene in the human genome. Homologous cDNA products showing 90% sequence identity were observed in the epididymides of all mammalian species investigated. A cloning and sequencing strategy, combining approximately 3.7-kb cDNA fragments obtained by conventional cDNA library construction with overlapping 5' rapid amplification of cDNA ends (RACE) fragments, yielded total sequence information of 4.7 kb for the human mRNA. This sequence comprises a long open reading frame of 3.1 kb. A homology search for related sequences revealed highest similarity (25% amino acid identity) with the secretin/vasoactive intestinal peptide (VIP) superfamily of G-protein-coupled receptors. The predicted extracellular amino-terminal extension, however, was much longer than in the other members, and showed similarity to highly glycosylated mucin-like cell-surface molecules.

The cDNA sequence of HE1, a novel human epididymal gene product isolated by differential screening, predicts an abundant, small secretory glycopeptide. HE1 is encoded by a single copy gene and seems to be well conserved among mammals. The predicted HE1 peptide is identical to that of EPI-1, the chimpanzee homologous gene product, and to ESP14.6, the macaque homologous product. Its tentative N-terminus also shows similarity to that of a major porcine epididymal sperm-associated glycopeptide. Northern blot analysis of various bovine and rat tissues indicated that their HE1-homologous gene products showed highly increased expression in the epididymis, suggesting that the tissue distribution observed in humans is also conserved. With use of antipeptide antibodies, HE1-related antigen was shown by immunohistochemical methods to be present within the epithelium and lumen of the human corpus epididymidis. High amounts were also present in the lumen of the cauda epididymidis and deferent duct. Moreover, HE1 seemed to be associated with epididymal spermatozoa, and association with ejaculated spermatozoa remained doubtful. With Western blotting, an antiserum raised against the synthetic HE1 epitope specifically cross-reacted with proteins of human seminal plasma in the range of 25-27 kDa. An antiserum raised against chimpanzee EPI-1 recognized human seminal plasma antigens of the same apparent molecular size.

A novel human cDNA encoding a small secretory glycopeptide specifically expressed within the caput and proximal corpus regions of the human epididymis, HE2, has been cloned and sequenced. Southern hybridization analysis showed it to be derived from a single-copy gene. A subclone containing the coding region of the mature peptide was used to generate the putatively encoded peptide as a 10,000 M(r) recombinant fusion protein in a bacterial expression system. This recombinant HE2 protein, as well as an oligopeptide deduced from the open-reading frame, was used to raise polyclonal antisera. Immunohistochemistry and immunofluorescence showed that the predicted HE2 peptide is present in the epididymal epithelium and on the sperm surface, where it adopts a typical antigenic pattern with a subacrosomal equatorial distribution on the sperm head.

Three tissue-specific gene probes that had been isolated by differential screening from a human epididymal cDNA library-HE1, HE2, and HE5- were employed to investigate regional specializations in the human epididymis. All 3 cDNAs were derived from major transcripts of the epithelial cells lining the epididymal duct. Each mRNA species, however, exhibited a discrete longitudinal pattern of hybridization with maxima in different regions of this organ, suggesting regional specializations of gene expression. The HE5 mRNA, which was recently shown to encode the peptide backbone of the human leukocyte differentiation antigen CDw52, showed maximum levels in the distal corpus epididymidis and in the vas deferens, whereas the HE2 mRNA was found predominantly in the caput and proximal corpus sections of the epididymis. HE1 mRNA was found in high amounts in all parts of the epididymis, displaying a 2-peak expression pattern with maxima in the distal caput and distal corpus of the epididymal duct.

Differential cDNA screening identified four new genes that are expressed specifically in the human epididymis. Their corresponding mRNAs are relatively short, ranging from 0.7 to 1.0 kb, and belong to a class of abundant or moderately abundant transcripts. Tissue-specific expression was pronounced for all four sequences, which showed no hybridization signals with RNA from eight different human tissues. Cross-hybridization with epididymal RNAs from some non-rodent mammalian species was observed with two cDNAs; the remaining two transcripts failed to cross-hybridize with any of the animal epididymal RNAs tested.