HE5, a very abundant human epididymal gene product, was cloned by a differential screening procedure which employed testis as the primary negative control tissue. Sequencing of the HE5 cDNA showed it to be identical to that encoding the peptide backbone of the human leucocyte differentiation antigen CDw52. Since human genomic DNA contained only one cross-hybridizing fragment, this implies that both products were transcribed from the same gene. Northern blot analysis and in situ transcript hybridization revealed a highly tissue- and cell-type-specific expression pattern for HE5, showing the gene product to originate from epithelial cells of the epididymal and deferent duct. The possible identity of the HE5 product with the CDw52 antigen suggests a link between the immune and reproductive systems.

No abstract

The physiology of the epididymis is an integral part of the maturation process by which human spermatozoa acquire the ability to reach and fertilize an oocyte. Because of the high degree of species specificity exhibited by the epididymal proteins involved in sperm maturation, we have assessed tissue from several alternative species for their suitability as a model for human epididymal physiology. Of these, the dog appears to offer an appropriate system. Northern hybridization using cDNA probes specific for human epididymal genes established that, irrespective of dog breed, the canine equivalents of the epididymis-specific HE1, HE4 and HE5 mRNAs were expressed highly in the canine epididymis. cDNA cloning and sequencing confirmed that the canine gene products, CE1, CE4 and CE5 were indeed true structural homologues of their human counterparts. Finally, tissue culture conditions were established wherein all three specific canine genes remained up-regulated after 5 days of culture. Thus, the prerequisite criteria for the development of a system which models human epididymal physiology are to a large degree fulfilled by this canine culture system.

To study the role of scrotal vs. body temperature in epididymal function we established a simplified cell culture system from the dog epididymis in which the cells showed a pattern of gene expression similar to that in the human epididymis and retained many characteristics of epididymal epithelial cells. The cultured cells had an epithelial-type cytoskeleton, nuclear androgen receptor protein, and a striking temperature responsiveness. Exposure of the cells to a culture temperature of 37 C, compared to 33 C, had a fast and irreversibly suppressive effect on the levels of an abundant epididymal messenger RNA (mRNA), CE5, which represents the canine counterpart of the human CD52/HE5 mRNA, encoding a major glycosylphoshatidylinnositol (GPI)-anchored sperm membrane glycopeptide. The temperature effect on the mRNA was a direct and specific one, not mediated by temperature influences on the testis and not affecting other epididymal mRNAs. Exposure of the cells to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (25 micrograms/ml culture medium) and cycloheximide (2 micrograms/ml) suggested that the steady state levels of CD52/CE5 mRNA may be controlled posttranscriptionally by changing the half-life of this specific mRNA in response to an extracellular temperature stimulus.

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 viuo and in vitro studies it was concluded that androgen and temperature are principal factors synergistically modulating epididymal CD52 expression. The human counterparts of two wellknown 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.