Caroline Osterhoff

Function of human epididymal proteins in sperm maturation.

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.

Immortalization by large T-antigen of the adult epididymal duct epithelium.

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 degrees 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.

HE6/GPR64 adhesion receptor co-localizes with apical and subapical F-actin scaffold in male excurrent duct epithelia

A role for HE6/GPR64 in male excurrent ducts in the regulation of water balance was suggested from targeted gene mutation in the mouse. Results of the present immunolocalization study strengthen this hypothesis. Employing monospecific antibodies and laser confocal microscopy, we studied the localization of the receptor protein in the human and wild-type mouse ductuli efferentes and epididymis. We show that HE6/GPR64 is specifically associated with cell types and subcellular domains involved in the process of fluid reabsorption. In the mouse, dual labelling with anti-tubulin antibodies revealed that HE6/GPR64 was absent from the (kino-) cilia of ciliated cells. Instead, the receptor protein accumulated in the non-ciliated principal cells. Specifically, strong immunofluorescence was observed in the apical compartment of these cells. Dual labelling with phalloidin and anti-ezrin antibodies revealed that in the mouse the bulk amount of HE6/GPR64 protein co-localized with the F-actin-ezrin scaffold in brush border-like microvilli of ductuli efferentes and long stereocilia of the epididymis proper. In the ductuli efferentes, HE6/GPR64 also co-localized with the subapical F-actin network immediately below the microvilli. Comparable immunostaining patterns were observed in human and mouse; however, a specific feature of the human ductuli efferentes was an intense HE6/GPR64-related labelling of crypt-like grooves or furrows of hitherto unknown function.