Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.
Expression of a glutamate transporter (EAAC1), a lipocalin (MEP17) and beta-galactosidase (beta-Gal) in histological sections was used to monitor post-natal development of the murine epididymis. Three epithelia in the adult caput of wild-type mice were distinguished: I, the initial segment; II, the proximal caput; and III, the distal caput. The regions in which epithelia I, II and III were situated were called regions I, II and III, respectively. Regions I, II and III developed from a precursor epithelium present on day 14; from day 16, a presumptive region I epithelium was evident and, by day 21, epithelia characteristic of future regions II and III appeared. The relationship between the c-ros gene and the initial segment was studied by investigating the development of the caput epididymidis in transgenic homozygous c-ros knockout (-/-) mice that lack the initial segment, heterozygous (+/-) males and wild-type males in which the efferent ducts had been ligated prepubertally so that the initial segment failed to develop. In mice with prepubertally ligated efferent ducts, regions II and III developed normally but region I was missing in the adult and expression of c-ros was partially decreased. In (-/-) mice, the precursor epithelium was present, differentiation of epithelium II was delayed until day 32 and epithelium I never developed. Thus, caput region I develops before c-ros expression, high testosterone secretion and differentiation of regions II and III but not if the organ is deprived of the oncogene c-ros or testicular exocrine secretions. The caput of the knockout male lacks solely the initial segment so that the efferent ducts are in continuity with the post-initial segment, proximal caput region. The ligand for c-ros may be present in testicular fluid and both ligand and receptor may be necessary for differentiation of epithelia I and II.
The murine caput epididymidis responded to deprivation of luminal fluid from the testis by regression of the initial segment but maintenance of the adjacent proximal and distal caput regions, as judged by immunohistochemical staining of the glutamate transporter EAAC1 and the lipocalin MEP17 and enzymatic activity of beta-galactosidase (beta-Gal). Additional removal of circulating androgens by bilateral castration similarly led to loss of the initial segment and of the proximal caput but the distal caput was transformed into an epithelium containing more apical than principal cells staining for EAAC1; this epithelium resembled the precursor epithelium usually only seen in prepubertal juveniles. Administration of dihydrotestosterone (DHT) to the castrates maintained the proximal and distal caput epithelia and induced a proximal epithelium, which resembled the initial segment in its prominent staining for Golgi, EAAC1 and beta-Gal activity, although it was short and exhibited no MEP17 expression. DHT was present in the c-ros knockout caput epididymidis lacking the initial segment and in the heterozygous organ but the DHT concentration was lower in the knockout corpus. The maintenance of the full complement of epithelia in the murine caput epididymidis in the adult thus requires a combination of luminal fluid from the testis, tissue DHT and the presence of the c-ros oncogene.
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