Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation.

The mammalian epididymis plays a critical role in sperm maturation, a function dependent on testicular androgens. However, the function of the initial segment, the most proximal part of the epididymis, is also dependent on luminal factors of testicular origin. Efferent duct ligation (EDL), which prevents luminal testicular fluid from reaching the epididymis, results in changes in gene expression within this region. Cystatin-related epididymal spermatogenic (cres) gene and gamma-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5' site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between -530 and -681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.

Characterization of fibroblast growth factor receptors expressed in principal cells in the initial segment of the rat epididymis.

Studies from our laboratory support a model in which growth factors produced in the testis reach the epididymis via the luminal system and play an important role in maintaining the function of epithelial cells, particularly in the initial segment. Previous work showed that gamma-glutamyl transpeptidase (GGT) mRNA IV, which is highly expressed in the rat initial segment, may be under the control of luminal fibroblast growth factor 2 (FGF-2) from the testis. The current studies were undertaken to identify which fibroblast growth factor receptors (FGFRs) are present in the principal cells of the rat initial segment and to identify other potential ligands for these receptors in rat rete testis fluid (RTF). Immunoblot analysis revealed that FGFRs 1-4 were present, and reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that both the IIIb and IIIc splice variants of FGFRs 1-3 were expressed. However, RT-PCR using RNA isolated from principal cells collected by laser capture microdissection revealed only FGFR-1 IIIc. Additional PCR analysis established that both the alpha and beta forms of FGFR-1 IIIc were expressed in principal cells. Both FGF-4 and FGF-8 were present in rat RTF, as determined by immunoblotting. Thus, FGF-2, -4, and -8, found in RTF, may act upon FGFR-1 IIIc in the principal cells of the initial segment to regulate GGT mRNA IV expression.

Organic cation/carnitine transporter, OCTN2, is differentially expressed in the adult rat epididymis.

L-Carnitine must be transported against a substantial concentration gradient across the epididymal epithelium to achieve high intraluminal levels, approximately 50 mM in the cauda. Recently, an organic cation transporter, OCTN2, was cloned from rat intestinal epithelium and shown to transport L-carnitine in a sodium-dependent manner. To test the hypothesis that OCTN2 was present in the epididymis, primers were designed based on the published OCTN2 mRNA sequence. A 1.9-kilobase OCTN2 cDNA from rat epididymis was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned. Northern analysis demonstrated the presence of OCTN2 transcripts in the epididymis, with highest expression in the distal caput and corpus. To localize the protein, an antibody raised against a carboxy-terminal peptide of OCTN2 was produced in rabbits and used for Western blot analysis and immunohistochemistry. The antibody recognized a band of approximately 65 kDa in Western blots using epididymal lysates. Immunohistochemical studies demonstrate that OCTN2 is present in the basolateral membrane of epithelial cells in the distal caput, corpus, and proximal cauda epididymides. In conclusion, OCTN2 is present in the rat epididymis in a region-dependent manner and is likely to be responsible for the transport of L-carnitine into the cells of the epididymal epithelium.