Expression and distribution of OCTN2 in mouse epididymis and its association with obstructive azoospermia in juvenile visceral steatosis mice.

AIM: L-carnitine, an essential cofactor for mitochondrial, beta-oxidation of long-chain fatty acids, is known to play important roles in sperm maturation and metabolism when spermatozoa pass and acquire motility in the epididymis. We reported that obstructive azoospermia occurred in the epididymis in the juvenile visceral steatosis (JVS) mice, which are OCTN2 dysfunction mice caused by mutations in the gene encoding OCTN2, have been used for animal models of primary systemic carnitine deficiency. The aim of present study is to investigate the expression of OCTN2 protein in the mouse epididymis and its relation between the localization of OCTN2 and obstructive azoospermia in JVS mice as animal models for human male infertility. METHODS: Animals used in this study were wild-type (C57BL/6 J) mice (n = 4) and JVS mice (n = 4). We made a specific polyclonal antibody against OCTN2 and examined immunohistochemically the localization of OCTN2 in the mouse epididymis. RESULTS: OCTN2 was localized on the apical membrane of the principal cells of distal corpus and cauda epididymides. Immunocytochemistry demonstrated that OCTN2 was localized on the surface of microvillus upon the principal cells. In JVS mice, immunoreactivity started in a region immediately distal to where the sperm obstruction occurred. CONCLUSIONS: Our results suggest that OCTN2 functions as a carnitine transporter between the epithelium and the lumen in distal corpus and cauda epididymides and provides a clue as to why obstructive azoospermia is induced in distal parts of epididymis.

Expression of novel organic cation/carnitine transporter (OCTN2) in the mouse pancreas.

Among the organic cation transporters, OCTN2 is identified as the most important carnitine transporter owing to the ability to transport carnitine. Although the OCTN2 is previously found in various tissues, there have been no reports showing the OCTN2 in the pancreas. In this study, we examined the expression and localization of OCTN2 in the mouse pancreas by the aid of an in situ hybridization technique and immunohistochemistry with anti-OCTN2 antibody. As a result, the OCTN2 expression was found in the A-cells for the first time. OCTN2 was not expressed in B-cells, notwithstanding that the metabolism of long-chain fatty acids, which are transported into the mitochondria with the help of carnitine, was expected for fatty acid-stimulated insulin secretion. Thus, this study suggests the possibility of carnitine uptake in the pancreatic A-cells through OCTN2 and implies the presence of carnitine transporter(s) other than OCTN2 in the B-cell.

Hepatocyte growth factor activator inhibitor types 1 and 2 are expressed by tubular epithelium in kidney and down-regulated in renal cell carcinoma.

PURPOSE: Hepatocyte growth factor (HGF) and its receptor MET have been implicated in kidney development and renal cell carcinoma (RCC) progression. HGF is secreted as an inactive proform and it must be activated to initiate MET signaling. HGF activator (HGFA) activates pro-HGF in injured tissue. We evaluated the expression of HGFA and its endogenous inhibitors HAI-1 and HAI-2 in normal kidney and RCC. MATERIALS AND METHODS: We examined the gene expression of HGFA, HAI-1, HAI-2, HGF and MET in a normal kidney by laser captured microdissection, followed by reverse transcriptase-polymerase chain reaction. We also quantified the mRNA levels of these proteins in 14 RCC cases by real-time reverse transcriptase-polymerase chain reaction. RESULTS: HAI-1 and HAI-2 were abundant in the normal kidney. The uriniferous tubules showed the highest levels of HAI-1 and HAI-2 mRNA. HGFA was hardly detectable in the normal kidney. However, in the kidney with RCC a low but distinct level of HGFA mRNA became detectable in the tumor and adjacent renal tissue. The HAI-1 mRNA level was significantly and consistently down-regulated in RCC relative to normal tissue. HAI-2 mRNA was also significantly low in the advanced stage of RCC. MET was up-regulated in most cases of RCC. CONCLUSIONS: HAI-1 and HAI-2 were expressed in renal tubular epithelial cells. The expression of the 2 HAIs was significantly down-regulated in RCC, whereas HGFA expression was enhanced in the diseased kidney, suggesting an imbalance between HAI and its target proteinases, including HGFA, in favor of proteinase activities in RCC.

Expression of hepatocyte growth factor activator inhibitor type 2 (HAI-2) in human testis: identification of a distinct transcription start site for the HAI-2 gene in testis.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are recently identified integral membrane Kunitz-type proteinase inhibitors. They have important regulatory roles in pericellular activation of hepatocyte growth factor/scatter factor (HGF/SF) which is critically involved in the development and regeneration of various tissues. Recent reports suggest that HGF/SF is also involved in testicular development and spermatogenesis. In this study, we analyzed the expression of HAIs in the testis. In human testis, HAI-2 was strongly expressed whereas HAI-1 mRNA was hardly detectable. Of interest was the observation that the mRNA size of HAI-2 was shorter in the testis (1.2 kb) than those in the other tissues such as placenta (1.5 kb). Subsequent experiments revealed that there are two major transcription start sites of the HAI-2 gene, which are -30 bp and -360 bp upstream from the translation initiation ATG codon. Although the latter site appeared to be mainly used in the placenta and other non-testicular organs, only the former site is used in testis, resulting in the -300 bp shorter mRNA. An immunohistochemical study using a specific monoclonal antibody raised against human HAI-2 protein indicated that HAI-2 is expressed exclusively in primary spermatocytes. These results suggest a distinct regulation of HAI-2 gene expression in testis and that HAI-2 may play a role in the process of spermatogenesis.