Expression and regulation of connexin43 in rat Leydig cells

Gap junctions are intercellular protein channels which provide a pathway fo r the exchange of ions and small molecules. This exchange of materials allo ws metabolic coupling of cells. Gap junction channels are made up of connex ins, integral membrane proteins encoded by a multigene family. Rat testes c ontain mRNAs for at least five different connexins: Cx26, Cx32, Cx33, Cx37 and Cx43. Immunocytochemical studies have shown chat Cx43 assembles gap jun ctions between Leydig cells. The present study investigated the expression and regulation of the Cx43 gene in rat Leydig cells. Purified Leydig cells were obtained from 40- to 80-day-old Sprague-Dawley rats using a combinatio n of arterial perfusion, collagenase digestion, centrifugal elutriation and Percoll. gradient centrifugation. Leydig cells from 20- and 30-day-old rat s were isolated without arterial perfusion or centrifugal elutriation. Cx43 mRNA was present in 20-day-old rat Leydig cells, reached a plate au at day 40, and remained at high levels in 65- and 80-day-old rat Leydig cells. To evaluate the regulation of Cx43 gene expression, Leydig cells were culture d overnight and then treated with human chorionic gonadotropin (IICG) for v ariable periods of time. Addition of hCG (10 ng/ml) increased cytochrome P4 50 side-chain cleavage and steroidogenic acute regulatory protein mRNA leve ls and testosterone formation, However, Cx43 mRNA levels were inhibited by hCG in a time- and dose-dependent manner. Cx43 mRNA levels decreased 27% as early as 2h after the addition of hCG and decreased 60% by 24 h. Treatment of Leydig cells with 8-bromo-cAMP (0.1 mM) for 6 and 24 h also reduced Cx4 3 mRNA levels by 36 and 56% respectively. Primary cultured Leydig cells sta ined strongly positive with anti-Cx43 monoclonal antibody. Treatment with h CG for 24 h reduced Cx43 signals and caused Cx43 to redistribute to the per iphery of the cells. To evaluate the regulation of Cx43 in vitro, rats were treated with hCG (300 ng i.p.) and testes were removed 24 h later. Frozen section of testes revealed that these interstitial cells stained positive f or 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) by histochemical staini ng and were positive for Cx33 by immunofluorescence staining. The adjacent seminiferous tubules stained only weakly positive for Cx33. Twenty-four hou rs after hCG treatment, 3 beta-HSD activity increased while Cx43 immunostai ning of Leydig cells was reduced. In conclusion, gap junction channels of L eydig cells are regulated by hCG both in vivo and in vitro. hCG increased L eydig cell steroidogenesis and steroidogenic enzyme mRNA levels but caused a redistribution of Cx43.