LUTEINIZING AND HUMAN CHORIONIC-GONADOTROPIN HORMONES INCREASE INTERCELLULAR COMMUNICATION AND GAP-JUNCTIONS IN CULTURED MOUSE LEYDIG-CELLS

The effect of luteinizing (LH) and human chorionic gonadotropin (hCG) hormones on gap junctions (GJs) and intercellular communication (ic) w as evaluated in Leydig (interstitial) cells from mouse testes. Cell cu ltures enriched in Leydig cells were studied under control conditions and when maintained in the presence of 100 ng/mL LH, 10 ng/mL hCG, or 1 mM dibutiryl-cAMP (db-cAMP), for 8, 24, and 36 h. To monitor the ext ent of ic, Lucifer yellow (LY) was injected through a patch pipet into one cell of-small cell aggregates (6-10), and its transfer was evalua ted using fluorescent microscopy. The expression of GJs was monitored using immunofluorescent (IF) labeling of connexin 43 (Cx43) with a spe cific antibody. Testosterone secretion was determined by radioimmunoas say. At all culture times, testosterone levels in the medium were high er in treated than in control cell cultures. In cell cultures of 8 h, LY transferred to most of the neighboring cells (93%) and cell membran e appositions showed abundant Cx43; no difference was found between co ntrol and treated cells. In contrast, in control cell cultures of 24 a nd 36 h, LY transferred to a reduced fraction of neighboring cells (46 and 21 %, respectively) and Cx43 labeling was markedly decreased. Add ition of LH, hCG, or db-cAMP, to cell cultures for 24 and 36 h complet ely prevented the decrease in ic and Cx43 expression. Immunoblot studi es, from total protein homogenates of cell cultures of 36 h, showed th at relative levels of 40- and 43-kDa bands, characteristic of Cx43, we re higher in treated than in control cells. These results demonstrate that the expression of Cx43 and ic in Leydig cells is modulated by LH and hCG, and suggest that their effect is mediated by the second messe nger of these hormones, cAMP.