The Mouse Testis Is the Source of Various Serine
Proteases and Serine Proteinase Inhibitors (SERPINs):Serine Proteases and SERPINs Identified in Leydig Cells Are under Gonadotropin Regulation
Fanny Odet,Ade ′lie Verot,and Brigitte Le Magueresse-Battistoni
Institut National de la Sante ′et de la Recherche Me ′dicale,Unite ′418,Institut National de Recherche et de Se ′curite ′Unite ′Mixte de Recherche 1245,and Universite ′Lyon 1,Hopital Debrousse,69322Lyon cedex 05,France
The occurrence of various serine proteinases and serine pro-teinases inhibitors (SERPINs)was investigated by RT-PCR in whole testes of 1-,3-,and 8-wk-old mice in crude and enriched germ cell fractions,mouse Leydig tumor cells (mLTC-1),and primary cultures of 3-and 8-wk-old enriched fractions of Ley-dig cells and 3-wk-old Sertoli cells.New members were iden-tified in the testis protease repertoire.Within the Leydig rep-ertoire,a PCR product was found for plasminogen activators urokinase plasminogen activator (uPA)and tissue plasmino-gen activator (8-wk-old cells),matriptase-2(mLTC-1),kal-likrein-21,SERPINA5,SERPINB2(primary cultures),and serine peptidase inhibitor Kunitz type 2(SPINT2).The go-nadotropin regulation was explored by semiquantitative RT-PCR,using steroidogenic acute regulatory protein (StAR)as a positive control.Matriptase-2,kallikrein-21,SPINT2,and SERPINA5were down-regulated,whereas uPA and its recep-tor were up-regulated by human chorionic gonadotropin (hCG)via cAMP in the mLTC-1cells.Positive effects were observed transiently after 1–8h of hCG exposure,and nega-tive effects,first evidenced after 6h,lasted 48h.The hCG-induced effects were confirmed in primary cultures.In addi-tion,SERPINB2was augmented by hCG in primary cultures.Addition of either trypsin or protease inhibitors did not alter the hCG-induced surge of StAR.Because hCG regulated pro-teases and SERPINs (whereas testosterone did not),it could alter the proteolytic balance of Leydig cells and consequently the metabolism of extracellular matrix components.There-fore,even though a direct interplay between the early hCG-induced surge of uPA and StAR is unlikely,our data to-gether with the literature suggest that extracellular matrix proteins alter Leydig cell steroidogenesis.(Endocrinology 147:4374–4383,2006)
NUMBER OF important processes that regulate the activity and fate of many proteins are strictly depen-dent on proteolytic processing events.The serine proteinase family is one of the oldest characterized and largest families of proteolytic genes (227members in the mouse)(1,2),which has well-characterized roles in diverse cellular activities in-cluding blood coagulation,platelet activation,fibrinolysis and thrombolysis,extracellular matrix (ECM)remodeling,and cancer invasion (3).The serine proteinases can be further subdivided into 16families,among them the plasminogen activators,transmembrane serine proteinases,and kal-likreins.Serine proteinase activity is regulated by serine pro-teinase inhibitors (SERPINs).The SERPINs belong to an ex-panding superfamily of structurally similar but functionally diverse proteins,and most of them share a substrate suicide mechanism irreversibly inactivating the SERPINs (4–6).
The testis is a reproductive organ that serves two decisive functions,the production of spermatozoa taking place in the seminiferous tubules and the synthesis and secretion of tes-tosterone by the Leydig cells located in the interstitium.Spermatogenesis is hormonally regulated,pituitary gonad-otropins positively regulating Leydig and Sertoli cell secre-tions through LH and FSH,respectively.Testosterone,pro-duced as a result of LH action on Leydig cells,acts via androgen receptors localized on Sertoli,peritubular,and Leydig cells.Sertoli cells play several key roles in spermat-ogenesis.They are targets for FSH and testosterone,these hormones being responsible for the initiation and mainte-nance of spermatogenesis.Moreover,together with the peri-tubular cells,they also form the cytoarchitectural scaffolding of the tubule,providing structural and nutritional support for the developing germinal cells (7–10).
In an attempt to elucidate the role of proteinases and proteinase inhibitors in testicular physiology,we examined the presence of various serine proteinases and SERPINs in the whole testes of mice as well as isolated testicular cells.For this purpose,we used total RNA recovered from whole testes of 1-,3-,and 8-wk-old mice,from crude and enriched germ cell fractions,primary cultures of 3-wk-old Sertoli cells,3-and 8-wk-old enriched preparations of Leydig cells,and mouse Leydig tumor cells (mLTC-1).Because Leydig cells were found to express several proteinases and SERPINs,we determined whether they were under gonadotropin regula-tion.We also examined the influence of testosterone.
First Published Online June 1,2006
Abbreviations:aPC,Activated protein C;ECM,extracellular matrix;hCG,human chorionic gonadotropin;HGFA,hepatocyte growth factor activator;HPRT,hypoxanthine phosphoribosyl transferase;mLTC,mouse Leydig tumor cell;PA,plasminogen activator;PC,protein C;PCI,protein C inhibitor;SERPIN,serine proteinase inhibitor;SPINT1or 2,serine peptidase inhibitor Kunitz type 1or 2;StAR,steroidogenic acute regulatory protein;tPA,tissue plasminogen activator;uPA,urokinase plasminogen activator;uPAR,uPA receptor.
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