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A Novel Gonadotropin-regulated Testicular RNA Helicase

1999; Elsevier BV; Volume: 274; Issue: 53 Linguagem: Inglês

10.1074/jbc.274.53.37932

1083-351X

Pei‐Zhong Tang, Chon‐Hwa Tsai‐Morris, Maria Dufau,

Reproductive Biology and Fertility

A gonadotropin-regulated testicular RNA helicase (GRTH) was identified and characterized. GRTH cloned from rat Leydig cell, mouse testis, and human testis cDNA libraries is a novel member of the DEAD-box protein family. GRTH is transcriptionally up-regulated by chorionic gonadotropin via cyclic AMP-induced androgen formation in the Leydig cell. It has ATPase and RNA helicase activities and increases translation in vitro. This helicase is highly expressed in rat, mouse, and human testes and weakly expressed in the pituitary and hypothalamus. GRTH is produced in both somatic (Leydig cells) and germinal (meiotic spermatocytes and round haploid spermatids) cells and is developmentally regulated. GRTH predominantly localized in the cytoplasm may function as a translational activator. This novel helicase could be relevant to the control of steroidogenesis and the paracrine regulation of androgen-dependent spermatogenesis in the testis. A gonadotropin-regulated testicular RNA helicase (GRTH) was identified and characterized. GRTH cloned from rat Leydig cell, mouse testis, and human testis cDNA libraries is a novel member of the DEAD-box protein family. GRTH is transcriptionally up-regulated by chorionic gonadotropin via cyclic AMP-induced androgen formation in the Leydig cell. It has ATPase and RNA helicase activities and increases translation in vitro. This helicase is highly expressed in rat, mouse, and human testes and weakly expressed in the pituitary and hypothalamus. GRTH is produced in both somatic (Leydig cells) and germinal (meiotic spermatocytes and round haploid spermatids) cells and is developmentally regulated. GRTH predominantly localized in the cytoplasm may function as a translational activator. This novel helicase could be relevant to the control of steroidogenesis and the paracrine regulation of androgen-dependent spermatogenesis in the testis. luteinizing hormone human chorionic gonadotropin gonadotropin-regulated testicular RNA helicase polymerase chain reaction fluorescence in situ hybridization base pair glutathione S-transferase polyacrylamide gel electrophoresis bovine serum albumin kilobase pairs nucleotide EGFP, enhanced green fluorescence protein green fluorescence protein The closely related gonadotropins, luteinizing hormone (LH)1 and human chorionic gonadotropin (hCG), exert their functions through specific G protein-coupled receptors in gonadal cells. Physiological concentrations of gonadotropins maintain the steroidogenic function of the Leydig cells of the testis (see Ref. 1Dufau M.L. Annu. Rev. Physiol. 1988; 50: 483-508Crossref PubMed Scopus (223) Google Scholar for review). However, high concentrations of gonadotropins cause desensitization of steroidogenic enzymes of the androgen pathway, with consequent reduction of testosterone formation (2Dufau M.L. Cigorraga S.B. Baukal A.J. Bator J.M. Sorrell S.H. Neubauer J.F. Catt K.J. J. Steroid Biochem. 1979; 11: 193-199Crossref PubMed Scopus (33) Google Scholar, 3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar, 4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar, 5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar). The attenuation of steroidogenesis results from receptor activation by the gonadotropic hormone but is independent of the subsequent phase of receptor down-regulation. The control of the enzymes involved in this regulation including 3β-hydroxysteroid dehydrogenase types I and II (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar), 17α-hydroxylase/17, 20 lyase (5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar), and 17β-hydroxysteroid dehydrogenase type III (4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar) operates at the transcriptional level. Because this process affects several enzymes, the direct or indirect involvement of a master switch has been proposed (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar). Likely candidates for this regulation include the active steroid metabolites that are produced during gonadotropic stimulation, i.e. androgen (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar) and estrogen (6Dufau M.L. Catt K.J. Vitam. Horm. 1978; 36: 461-592Crossref PubMed Scopus (131) Google Scholar, 7Nozu K. Dehejia A. Zawistowich L. Catt K.J. Dufau M.L. J. Biol. Chem. 1981; 256: 12875-12882Abstract Full Text PDF PubMed Google Scholar, 8Nozu K. Matsuura S. Catt K.J. Dufau M.L. J. Biol. Chem. 1981; 256: 10012-10017Abstract Full Text PDF PubMed Google Scholar).To identify potentially relevant proteins that participate in gonadotropin up- or down-regulation of steroidogenic enzymes at the transcriptional or post-transcriptional level, we utilized differential display analysis of RNA from testicular Leydig cells subjected to a single exposure of gonadotropin in vivo. We report the demonstration of a novel gonadotropin-regulated testicular RNA helicase (GRTH) that belongs to the family of DEAD-box proteins and is predominantly expressed in Leydig and germinal meiotic cells of the testis. This protein was found to be markedly up-regulated in the Leydig cell by hCG doses that cause steroidogenic desensitization. It is likely that GRTH serves in general to maintain receptors, enzymes, and factors that support testicular functions and spermatogenesis.DISCUSSIONIn this study, we have cloned and characterized a novel gonadotropin-regulated and developmentally expressed testicular RNA helicase (GRTH). This protein, which is expressed in the Leydig and germ cells of the testis, belongs to the DEAD (Asp-Glu-Ala-Asp)-box family within the superfamily of RNA helicases (28Schmid S.R. Linder P. Mol. Microbiol. 1992; 6: 283-291Crossref PubMed Scopus (448) Google Scholar) and contains all the conserved domains of the family. GRTH differs from other members of this family in having high intrinsic ATPase activity in the absence of RNA but resembles several members (19Pause A. Sonenberg N. EMBO J. 1992; 11: 2643-2654Crossref PubMed Scopus (529) Google Scholar, 21Tseng S.S. Weaver P.L. Liu Y. Hitomi M. Tartakoff A.M. Chang T.H. EMBO J. 1998; 17: 2651-2662Crossref PubMed Scopus (220) Google Scholar, 27Hirling H. Scheffner M. Restle T. Stahl H. Nature. 1989; 339: 562-564Crossref PubMed Scopus (240) Google Scholar), in that its ATPase activity was remarkably enhanced in presence of mRNA, synthetic poly(A), and DNA. Thus, GRTH not only catalyzed ATP hydrolysis to supply energy for RNA processes but perhaps also for some other biological processes. The DNA-enhanced ATP hydrolysis is not utilized for the process of unwinding DNA duplexes. Bidirectional unwinding of RNA duplexes suggested that GRTH is not only a less restricting ATPase and but also a less restricting RNA helicase. These functional features indicate that GRTH could be involved in a variety of biological processes in the target tissues. It is very likely that GRTH participates in poly(A)+-related mRNA processes including melting the secondary structure of mRNA and initiation of translation.GRTH is the first protein of the DEAD-box family reported to be regulated by a hormone. The initial goal of the study was to identify factor(s) responsible for gonadotropin down-regulation of gonadal receptors and steroidogenic enzymes of testicular Leydig cells that would consequently reflect the production of androgen. On the other hand, for maintenance of the expression of relevant and/or general cellular genes and the restoration of the cellular down-regulated functions, some activators may be induced or enhanced during the initial hormonal stimulus or early in the desensitization process. GRTH was the only displayed fragment that was verified as an hCG up-regulated gene. However, the regulation of GRTH increase by hCG did not precede the reduction of these enzymes (2Dufau M.L. Cigorraga S.B. Baukal A.J. Bator J.M. Sorrell S.H. Neubauer J.F. Catt K.J. J. Steroid Biochem. 1979; 11: 193-199Crossref PubMed Scopus (33) Google Scholar, 3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar, 4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar, 5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar). This suggested that GRTH does not play a role in the down-regulation phase of the desensitizing process but perhaps in other gonadotropin-regulated cellular functions, including maintenance of steroidogenic enzymes and receptors and their recovery from desensitization. In this regard, the return of GRTH expression to control correlated with the temporal recovery of enzymes and receptors (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar, 4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar, 5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar).We also demonstrated that the up-regulation of GRTH gene expression results from direct and/or indirect actions at the transcriptional level of signaling molecules or metabolic products induced by hCG. The regulation of GRTH could share aspects of the control mechanism(s) responsible for hCG-induced down-regulation of steroidogenic enzymes and receptors. Our cotransfection studies excluded the involvement of GRTH as a transcriptional inhibitor responsible for hCG-induced desensitization of receptors and steroidogenic enzymes (Fig. 6 A). The similar GRTH-induced increases of reporter gene activities driven by different promoters in the absence of mRNA changes implied that the stimulating action of GRTH was through a common mechanism at the post-transcriptional level. The increasedin vitro translational activity induced by GRTH and the predominant localization of GRTH-GFP in the cytoplasm of transfected cells (Fig. 6, B and C) support its involvement in the translational process. However, its participation in other biological processes cannot be excluded, since weak nuclear localization was observed, and both RNA and DNA can stimulate ATPase activity of GRTH. Thus, it is conceivable that the up-regulation of the GRTH gene product induced by the hormonal stimuli may contribute to the translatability of hormone receptors and steroidogenic enzymes and other proteins.GRTH is specifically expressed in male but not in female gonadal tissue. In addition to its expression in the Leydig cells of the interstitial compartment of the testis, GRTH is also highly expressed in meiotic and round haploid germinal cells (Fig. 3). Differentiation in germ cells requires enhanced activation of transcription and translation of specific genes in which GRTH may play important roles. Interestingly, some members of the RNA helicase family were expressed differentially during development or restrictively distributed in germ cells (24de Valoir T. Tucker M.A. Belikoff E.J. Camp L.A. Bolduc C. Beckingham K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2113-2117Crossref PubMed Scopus (63) Google Scholar, 25Maekawa H. Nakagawa T. Uno Y. Kitamura K. Shimoda C. Mol. Gen. Genet. 1994; 244: 456-464Crossref PubMed Scopus (54) Google Scholar, 26Leroy P. Alzari P. Sassoon D. Wolgemuth D. Fellous M. Cell. 1989; 57: 549-559Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 29Gururajan R. Perry O.K.H. Melton D.A. Weeks D.L Nature. 1991; 349: 717-719Crossref PubMed Scopus (70) Google Scholar), and it has been suggested that these proteins may actively participate in sexual development (25Maekawa H. Nakagawa T. Uno Y. Kitamura K. Shimoda C. Mol. Gen. Genet. 1994; 244: 456-464Crossref PubMed Scopus (54) Google Scholar) and in the process of oogenesis (24de Valoir T. Tucker M.A. Belikoff E.J. Camp L.A. Bolduc C. Beckingham K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2113-2117Crossref PubMed Scopus (63) Google Scholar, 29Gururajan R. Perry O.K.H. Melton D.A. Weeks D.L Nature. 1991; 349: 717-719Crossref PubMed Scopus (70) Google Scholar) and spermatogenesis (26Leroy P. Alzari P. Sassoon D. Wolgemuth D. Fellous M. Cell. 1989; 57: 549-559Abstract Full Text PDF PubMed Scopus (141) Google Scholar). The structure and function of GRTH links this new helicase with other members of the RNA helicase family, including eIF4A (22Jaramillo M. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1991; 11: 5992-5997Crossref PubMed Scopus (86) Google Scholar, 30Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Crossref PubMed Scopus (497) Google Scholar), Ded1p (31Chuang R.Y. Weaver P.L. Liu Z. Chang T.H. Science. 1997; 275: 1468-1471Crossref PubMed Scopus (266) Google Scholar), and PL10 proteins (26Leroy P. Alzari P. Sassoon D. Wolgemuth D. Fellous M. Cell. 1989; 57: 549-559Abstract Full Text PDF PubMed Scopus (141) Google Scholar).The male gonad-specific expression of GRTH and the striking increase of its mRNA in pubertal and post-pubertal rodents suggest that GRTH is functionally related to gonadal maturation and spermatogenesis. The developmental increase in GRTH in the pubertal rodent testis occurs at the same time as gonadotropin-induced androgen responses appear in the Leydig cell (32Huhtaniemi I.T. Nozu K. Warren D.W. Dufau M.L. Catt K.J. Endocrinology. 1982; 111: 1711-1720Crossref PubMed Scopus (82) Google Scholar), and when the differentiation of germ cells that ultimately leads to the development of haploid spermatids begins to occur (33Clermont Y. Perey B. Am. J. Anat. 1957; 100: 241-268Crossref PubMed Scopus (529) Google Scholar). It is reasonable to propose that the steroid hormone produced in the gonadotropin-stimulated testis could not only exert intracrine or autocrine actions on GRTH gene expression in the Leydig cell but may also influence GRTH gene transcription within the seminiferous tubule. The notably high expression of the helicase in pachytene spermatocytes indicates that GRTH may be one of the genes involved in the meiotic process that has a crucial role in spermatogenesis (34Edelmann W. Cohen P.E. Kane M. Lau K. Morrow B. Bennett S. Umar A. Kunkel T. Cattoretti G. Chaganti R. Pollard J.W. Kolodner R.D. Kucherlapati R. Cell. 1996; 85: 1125-1134Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar).The specific expression of GRTH in the testis, but not in ovary, implies that its gene is either suppressed by female-specific or induced by male-specific gonadal factor(s). Since estrogen is produced in both male and female gonads, it is unlikely to be involved in the induction of GRTH gene expression. Consistent with this, estradiol did not affect GRTH mRNA in cultured rat Leydig cells. In contrast, the non-metabolizable androgen, dihydrotestosterone, increased the expression of GRTH mRNA in Leydig cells. The stimulation effects of hCG in vivo on the GRTH gene expression were also observedin vitro, and cAMP, the second messenger of hCG action, had comparable effects. However, no stimulation was observed after inhibition of steroid biosynthetic enzymes, which abolished hCG- and cAMP-stimulated increases of testosterone in Leydig cells. The absence of a rapid change in basal GRTH mRNA levels during androgen suppression in vitro is not unexpected, since incubations could only be conducted with short term primary cultures and the GRTH mRNA is long-lived (Fig. 4 C). Androgen induced by gonadotropin could act through its cognate receptors in the Leydig cell to increase GRTH gene expression. It could also exert actions in the germinal cells of the seminiferous epithelium through androgen receptors present in Sertoli cells, since germinal cells do not possess androgen receptors (35Bremner W.J. Millar M.R. Sharpe R.M. Saunders P.T. Endocrinology. 1994; 135: 1227-1234Crossref PubMed Scopus (310) Google Scholar). Although androgens are not present in the pituitary, hypothalamus, and brain, where low GRTH mRNA levels are found, androgen receptors are present in these tissues. It is conceivable that androgen from the circulation may induce GRTH expression at these sites. Major developmental changes in GRTH mRNA were observed during puberty when predominant expression of GRTH mRNA was observed in the Leydig cell and germinal epithelium. Our findings suggest that this novel testicular helicase could serve to maintain testicular functions related to steroidogenesis and spermatogenesis. The closely related gonadotropins, luteinizing hormone (LH)1 and human chorionic gonadotropin (hCG), exert their functions through specific G protein-coupled receptors in gonadal cells. Physiological concentrations of gonadotropins maintain the steroidogenic function of the Leydig cells of the testis (see Ref. 1Dufau M.L. Annu. Rev. Physiol. 1988; 50: 483-508Crossref PubMed Scopus (223) Google Scholar for review). However, high concentrations of gonadotropins cause desensitization of steroidogenic enzymes of the androgen pathway, with consequent reduction of testosterone formation (2Dufau M.L. Cigorraga S.B. Baukal A.J. Bator J.M. Sorrell S.H. Neubauer J.F. Catt K.J. J. Steroid Biochem. 1979; 11: 193-199Crossref PubMed Scopus (33) Google Scholar, 3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar, 4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar, 5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar). The attenuation of steroidogenesis results from receptor activation by the gonadotropic hormone but is independent of the subsequent phase of receptor down-regulation. The control of the enzymes involved in this regulation including 3β-hydroxysteroid dehydrogenase types I and II (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar), 17α-hydroxylase/17, 20 lyase (5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar), and 17β-hydroxysteroid dehydrogenase type III (4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar) operates at the transcriptional level. Because this process affects several enzymes, the direct or indirect involvement of a master switch has been proposed (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar). Likely candidates for this regulation include the active steroid metabolites that are produced during gonadotropic stimulation, i.e. androgen (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar) and estrogen (6Dufau M.L. Catt K.J. Vitam. Horm. 1978; 36: 461-592Crossref PubMed Scopus (131) Google Scholar, 7Nozu K. Dehejia A. Zawistowich L. Catt K.J. Dufau M.L. J. Biol. Chem. 1981; 256: 12875-12882Abstract Full Text PDF PubMed Google Scholar, 8Nozu K. Matsuura S. Catt K.J. Dufau M.L. J. Biol. Chem. 1981; 256: 10012-10017Abstract Full Text PDF PubMed Google Scholar). To identify potentially relevant proteins that participate in gonadotropin up- or down-regulation of steroidogenic enzymes at the transcriptional or post-transcriptional level, we utilized differential display analysis of RNA from testicular Leydig cells subjected to a single exposure of gonadotropin in vivo. We report the demonstration of a novel gonadotropin-regulated testicular RNA helicase (GRTH) that belongs to the family of DEAD-box proteins and is predominantly expressed in Leydig and germinal meiotic cells of the testis. This protein was found to be markedly up-regulated in the Leydig cell by hCG doses that cause steroidogenic desensitization. It is likely that GRTH serves in general to maintain receptors, enzymes, and factors that support testicular functions and spermatogenesis. DISCUSSIONIn this study, we have cloned and characterized a novel gonadotropin-regulated and developmentally expressed testicular RNA helicase (GRTH). This protein, which is expressed in the Leydig and germ cells of the testis, belongs to the DEAD (Asp-Glu-Ala-Asp)-box family within the superfamily of RNA helicases (28Schmid S.R. Linder P. Mol. Microbiol. 1992; 6: 283-291Crossref PubMed Scopus (448) Google Scholar) and contains all the conserved domains of the family. GRTH differs from other members of this family in having high intrinsic ATPase activity in the absence of RNA but resembles several members (19Pause A. Sonenberg N. EMBO J. 1992; 11: 2643-2654Crossref PubMed Scopus (529) Google Scholar, 21Tseng S.S. Weaver P.L. Liu Y. Hitomi M. Tartakoff A.M. Chang T.H. EMBO J. 1998; 17: 2651-2662Crossref PubMed Scopus (220) Google Scholar, 27Hirling H. Scheffner M. Restle T. Stahl H. Nature. 1989; 339: 562-564Crossref PubMed Scopus (240) Google Scholar), in that its ATPase activity was remarkably enhanced in presence of mRNA, synthetic poly(A), and DNA. Thus, GRTH not only catalyzed ATP hydrolysis to supply energy for RNA processes but perhaps also for some other biological processes. The DNA-enhanced ATP hydrolysis is not utilized for the process of unwinding DNA duplexes. Bidirectional unwinding of RNA duplexes suggested that GRTH is not only a less restricting ATPase and but also a less restricting RNA helicase. These functional features indicate that GRTH could be involved in a variety of biological processes in the target tissues. It is very likely that GRTH participates in poly(A)+-related mRNA processes including melting the secondary structure of mRNA and initiation of translation.GRTH is the first protein of the DEAD-box family reported to be regulated by a hormone. The initial goal of the study was to identify factor(s) responsible for gonadotropin down-regulation of gonadal receptors and steroidogenic enzymes of testicular Leydig cells that would consequently reflect the production of androgen. On the other hand, for maintenance of the expression of relevant and/or general cellular genes and the restoration of the cellular down-regulated functions, some activators may be induced or enhanced during the initial hormonal stimulus or early in the desensitization process. GRTH was the only displayed fragment that was verified as an hCG up-regulated gene. However, the regulation of GRTH increase by hCG did not precede the reduction of these enzymes (2Dufau M.L. Cigorraga S.B. Baukal A.J. Bator J.M. Sorrell S.H. Neubauer J.F. Catt K.J. J. Steroid Biochem. 1979; 11: 193-199Crossref PubMed Scopus (33) Google Scholar, 3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar, 4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar, 5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar). This suggested that GRTH does not play a role in the down-regulation phase of the desensitizing process but perhaps in other gonadotropin-regulated cellular functions, including maintenance of steroidogenic enzymes and receptors and their recovery from desensitization. In this regard, the return of GRTH expression to control correlated with the temporal recovery of enzymes and receptors (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar, 4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar, 5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar).We also demonstrated that the up-regulation of GRTH gene expression results from direct and/or indirect actions at the transcriptional level of signaling molecules or metabolic products induced by hCG. The regulation of GRTH could share aspects of the control mechanism(s) responsible for hCG-induced down-regulation of steroidogenic enzymes and receptors. Our cotransfection studies excluded the involvement of GRTH as a transcriptional inhibitor responsible for hCG-induced desensitization of receptors and steroidogenic enzymes (Fig. 6 A). The similar GRTH-induced increases of reporter gene activities driven by different promoters in the absence of mRNA changes implied that the stimulating action of GRTH was through a common mechanism at the post-transcriptional level. The increasedin vitro translational activity induced by GRTH and the predominant localization of GRTH-GFP in the cytoplasm of transfected cells (Fig. 6, B and C) support its involvement in the translational process. However, its participation in other biological processes cannot be excluded, since weak nuclear localization was observed, and both RNA and DNA can stimulate ATPase activity of GRTH. Thus, it is conceivable that the up-regulation of the GRTH gene product induced by the hormonal stimuli may contribute to the translatability of hormone receptors and steroidogenic enzymes and other proteins.GRTH is specifically expressed in male but not in female gonadal tissue. In addition to its expression in the Leydig cells of the interstitial compartment of the testis, GRTH is also highly expressed in meiotic and round haploid germinal cells (Fig. 3). Differentiation in germ cells requires enhanced activation of transcription and translation of specific genes in which GRTH may play important roles. Interestingly, some members of the RNA helicase family were expressed differentially during development or restrictively distributed in germ cells (24de Valoir T. Tucker M.A. Belikoff E.J. Camp L.A. Bolduc C. Beckingham K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2113-2117Crossref PubMed Scopus (63) Google Scholar, 25Maekawa H. Nakagawa T. Uno Y. Kitamura K. Shimoda C. Mol. Gen. Genet. 1994; 244: 456-464Crossref PubMed Scopus (54) Google Scholar, 26Leroy P. Alzari P. Sassoon D. Wolgemuth D. Fellous M. Cell. 1989; 57: 549-559Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 29Gururajan R. Perry O.K.H. Melton D.A. Weeks D.L Nature. 1991; 349: 717-719Crossref PubMed Scopus (70) Google Scholar), and it has been suggested that these proteins may actively participate in sexual development (25Maekawa H. Nakagawa T. Uno Y. Kitamura K. Shimoda C. Mol. Gen. Genet. 1994; 244: 456-464Crossref PubMed Scopus (54) Google Scholar) and in the process of oogenesis (24de Valoir T. Tucker M.A. Belikoff E.J. Camp L.A. Bolduc C. Beckingham K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2113-2117Crossref PubMed Scopus (63) Google Scholar, 29Gururajan R. Perry O.K.H. Melton D.A. Weeks D.L Nature. 1991; 349: 717-719Crossref PubMed Scopus (70) Google Scholar) and spermatogenesis (26Leroy P. Alzari P. Sassoon D. Wolgemuth D. Fellous M. Cell. 1989; 57: 549-559Abstract Full Text PDF PubMed Scopus (141) Google Scholar). The structure and function of GRTH links this new helicase with other members of the RNA helicase family, including eIF4A (22Jaramillo M. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1991; 11: 5992-5997Crossref PubMed Scopus (86) Google Scholar, 30Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Crossref PubMed Scopus (497) Google Scholar), Ded1p (31Chuang R.Y. Weaver P.L. Liu Z. Chang T.H. Science. 1997; 275: 1468-1471Crossref PubMed Scopus (266) Google Scholar), and PL10 proteins (26Leroy P. Alzari P. Sassoon D. Wolgemuth D. Fellous M. Cell. 1989; 57: 549-559Abstract Full Text PDF PubMed Scopus (141) Google Scholar).The male gonad-specific expression of GRTH and the striking increase of its mRNA in pubertal and post-pubertal rodents suggest that GRTH is functionally related to gonadal maturation and spermatogenesis. The developmental increase in GRTH in the pubertal rodent testis occurs at the same time as gonadotropin-induced androgen responses appear in the Leydig cell (32Huhtaniemi I.T. Nozu K. Warren D.W. Dufau M.L. Catt K.J. Endocrinology. 1982; 111: 1711-1720Crossref PubMed Scopus (82) Google Scholar), and when the differentiation of germ cells that ultimately leads to the development of haploid spermatids begins to occur (33Clermont Y. Perey B. Am. J. Anat. 1957; 100: 241-268Crossref PubMed Scopus (529) Google Scholar). It is reasonable to propose that the steroid hormone produced in the gonadotropin-stimulated testis could not only exert intracrine or autocrine actions on GRTH gene expression in the Leydig cell but may also influence GRTH gene transcription within the seminiferous tubule. The notably high expression of the helicase in pachytene spermatocytes indicates that GRTH may be one of the genes involved in the meiotic process that has a crucial role in spermatogenesis (34Edelmann W. Cohen P.E. Kane M. Lau K. Morrow B. Bennett S. Umar A. Kunkel T. Cattoretti G. Chaganti R. Pollard J.W. Kolodner R.D. Kucherlapati R. Cell. 1996; 85: 1125-1134Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar).The specific expression of GRTH in the testis, but not in ovary, implies that its gene is either suppressed by female-specific or induced by male-specific gonadal factor(s). Since estrogen is produced in both male and female gonads, it is unlikely to be involved in the induction of GRTH gene expression. Consistent with this, estradiol did not affect GRTH mRNA in cultured rat Leydig cells. In contrast, the non-metabolizable androgen, dihydrotestosterone, increased the expression of GRTH mRNA in Leydig cells. The stimulation effects of hCG in vivo on the GRTH gene expression were also observedin vitro, and cAMP, the second messenger of hCG action, had comparable effects. However, no stimulation was observed after inhibition of steroid biosynthetic enzymes, which abolished hCG- and cAMP-stimulated increases of testosterone in Leydig cells. The absence of a rapid change in basal GRTH mRNA levels during androgen suppression in vitro is not unexpected, since incubations could only be conducted with short term primary cultures and the GRTH mRNA is long-lived (Fig. 4 C). Androgen induced by gonadotropin could act through its cognate receptors in the Leydig cell to increase GRTH gene expression. It could also exert actions in the germinal cells of the seminiferous epithelium through androgen receptors present in Sertoli cells, since germinal cells do not possess androgen receptors (35Bremner W.J. Millar M.R. Sharpe R.M. Saunders P.T. Endocrinology. 1994; 135: 1227-1234Crossref PubMed Scopus (310) Google Scholar). Although androgens are not present in the pituitary, hypothalamus, and brain, where low GRTH mRNA levels are found, androgen receptors are present in these tissues. It is conceivable that androgen from the circulation may induce GRTH expression at these sites. Major developmental changes in GRTH mRNA were observed during puberty when predominant expression of GRTH mRNA was observed in the Leydig cell and germinal epithelium. Our findings suggest that this novel testicular helicase could serve to maintain testicular functions related to steroidogenesis and spermatogenesis. In this study, we have cloned and characterized a novel gonadotropin-regulated and developmentally expressed testicular RNA helicase (GRTH). This protein, which is expressed in the Leydig and germ cells of the testis, belongs to the DEAD (Asp-Glu-Ala-Asp)-box family within the superfamily of RNA helicases (28Schmid S.R. Linder P. Mol. Microbiol. 1992; 6: 283-291Crossref PubMed Scopus (448) Google Scholar) and contains all the conserved domains of the family. GRTH differs from other members of this family in having high intrinsic ATPase activity in the absence of RNA but resembles several members (19Pause A. Sonenberg N. EMBO J. 1992; 11: 2643-2654Crossref PubMed Scopus (529) Google Scholar, 21Tseng S.S. Weaver P.L. Liu Y. Hitomi M. Tartakoff A.M. Chang T.H. EMBO J. 1998; 17: 2651-2662Crossref PubMed Scopus (220) Google Scholar, 27Hirling H. Scheffner M. Restle T. Stahl H. Nature. 1989; 339: 562-564Crossref PubMed Scopus (240) Google Scholar), in that its ATPase activity was remarkably enhanced in presence of mRNA, synthetic poly(A), and DNA. Thus, GRTH not only catalyzed ATP hydrolysis to supply energy for RNA processes but perhaps also for some other biological processes. The DNA-enhanced ATP hydrolysis is not utilized for the process of unwinding DNA duplexes. Bidirectional unwinding of RNA duplexes suggested that GRTH is not only a less restricting ATPase and but also a less restricting RNA helicase. These functional features indicate that GRTH could be involved in a variety of biological processes in the target tissues. It is very likely that GRTH participates in poly(A)+-related mRNA processes including melting the secondary structure of mRNA and initiation of translation. GRTH is the first protein of the DEAD-box family reported to be regulated by a hormone. The initial goal of the study was to identify factor(s) responsible for gonadotropin down-regulation of gonadal receptors and steroidogenic enzymes of testicular Leydig cells that would consequently reflect the production of androgen. On the other hand, for maintenance of the expression of relevant and/or general cellular genes and the restoration of the cellular down-regulated functions, some activators may be induced or enhanced during the initial hormonal stimulus or early in the desensitization process. GRTH was the only displayed fragment that was verified as an hCG up-regulated gene. However, the regulation of GRTH increase by hCG did not precede the reduction of these enzymes (2Dufau M.L. Cigorraga S.B. Baukal A.J. Bator J.M. Sorrell S.H. Neubauer J.F. Catt K.J. J. Steroid Biochem. 1979; 11: 193-199Crossref PubMed Scopus (33) Google Scholar, 3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar, 4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar, 5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar). This suggested that GRTH does not play a role in the down-regulation phase of the desensitizing process but perhaps in other gonadotropin-regulated cellular functions, including maintenance of steroidogenic enzymes and receptors and their recovery from desensitization. In this regard, the return of GRTH expression to control correlated with the temporal recovery of enzymes and receptors (3Tang P.Z. Tsai-Morris C.H. Dufau M.L. Endocrinology. 1998; 139: 4496-4505Crossref PubMed Scopus (34) Google Scholar, 4Tsai-Morris C.H. Khanum A. Tang P.-Z. Dufau M.L. Endocrinology. 1999; 140: 3534-3542Crossref PubMed Scopus (40) Google Scholar, 5Nishihara M. Winters C.A. Buzko E. Waterman M.R. Dufau M.L. Biochem. Biophys. Res. Commun. 1988; 154: 151-158Crossref PubMed Scopus (36) Google Scholar). We also demonstrated that the up-regulation of GRTH gene expression results from direct and/or indirect actions at the transcriptional level of signaling molecules or metabolic products induced by hCG. The regulation of GRTH could share aspects of the control mechanism(s) responsible for hCG-induced down-regulation of steroidogenic enzymes and receptors. Our cotransfection studies excluded the involvement of GRTH as a transcriptional inhibitor responsible for hCG-induced desensitization of receptors and steroidogenic enzymes (Fig. 6 A). The similar GRTH-induced increases of reporter gene activities driven by different promoters in the absence of mRNA changes implied that the stimulating action of GRTH was through a common mechanism at the post-transcriptional level. The increasedin vitro translational activity induced by GRTH and the predominant localization of GRTH-GFP in the cytoplasm of transfected cells (Fig. 6, B and C) support its involvement in the translational process. However, its participation in other biological processes cannot be excluded, since weak nuclear localization was observed, and both RNA and DNA can stimulate ATPase activity of GRTH. Thus, it is conceivable that the up-regulation of the GRTH gene product induced by the hormonal stimuli may contribute to the translatability of hormone receptors and steroidogenic enzymes and other proteins. GRTH is specifically expressed in male but not in female gonadal tissue. In addition to its expression in the Leydig cells of the interstitial compartment of the testis, GRTH is also highly expressed in meiotic and round haploid germinal cells (Fig. 3). Differentiation in germ cells requires enhanced activation of transcription and translation of specific genes in which GRTH may play important roles. Interestingly, some members of the RNA helicase family were expressed differentially during development or restrictively distributed in germ cells (24de Valoir T. Tucker M.A. Belikoff E.J. Camp L.A. Bolduc C. Beckingham K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2113-2117Crossref PubMed Scopus (63) Google Scholar, 25Maekawa H. Nakagawa T. Uno Y. Kitamura K. Shimoda C. Mol. Gen. Genet. 1994; 244: 456-464Crossref PubMed Scopus (54) Google Scholar, 26Leroy P. Alzari P. Sassoon D. Wolgemuth D. Fellous M. Cell. 1989; 57: 549-559Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 29Gururajan R. Perry O.K.H. Melton D.A. Weeks D.L Nature. 1991; 349: 717-719Crossref PubMed Scopus (70) Google Scholar), and it has been suggested that these proteins may actively participate in sexual development (25Maekawa H. Nakagawa T. Uno Y. Kitamura K. Shimoda C. Mol. Gen. Genet. 1994; 244: 456-464Crossref PubMed Scopus (54) Google Scholar) and in the process of oogenesis (24de Valoir T. Tucker M.A. Belikoff E.J. Camp L.A. Bolduc C. Beckingham K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2113-2117Crossref PubMed Scopus (63) Google Scholar, 29Gururajan R. Perry O.K.H. Melton D.A. Weeks D.L Nature. 1991; 349: 717-719Crossref PubMed Scopus (70) Google Scholar) and spermatogenesis (26Leroy P. Alzari P. Sassoon D. Wolgemuth D. Fellous M. Cell. 1989; 57: 549-559Abstract Full Text PDF PubMed Scopus (141) Google Scholar). The structure and function of GRTH links this new helicase with other members of the RNA helicase family, including eIF4A (22Jaramillo M. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1991; 11: 5992-5997Crossref PubMed Scopus (86) Google Scholar, 30Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Crossref PubMed Scopus (497) Google Scholar), Ded1p (31Chuang R.Y. Weaver P.L. Liu Z. Chang T.H. Science. 1997; 275: 1468-1471Crossref PubMed Scopus (266) Google Scholar), and PL10 proteins (26Leroy P. Alzari P. Sassoon D. Wolgemuth D. Fellous M. Cell. 1989; 57: 549-559Abstract Full Text PDF PubMed Scopus (141) Google Scholar). The male gonad-specific expression of GRTH and the striking increase of its mRNA in pubertal and post-pubertal rodents suggest that GRTH is functionally related to gonadal maturation and spermatogenesis. The developmental increase in GRTH in the pubertal rodent testis occurs at the same time as gonadotropin-induced androgen responses appear in the Leydig cell (32Huhtaniemi I.T. Nozu K. Warren D.W. Dufau M.L. Catt K.J. Endocrinology. 1982; 111: 1711-1720Crossref PubMed Scopus (82) Google Scholar), and when the differentiation of germ cells that ultimately leads to the development of haploid spermatids begins to occur (33Clermont Y. Perey B. Am. J. Anat. 1957; 100: 241-268Crossref PubMed Scopus (529) Google Scholar). It is reasonable to propose that the steroid hormone produced in the gonadotropin-stimulated testis could not only exert intracrine or autocrine actions on GRTH gene expression in the Leydig cell but may also influence GRTH gene transcription within the seminiferous tubule. The notably high expression of the helicase in pachytene spermatocytes indicates that GRTH may be one of the genes involved in the meiotic process that has a crucial role in spermatogenesis (34Edelmann W. Cohen P.E. Kane M. Lau K. Morrow B. Bennett S. Umar A. Kunkel T. Cattoretti G. Chaganti R. Pollard J.W. Kolodner R.D. Kucherlapati R. Cell. 1996; 85: 1125-1134Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). The specific expression of GRTH in the testis, but not in ovary, implies that its gene is either suppressed by female-specific or induced by male-specific gonadal factor(s). Since estrogen is produced in both male and female gonads, it is unlikely to be involved in the induction of GRTH gene expression. Consistent with this, estradiol did not affect GRTH mRNA in cultured rat Leydig cells. In contrast, the non-metabolizable androgen, dihydrotestosterone, increased the expression of GRTH mRNA in Leydig cells. The stimulation effects of hCG in vivo on the GRTH gene expression were also observedin vitro, and cAMP, the second messenger of hCG action, had comparable effects. However, no stimulation was observed after inhibition of steroid biosynthetic enzymes, which abolished hCG- and cAMP-stimulated increases of testosterone in Leydig cells. The absence of a rapid change in basal GRTH mRNA levels during androgen suppression in vitro is not unexpected, since incubations could only be conducted with short term primary cultures and the GRTH mRNA is long-lived (Fig. 4 C). Androgen induced by gonadotropin could act through its cognate receptors in the Leydig cell to increase GRTH gene expression. It could also exert actions in the germinal cells of the seminiferous epithelium through androgen receptors present in Sertoli cells, since germinal cells do not possess androgen receptors (35Bremner W.J. Millar M.R. Sharpe R.M. Saunders P.T. Endocrinology. 1994; 135: 1227-1234Crossref PubMed Scopus (310) Google Scholar). Although androgens are not present in the pituitary, hypothalamus, and brain, where low GRTH mRNA levels are found, androgen receptors are present in these tissues. It is conceivable that androgen from the circulation may induce GRTH expression at these sites. Major developmental changes in GRTH mRNA were observed during puberty when predominant expression of GRTH mRNA was observed in the Leydig cell and germinal epithelium. Our findings suggest that this novel testicular helicase could serve to maintain testicular functions related to steroidogenesis and spermatogenesis. We are grateful to Dr. Kenneth S. K. Tung (Department of Pathology, University of Virginia Medical School, Charlottesville, VA) for valuable advice on histological interpretation.