Steroid biosynthetic lesions in gonadotropin-desensitized Leydig cells

Dufau, Maria L.; Cigorraga, Selva Beatriz; Báukal, Albert J.; Bator, Jenny M.; Sorrell, Susan H.; Neubauer, Jolanta; Catt, Kevin J.

doi:10.1016/0022-4731(79)90296-6

Cited by 43 publications

(6 citation statements)

References 23 publications

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“…Furthermore, transfection of GR-LACS cDNA into cells did not affect the activity of cotransfected receptor and steroidogenic enzyme promoters (not shown). Thus, the available evidence indicates that the observed changes on GR-LACS do not contribute to the down-regulation of steroidogenic enzymes and receptors (2,4,6,7). The transcriptional down-regulation of GR-LACS induced by hCG may result from the direct or indirect nuclear actions of mediators of the trophic hormone stimulus that precede the phase of down-regulation.…”

Section: Discussionmentioning

confidence: 97%

“…Mediators of LH action exert homologous regulation of receptors, steroidogenic enzymes, and other genes of the Leydig cell. In contrast to the physiological concentrations of gonadotropins that maintain steroidogenesis and gonadal LH͞human chorionic gonadotropin (hCG) and prolactin receptors, high exogenous concentrations of hCG down-regulate receptors and steroidogenic enzymes of Leydig cells at steps beyond pregnenolone formation (2)(3)(4)(5)(6)(7). The impairment of steroidogenesis that follows the initial receptormediated activation is independent of receptor down-regulation and results from changes at the transcriptional levels that cause a marked reduction of enzyme expression.…”

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confidence: 99%

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Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase

Tang¹,

Tsai‐Morris²,

Dufau³

2001

Proc. Natl. Acad. Sci. U.S.A.

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L uteinizing hormone (LH) supports steroidogenesis and maintains testicular and ovarian function (1). Mediators of LH action exert homologous regulation of receptors, steroidogenic enzymes, and other genes of the Leydig cell. In contrast to the physiological concentrations of gonadotropins that maintain steroidogenesis and gonadal LH͞human chorionic gonadotropin (hCG) and prolactin receptors, high exogenous concentrations of hCG down-regulate receptors and steroidogenic enzymes of Leydig cells at steps beyond pregnenolone formation (2-7). The impairment of steroidogenesis that follows the initial receptormediated activation is independent of receptor down-regulation and results from changes at the transcriptional levels that cause a marked reduction of enzyme expression. Recently, we cloned a gonadotropin-regulated testicular RNA helicase that is present in Leydig cells and meiotic germinal cells of the testis (8). This protein was found to be markedly up-regulated by hCG by means of cAMP-induced androgen formation in Leydig cells at hCG doses that cause down-regulation of steroidogenic enzymes and receptors.In a further search for genes regulated by gonadotropin, we have isolated a previously uncharacterized protein that is downregulated by hCG and is termed gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS). This member of the LACS family is highly abundant in the Leydig cell and is distinct from the previously characterized mammalian forms of the enzyme (9-13). Its constitutive expression in Leydig cells is negatively regulated by the receptor-mediated action of LH. In addition to its potential contributions to energy production and testicular steroidogenesis, GR-LACS could provide long chain acyl-CoA esters with regulatory effects on enzyme activity, membrane function, and gene expression. Materials and Methods Animal Treatment and Cell Preparation. The gonadotropin-induced down-regulation of LH receptor and steroidogenic enzymes inLeydig cells was produced as described (3) by administration of an s.c. injection of 2.5 g of hCG to adult male rats. Animals were killed at various times after treatment, and the testes were removed. Leydig cells were prepared by collagenase dispersion and purified by centrifugal elutriation (14). The seminiferous tubules were minced, and the germ cells were collected by centrifugation. Cells were processed for RNA and protein analysis.Differential Display Analysis, Isolation, and Cloning of GR-LACS. Total RNA samples prepared from Leydig cells of control and hCGtreated rats were analyzed by differential display technology (8). PCR products with incorporated [ 33 P]dATP (2,000 Ci͞mmol, 1 Ci ϭ 37 GBq; DuPont͞NEN) were separated in nondenatured 6% sequencing gels. A 3Ј-untranslated region fragment derived from differential display analysis was used as a probe for screening a rat Leydig cell cDNA library in ZAP Express vector (Stratagene) to obtain full-length cDNA.Chromosomal Localization. Chromosomal mapping (rat, mouse, and human lymphocytes) was performed by fluores...

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Section: Discussionmentioning

confidence: 97%

mentioning

confidence: 99%

Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase

Tang¹,

Tsai‐Morris²,

Dufau³

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Animal Treatment and Cell Preparation-Gonadotropin-induced LH receptor and steroidogenic enzymes down-regulation of Leydig cells was produced as described previously (2) by administration of a single 2.5-g subcutaneous injection of hCG (Pregnyl, Organon) to adult male rats (200 -250 g) (Charles River Laboratories Inc., Wilmington, MA). Animals were sacrificed 24 h after hCG treatment, and testes were removed.…”

Section: Methodsmentioning

confidence: 99%

“…1 for review). However, high concentrations of gonadotropins cause desensitization of steroidogenic enzymes of the androgen pathway, with consequent reduction of testosterone formation (2)(3)(4)(5). The attenuation of steroidogenesis results from receptor activation by the gonadotropic hormone but is independent of the subsequent phase of receptor down-regulation.…”

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confidence: 99%

A Novel Gonadotropin-regulated Testicular RNA Helicase

Tang

Tsai‐Morris

Dufau

1999

Journal of Biological Chemistry

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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.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. 1 for review). However, high concentrations of gonadotropins cause desensitization of steroidogenic enzymes of the androgen pathway, with consequent reduction of testosterone formation (2-5). 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 3␤-hydroxysteroid dehydrogenase types I and II (3), 17␣-hydroxylase/17, 20 lyase (5), and 17␤-hydroxysteroid dehydrogenase type III (4) operates at the transcriptional level. Because this process affects several enzymes, the direct or indirect involvement of a master switch has been proposed (3).Likely candidates for this regulation include the active steroid metabolites that are produced during gonadotropic stimulation, i.e. androgen (3) and estrogen (6 -8).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. (2) by administration of a single 2.5-g subcutaneous injection of hCG (Pregnyl, Organon) to adult male rats (200 -250 g) (Ch...

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“…Leydig cells in primary culture have been used to study the mechanism of action of gonadotropin, but testosterone production is not sustained more than a few days (2)(3)(4)(5)(6). Clonal cell lines derived from mouse (7)(8)(9) and rat (10) Leydig cell tumors, although they may retain LH/hCG receptors, are unable to pursue steroidogenesis beyond progesterone synthesis.…”

Section: For Review)mentioning

confidence: 99%

Construction of a Leydig cell line synthesizing testosterone under gonadotropin stimulation: a complex endocrine function immortalized by cell hybridization.

Finaz¹,

Lefèvre²,

Dampfhoffer³

1987

Proc. Natl. Acad. Sci. U.S.A.

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Hybridization between a mouse Leydig tumor cell line, MA-l0, which produces cyclic AMP and progesterone under human chorionic gonadotropin (hCG) stimulation, and freshly isolated mouse Leydig cells gave rise to 54 hybrid clones, one of which, LK17, was capable of hCGstimulated testosterone production. Subcloning of this hybrid resulted in the emergence of a subclone, K9, whose testosterone production is more than 10 times that of parent clone LK17, after hCG stimulation, with an ED50 of 37 pM. Testosterone synthesis by K9 cells was multiplied by 25 after gonadotropin stimulation, and binding of hCG declined after prolonged exposure to the hormone. These similarities with murine Leydig cells in primary culture make the K9 clone an attractive alternative for physiological studies.Production of testosterone by Leydig cells is the outcome of a complex steroidogenic pathway, initiated by the cleavage of the side chain of cholesterol. This step is triggered by the binding of luteinizing gonadotropin to luteinizing hormone! human chorionic gonadotropin (LH/hCG) receptors located on the cell plasma membrane, followed by the activation of adenylate cyclase and protein kinase (see ref. 1 for review). Leydig cells in primary culture have been used to study the mechanism of action of gonadotropin, but testosterone production is not sustained more than a few days (2-6). Clonal cell lines derived from mouse (7-9) and rat (10) Leydig cell tumors, although they may retain LH/hCG receptors, are unable to pursue steroidogenesis beyond progesterone synthesis. Using somatic cell hybridization between the MA-10 mouse Leydig tumor cell line obtained by Ascoli (11) and freshly isolated mouse Leydig cells, we have immortalized the full steroidogenic function of the latter. The strategy used for obtaining this testosterone-producing gonadotropin-sensitive permanent cell line is described below. MATERIALS AND METHODSParental Cells. A clonal mouse Leydig tumor cell line, , was kindly provided by Mario Ascoli (Nashville, TN). This cell line responds to hCG stimulation by an increased secretion of progesterone and 20a-dehydroprogesterone. It was made hypoxanthine/guanine phosphoribosyltransferase negative (HGPRT-) by mutation using ethyl methanesulfonate, added at 300 ,4g/ml for 14 hr to cells grown to confluence in Dulbecce's modified Eagle's medium containing 15% fetal calf serum. The treated cells were allowed to recover 7 days in the initial medium, and HGPRT-cells were then selected by addition of 100 ,uM 6-thioguanine. Mutant colonies appeared 3 weeks later. A mutant clone, LK, was chosen for its endocrinological similarity to the MA-10 parent cell line-namely, the ability to respond to hCG stimulation by an increased progesterone production.Fresh Leydig cells, isolated from the testes of 6-week-old BALB/c mice, were purified and maintained in primary culture as previously described (6, 12). Ouabain at 20,4M will kill fresh Leydig cells but not the LK clone.Cell Hybridization and Subcloning. Twelve hours after plating of the ...

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Steroid biosynthetic lesions in gonadotropin-desensitized Leydig cells

Cited by 43 publications

References 23 publications

Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase

Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase

A Novel Gonadotropin-regulated Testicular RNA Helicase

Construction of a Leydig cell line synthesizing testosterone under gonadotropin stimulation: a complex endocrine function immortalized by cell hybridization.

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