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

Abstract

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...

Revisiting Rat Spermatogenesis with MALDI Imaging at 20-μm Resolution

“…However, our IMS data suggest ACSL- (19 -48) to be present at the periphery of seminiferous tubules. This is in accordance with previous work showing that the protein is expressed in Leydig cells and to a lesser extent in Sertoli cells and spermatogonia (53). In the rat testis, Leydig cells are isolated or clustered into small groups throughout the interstitial compartment.…”

“…The corresponding 79-kDa cytoplasmic progenitor protein is expressed in rodent gonads and brain. In the testis, it may participate in steroidogenesis and, through the multiple functions of acyl-CoA, in the regulation of the male gonad (53). This is another example for the ability of the top-down imaging strategy to specifically detect and identify …”

Cell-specific and Hormone-regulated Expression of Gonadotropin-regulated Testicular RNA Helicase Gene (GRTH/Ddx25) Resulting from Alternative Utilization of Translation Initiation Codons in the Rat Testis

“…The testicular cell-specific and hormonal-dependent usage of translation codons for GRTH protein synthesis add complexity and diversity to regulatory mechanism(s) that may participate during testicular development. The up-regulation of GRTH protein by gonadotropin in the Leydig cells follows closely the down-regulation of receptors, steroidogenic enzymes, and acyl-CoA synthetase (gonadotropin-regulated long chain acyl-CoA synthetase) (4,23), and its return to control levels is concomitant to its recovery from down-regulation. These changes could be reflective of the existence of a regulated mechanism responsible for recovery of down-regulated transcriptional events through increases in translatability of the reduced relevant messages.…”

The Acyl-CoA Synthetase “Bubblegum” (Lipidosin)

“…14 C]Palmitic acid (C16:0), [1-14 C]arachidonic acid (C20:4), [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]docosahexaenoic acid (C22:6), and [1-14 C]lignoceric acid (C24:0) were obtained from Moravek, Inc. [1-14 C]Myristic acid (C14:0), [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]stearic acid (C18:0), [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]oleic acid (C18:1), and [1][2][3][4][5][6][7]…”

“…Subsequently, Moriya-Sato et al (7) reported that in the mouse, BG1 was expressed in adrenal gland and testis as well and Tang et al (8) noted that rat BG1 was also expressed in ovary. A Northern blot prepared using total RNA from tissues of 3-month-old 129SvEv mice and probed with an mBG1-specific probe indicated that mBG1 was expressed in brain, adrenal gland, testis, ovary, and spleen ( Fig.…”