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...

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...

The apparent decline in human male fertility and the concomitant increase in testicular pathology have prompted discussion of the underlying molecular mechanisms which may underpin these observations. While monitoring the expression of protamine-2 genes in the human ejaculate, we found a representative complement of sperm mRNAs following sequence-independent amplification of reverse-transcribed cDNAs with the polymerase chain reaction (RT-PCR). The revelation of unique sperm-derived PCR products using this method suggests that it should now be possible to investigate gene expression in human spermatogenesis by differential RNA fingerprinting of ejaculate spermatozoa. The identification of molecular markers and the corresponding genes associated with male infertility will be considerably enhanced by these investigations while obviating the requirement for invasive biopsy.

f Managing ecosystems to maintain biodiversity may be one approach to ensuring their dynamic stability, productivity, and delivery of vital services. The applicability of this approach to industrial ecosystems that harness the metabolic activities of microbes has been proposed but has never been tested at relevant scales. We used a tag-sequencing approach with bacterial small subunit rRNA (16S) genes and eukaryotic internal transcribed spacer 2 (ITS2) to measuring the taxonomic composition and diversity of bacteria and eukaryotes in an open pond managed for bioenergy production by microalgae over a year. Periods of high eukaryotic diversity were associated with high and more-stable biomass productivity. In addition, bacterial diversity and eukaryotic diversity were inversely correlated over time, possibly due to their opposite responses to temperature. The results indicate that maintaining diverse communities may be essential to engineering stable and productive bioenergy ecosystems using microorganisms.

3Beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerases (3beta-HSD) are enzymes that catalyze the conversion of delta5 to delta4 steroids in the gonads and adrenal for the biosynthesis of sex steroid and corticoids. In gonadotropin-desensitized Leydig cells, from rats treated with high doses of human CG (hCG), testosterone production is markedly reduced, a finding that was attributed in part to reduction of CYP17 expression. In this study, we present evidence for an additional steroidogenic lesion induced by gonadotropin. Using differential display analysis of messenger RNA (mRNA) from Leydig cells of rats treated with a single desensitizing dose of hCG (2.5 microg), we found that transcripts for type I and type II 3beta-HSD were substantially (5- to 8-fold) down-regulated. This major reduction, confirmed by RNase protection assay, was observed at the high hCG dose (2.5 microg), whereas minor or no change was found at lower doses (0.01 and 0.1 microg). In contrast, 3beta-HSD mRNA transcripts were not changed in luteinized ovaries of pseudopregnant rats treated with 2.5 microg hCG. The down-regulation of 3beta-HSD mRNA in the Leydig cell resulted from changes at the transcriptional level. Western blot analysis showed 3beta-HSD protein was significantly reduced by hCG treatment, with changes that were coincidental with the reduction of enzyme activity and temporally consistent with the reduction of 3beta-HSD mRNA but independent of LH receptor down-regulation. The reduction of 3beta-HSD mRNA resulting from transcriptional inhibition of gene expression, and the consequent reduction of 3beta-HSD activity could contribute to the inhibition of androgen production in gonadotropin-induced steroidogenic desensitization of Leydig cells. The gender-specific regulation of 3beta-HSD by hCG reflects differential transcriptional regulation of the enzymes to accommodate physiological hormonal requirements and reproductive function.