Tu Lin

Regulation of insulin-like growth factor-I messenger ribonucleic acid expression in Leydig cells

In the present study, we evaluated insulin-like growth factor-I (IGF-I) messenger RNA expression in the rat testis. Crude interstitial cells were separated into three distinct bands on 15-60% Percoll density gradients. IGF-I mRNA was mainly localized in the Leydig cell-enriched fraction (band 3), while band 1 and band 2 cells did not contain significant amounts of IGF-I mRNA. Leydig cell IGF-I mRNA consisted of multiple species varying from 0.8 to 7.5 kb and was present in rat Leydig cells all ages examined, from 25 to 55 days old. To further document that IGF-I mRNAs are present in Leydig cells, the method of Klinefelter et al. (Biol. Reprod. (1987) 36, 769-783) was used to isolate highly purified (greater than 98% pure) Leydig cells. Most of the IGF-I mRNA was localized in these Leydig cells, while there was no detectable IGF-I mRNA in the whole testis or other interstitial cells. Furthermore, IGF-I mRNA in Leydig cells was increased more than 2-fold by growth hormone (GH) administration in vivo. This suggests that IGF-I mRNA in Leydig cells is also GH dependent. Interstitial IGF-I produced in Leydig cells may have both autocrine and paracrine effects in the testis.

Interferon-γ Inhibits the Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid Expression and Protein Levels in Primary Cultures of Rat Leydig Cells1

Interferon-γ (IFNγ) is an immunomodulating cytokine that has profound effects on reproductive function. IFNγ inhibits steroidogenesis both in vivo and in vitro. The mechanism by which IFNγ inhibits Leydig cell steroidogenesis remains unclear. In the present study, we evaluated the effect of IFNγ on the expression and regulation of the steroidogenic acute regulatory protein (StAR) gene in primary cultures of rat Leydig cells. StAR facilitates the efficient production of steroid hormone by regulating the translocation of cholesterol from the outer to the inner mitochondrial membrane, the site of the cytochrome P450 side-chain cleavage (P450scc) enzyme system that converts cholesterol to pregnenolone. IFNγ inhibited hCG-induced StAR messenger RNA (mRNA) levels in a dose-dependent manner. The addition of IFNγ in a concentration of 500 U/ml decreased hCG-induced 3.8- and 1.7-kilobase StAR mRNA by 78% and 70%, respectively. IFNγ also reduced hCG-stimulated P450scc mRNA levels by 69%. The inhibitory effects of I...

Differential effects of recombinant interleukin-1α and β on Leydig cell function

Abstract Previously we have reported that human chorionic gonadotropin(hCG)-stimulated testosterone biosynthesis was markedly inhibited by purified natural human interleukin-1(IL-1). In the present study we evaluated the effects of human and murine recombinant IL-1 (rIL-1) on Leydig cell steroidogenesis in primary culture. Human rIL-1β caused a dose-dependent inhibition of hCG-, 8-bromo cyclic AMP-, and forskolin-induced testosterone formation. In contrast, human rIL-1α was considerably less potent. When the effects of the cytokines were corrected for their biological potencies, human rIL-1 β and murine rIL-1α were still more effective than human rIL-1α in inhibiting testosterone production (at least 100-fold more potent). Thus, even though IL-1α and IL-1β bind to the same receptors on T cells, Leydig cells exhibit differential sensitivity in response to rIL-1α and rIL-1β which is partly species dependent.

Tumor necrosis factor-α enhances inhibitory effects of interleukin-1β on Leydig cell steroidogenesis

Abstract Human recombinant tumor necrosis factor-α (rTNFα) alone (up to 1000 units/ml) did not alter either basal or human chorionic gonadotropin (hCG)-Induced testosterone formation in primary culture of rat Leydig cells. However, concomitant addition of rTNFα with human recombinant interleukin-1β (rIL-1β) enhanced the inhibitory effects of rIL-1β. The rIL-1β dose response curve was shifted to the left (IC50 changed from 1 ng/ml to 0.3 ng/ml). Even though rTNFα had no effect on testosterone formation, hCG-stimulated cyclic AMP formation was inhibited by rTNFα in a dose dependent manner. In the presence of both rTNFα and rIL-1β, hCG-induced cyclic AMP formation and binding of [ 125 I]-hCG to Leydig cells were further inhibited. Testicular macrophages represent about 20% of the interstitial cells. TNFα and IL-1 may by produced locally by interstitial macrophages and have paracrine effects on Leydig cell function.

Human chorionic gonadotropin induces interleukin-1 gene expression in rat Ley dig cells in vivo

Abstract Luteinizing hormone (LH)/human chorionic gonadotropin (hCG) causes inflammatory-type responses in the testis. In the present study, we evaluated the effects of hCG on Leydig cell interleukin-1 (IL-1) gene expression. Using monoclonal antibody (ED2) staining for macrophages, our Leydig cell preparations had no significant contamination with macrophages. When purified Leydig cells from normal rats were cultured for 24 h and then treated with IL-1β (1–100 ng/ml) for 6 h, IL-1β induced dose-dependent increases in IL-1β mRNA levels. IL-1β also induced IL-lα mRNA accumulation; however, the level of IL-1α mRNA was much lower than that of IL-1β mRNA. When rats were treated with either saline or hCG (5 units i.p.), hCG markedly induced IL-1β mRNA accumulation in purified Leydig cells at 6 h which persisted for over 24 h. However, hCG had no direct effect on purified Leydig cell or crude interstitial cell IL-1 mRNA levels. Our results suggest that inflammatory effects of hCG in vivo may be mediated by increased IL-1 gene expression in Leydig cells.

Recombinant murine tumor necrosis factor-alpha inhibits cholesterol side-chain cleavage cytochrome P450 and insulin-like growth factor-I gene expression in rat Leydig cells

The purpose of the present study was to evaluate the effects of murine recombinant tumor necrosis factor-alpha (TNF-alpha) on rat Leydig cell function. In primary cultures of Leydig cells, we found that in the presence of hCG (10 ng/ml), testosterone levels were markedly elevated, 69.3 +/- 3.1 ng/10(6) cells/h (mean + SE). TNF-alpha in a concentration of 1 ng/ml markedly inhibited testosterone biosynthesis (a 69% reduction; p < 0.01) and 100 ng/ml of TNF-alpha almost completely inhibited testosterone formation (p < 0.001). TNF-alpha (10 ng/ml) inhibited hCG (0.1, 1 and 10 ng/ml)-induced testosterone formation by 63%, 67% and 61%, respectively. TNF-alpha (10 ng/ml) also markedly inhibited 8-bromo cAMP-induced testosterone formation from 76 +/- 9 ng/10(6) cells/h to 4.9 ng/10(6) cells/h. This indicates that the major effect of TNF-alpha is at steps beyond LH receptor site. To further evaluate the site(s) of action of TNF-alpha, we evaluated its effect on the conversion of precursor steroids to testosterone. We found that the addition of 20-hydroxy-cholesterol could not reverse inhibitory effects of TNF-alpha on hCG-induced testosterone formation. TNF-alpha had no effect on the conversions of pregnenolone, 17-OH-pregnenolone, DHEA and androstenedione to testosterone. This indicates that the major effect of TNF-alpha is at the key steroidogenic enzyme, P450scc. We reported previously that human recombinant TNF-alpha had no effect on hCG-induced testosterone formation but did enhance the inhibitory effects of human recombinant IL-1beta. In the present study, we demonstrated that both murine TNF-alpha and human IL-1beta were potent inhibitors of hCG-induced testosterone formation. IL-1beta alone in concentrations of 0.1, 1 and 10 ng/ml inhibited testosterone formation by 45%, 62% and 91%, respectively, in the presence of TNF-alpha (10 ng/ml), IL-1beta in a concentration as low as 0.1 ng/ml completely blocked hCG-induced testosterone formation. We next evaluated the effect of TNF-alpha on P450scc gene expression. There was no constitutively expressed P450scc mRNA in Leydig cells after 24 h in culture. In response to hCG, there was a 33-fold increase in the P450scc mRNA level. Both TNF-alpha and IL-1beta inhibited hCG-induced expression of P450scc mRNA. Finally, the effect of TNF-alpha on IGF-I gene expression was investigated since IGF-I enhances Leydig cell androgen formation and IGF-I gene is expressed in high levels in Leydig cells. TNF-alpha inhibited both large (7.4 kb) and small species (0.8-1.2 kb) IGF-I mRNA levels in a dose-dependent manner. In conclusion, murine TNF-alpha is a potent inhibitor of Leydig cell function. TNF-alpha inhibited both P450scc and IGF-I mRNA gene expression.

Expression and Regulation of Interferon-γ-Inducible Protein 10 Gene in Rat Leydig Cells1

In the present study, we report the cloning of a gene that is differentially expressed in normal adult rat Leydig cells and whose expression is inhibited by hCG but is induced by interferon-γ (IFNγ). DNA sequence analysis identified this gene as rat IFNγ-inducible protein 10 (IP-10), a member of the -C-X-C- chemokine superfamily of proinflammatory cytokines. High levels of IP-10 messenger RNA (mRNA) were constitutively expressed in freshly isolated and primary cultured Leydig cells. hCG inhibited this expression in a dose-dependent manner. The addition of 1 ng/ml hCG inhibited IP-10 mRNA levels more than 80%. Conversely, IP-10 mRNA levels were markedly increased in response to murine interleukin-1α, murine tumor necrosis factor-α, and murine IFNγ by 3.3-, 10-, and 26-fold, respectively. Concomitant addition of murine interleukin-1α, murine tumor necrosis factor-α, and murine IFNγ synergistically increased IP-10 mRNA levels by 58-fold. Furthermore, in addition to one previously described rat IP-10 mRNA tra...

Recombinant monocyte-derived interleukin-1 receptor antagonist reverses inhibitory effects of interleukin-1 on Leydig cell steroidogenesis.

Previously we have reported that interleukin-1 (IL-1) is a potent inhibitor of Leydig cell function. Most recently, IL-1 receptor antagonist (IL-1ra) has been purified, sequenced and cloned. In the present study, we evaluated the recombinant monocyte-derived IL-1ra on the inhibitory effects of IL-1. The addition of recombinant human IL-1ra up to 1000 ng/ml has no discernible effects on human chorionic gonadotropin (hCG)-stimulated testosterone formation in primary cultures of rat Leydig cells. Similar to that reported previously, IL-1 beta caused a dose-dependent inhibition of hCG-induced testosterone. The inhibitory effect of IL-1 beta could be reversed by the concomitant addition of IL-1ra. The amounts of IL-1ra required to reverse the effect of IL-1 were 25-fold higher. Our results suggest that IL-1 is important in modulating Leydig cell function and its effect most likely is mediated by specific IL-1 receptors.