Exogenous administration of protease-resistant, non-matrix-binding IGFBP-2 inhibits tumour growth in a murine model of breast cancer.

BACKGROUND: Insulin-like growth factors (IGF-I and IGF-II) signal via the type 1 IGF receptor (IGF-1R) and IGF-II also activates the insulin receptor isoform A (IR-A). Signalling via both receptors promotes tumour growth, survival and metastasis. In some instances IGF-II action via the IR-A also promotes resistance to anti-IGF-1R inhibitors. This study assessed the efficacy of two novel modified IGF-binding protein-2 (IGFBP-2) proteins that were designed to sequester both IGFs. The two modified IGFBP-2 proteins were either protease resistant alone or also lacked the ability to bind extracellular matrix (ECM). METHODS: The modified IGFBP-2 proteins were tested in vitro for their abilities to inhibit cancer cell proliferation and in vivo to inhibit MCF-7 breast tumour xenograft growth. RESULTS: Both mutants retained low nanomolar affinity for IGF-I and IGF-II (0.8-2.1-fold lower than IGFBP-2) and inhibited cancer cell proliferation in vitro. However, the combined protease resistant, non-matrix-binding mutant was more effective in inhibiting MCF-7 tumour xenograft growth and led to inhibition of angiogenesis. CONCLUSIONS: By removing protease cleavage and matrix-binding sites, modified IGFBP-2 was effective in inhibiting tumour growth and reducing tumour angiogenesis.

The TGF-ß-Smad3 pathway inhibits CD28-dependent cell growth and proliferation of CD4 T cells.

Transforming growth factor-ß (TGF-ß) maintains self-tolerance through a constitutive inhibitory effect on T-cell reactivity. In most physiological situations, the tolerogenic effects of TGF-ß depend on the canonical signaling molecule Smad3. To characterize how TGF-ß/Smad3 signaling contributes to maintenance of T-cell tolerance, we characterized the transcriptional landscape downstream of TGF-ß/Smad3 signaling in resting or activated CD4 T cells. We report that in the presence of TGF-ß, Smad3 modulates the expression of >400 transcripts. Notably, we identified 40 transcripts whose expression showed Smad3 dependence in both resting and activated cells. This 'signature' confirmed the non-redundant role of Smad3 in TGF-ß biology and identified both known and putative immunoregulatory genes. Moreover, we provide genomic and functional evidence that the TGF-ß/Smad3 pathway regulates T-cell activation and metabolism. In particular, we show that TGF-ß/Smad3 signaling dampens the effect of CD28 stimulation on T-cell growth and proliferation. The impact of TGF-ß/Smad3 signals on T-cell activation was similar to that of the mTOR inhibitor Rapamycin. Considering the importance of co-stimulation on the outcome of T-cell activation, we propose that TGF-ß-Smad3 signaling may maintain T-cell tolerance by suppressing co-stimulation-dependent mobilization of anabolic pathways.

Normoxic expression of hypoxia-inducible factor 1 in rat Leydig cells in vivo and in vitro.

Hypoxia-inducible factors (HIF) are transcription factors that serve essential regulatory roles in cellular and molecular responses to oxygen debt. HIFs are composed of hypoxia-dependent α subunits (1α, 2α, 3α) and an oxygen-independent ß subunit. Previously we demonstrated that HIF-1, the master regulator of hypoxic responses, is expressed in the adult rat testis. We hypothesized that HIF-1 is involved in regulating responses to oxygen tension in the testis. Goals of this study were to determine if HIF-2α and HIF-3α are expressed in rat testis, identify testis cell types that express HIF-1α, and examine patterns of testicular HIF-1α protein expression under conditions of ischemia and hypoxia in vivo and in vitro. Reverse transcriptase polymerase chain reaction revealed that mRNA for Hif-1α, Hif-2α, and Hif-3α is expressed in the testis. The HIF-1α protein is the predominant subunit in testis. HIF-1α protein was abundant in normoxic testis, and its levels remained unchanged following ischemia created by surgically induced testicular torsion and reperfusion. Immunoblot and immunocytochemical experiments demonstrated that Leydig cells are the major source of HIF-1α in normoxic and hypoxic testes. To examine potential mechanisms of testicular HIF-1 stabilization, nuclear proteins from Leydig cells cultured in 5% or 21% oxygen, or cells cultured with H2O2, were analyzed by immunoblotting. Levels of HIF-1α were significantly diminished in 5% or 21% oxygen cultures compared with freshly isolated cells. Treating Leydig cells with H2O2 as a source of reactive oxygen species did not affect HIF-1α levels. High levels of constitutively expressed HIF-1α in normoxic Leydig cells suggest potentially unique roles for HIF-1 in Leydig cell responsiveness to oxygen.

Modulation of 11beta-hydroxysteroid dehydrogenase expression by bombesin: a possible mechanism for glucocorticoid resistance in androgen independent prostate cancer.

Treatment with glucocorticoids is one of a limited number of options for androgen independent prostate cancer. Neuroendocrine differentiation has been shown to contribute to androgen-independent prostate cancer progression. To study the potential link between neuroendocrine differentiation and the glucocorticoid action, we investigated the effects of the product of neuroendocrine differentiation--bombesin on glucocorticoid metabolizing enzymes--11beta-hydroxysteroid dehydrogenases in PC-3 cells. Our Western analysis, RT-PCR, and activity assays demonstrate that while 18-hour exposure to bombesin reduces 11beta-hydroxy-steroid dehydrogenases-1 profiles (activities 25% less, protein level 29% lower, mRNA levels 45% lower), contrarily it increases 11beta-hydroxysteroid dehydrogenases-2 profiles (activities 34%, protein levels 100%, mRNA levels 120%). Blockade bombesin action with bombesin receptor antagonists and the enzyme degrading bombesin prevented these changes, suggesting the observed modulations were bombesin receptor-specific. In addition, bombesin increased the amounts of interleukin-8 and mRNA of vascular endothelial growth factor receptor 2, which were lowered in the presence of cortisol, suggesting that neuropeptide blockade may extend the benefits of glucocorticoids in treating androgen-independent prostate cancer.

Endogenous inhibitors (GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2: derivatives of adrenally produced corticosterone and cortisol.

Two isoforms of 11beta-HSD exist; 11beta-HSD1 is bi-directional (the reductase usually being predominant) and 11beta-HSD2 functions as a dehydrogenase, conferring kidney mineralocorticoid specificity. We have previously described endogenous substances in human urine, "glycyrrhetinic acid-like factors (GALFs)", which like licorice, inhibit the bi-directional 11beta-HSD1 enzyme as well as the dehydrogenase reaction of 11beta-HSD2. Many of the more potent GALFs are derived from two major families of adrenal steroids, corticosterone and cortisol. For example, 3alpha5alpha-tetrahydro-corticosterone, its derivative, 3alpha5alpha-tetrahydro-11beta-hydroxy-progesterone (produced by 21-deoxygenation of corticosterone in intestinal flora); 3alpha5alpha-tetrahydro-11beta-hydroxy-testosterone (produced by side chain cleavage of cortisol); are potent inhibitors of 11beta-HSD1 and 11beta-HSD2-dehydrogenase, with IC50's in range 0.26-3.0 microM, whereas their 11-keto-3alpha5alpha-tetrahydro-derivatives inhibit 11beta-HSD1 reductase, with IC50's in range 0.7-0.8 microM (their 3alpha5beta-derivatives being completely inactive). Inhibitors of 11beta-HSD2 increase local cortisol levels, permitting it to act as a mineralocorticoid in kidney. Inhibitors of 11beta-HSD1 dehydrogenase/11beta-HSD1 reductase serve to adjust the set point of local deactivation/reactivation of cortisol in vascular and other glucocorticoid target tissues, including adipose, vascular, adrenal tissue, and the eye. These adrenally derived 11-oxygenated C21- and C19 -steroidal substances may serve as 11beta-HSD1- or 11beta-HSD2-GALFs. We conclude that adrenally derived products are likely regulators of local cortisol bioactivity in humans.

Mucosal deformation from an impinging transonic gas jet and the ballistic impact of microparticles.

By means of a transonic gas jet, gene guns ballistically deliver microparticle formulations of drugs and vaccines to the outer layers of the skin or mucosal tissue to induce unique physiological responses for the treatment of a range of conditions. Reported high-speed imaging experiments show that the mucosa deforms significantly while subjected to an impinging gas jet from a biolistic device. In this paper, the effect of this tissue surface deformation on microparticle impact conditions is simulated with computational fluid dynamics (CFD) calculations. The microparticles are idealized as spheres of diameters 26.1, 39 and 99 microm and a density of 1050 kg m-3. Deforming surface calculations of particle impact conditions are compared directly with an immobile surface case. The relative velocity and obliquity of the deforming surface decrease the normal component of particle impact velocity by up to 30% at the outer edge of the impinging gas jet. This is qualitatively consistent with reported particle penetration profiles in the tissue. It is recommended that these effects be considered in biolistic studies requiring quantified particle impact conditions.

Detailed mapping of the ERG-ETS2 interval of human chromosome 21 and comparison with the region of conserved synteny on mouse chromosome 16.

We have carried out a detailed annotation of 550 kb of genomic DNA on human chromosome 21 containing the ERG and ETS2 genes. Comparative genomic analysis between this region and the interval of conserved synteny on mouse chromosome 16 indicated that the order and orientation of the ERG and ETS2 genes were conserved and revealed several regions containing potential conserved noncoding sequences. Four pseudogenes including those for small protein G, laminin receptor, human transposase protein and meningioma-expressed antigen were identified. A potentially novel gene (C21orf24) with alternative mRNA transcripts, consensus splice donor and acceptor sites, but no coding potential nor murine orthologue, was identified and found to be expressed in a range of human cell lines. We have identified four novel splice variants that arise from a previously undescribed 5' exon of the human ERG gene. Comparison of the cDNA sequences enabled us to determine the complete exon-intron structure of the ERG gene. We have also identified the presence of noncoding RNAs in the first and second introns of the ETS2 gene. Our studies have important implications for Down syndrome as this region contains multiple mRNA transcripts, both coding and potentially noncoding, that may play as yet undescribed roles in the pathogenesis of this disorder.

Effect of chronic administration of 7alpha-methyl-19-nortestosterone on serum testosterone, number of spermatozoa and fertility in adult male bonnet monkeys (Macaca radiata).

Hormonal approaches to male contraception that are based on the suppression of LH secretion require androgen replacement treatment to maintain sexual behaviour and secondary sexual characteristics. Androgen supplementation not only involves large and frequent doses of testosterone esters but also results in undesirable effects on the prostate gland. In an attempt to avoid such problems, a synthetic androgen, 7alpha-methyl-19-nortestosterone (MENT), which is much more potent than testosterone, has been developed. In the present study, MENT was administered at different doses (25, 50, 100, 300 and 1000 microg day(-1)) either alone or in combination with oestradiol via Silastic implants for a specified period to adult male bonnet monkeys (Macaca radiata). Blood and semen samples were collected at specific intervals and analysed for serum testosterone and seminal parameters, respectively. The results of the present study clearly indicate that administration of MENT at all doses tested results in suppression of the nocturnal surge of testosterone (by day 3), as well as a decrease in the number of spermatozoa (by day 45). Co-administration of oestradiol resulted in a reduction in the dose of MENT required to suppress the nocturnal surge. None of the male bonnet monkeys treated with MENT were able to impregnate females, clearly demonstrating the efficacy of MENT in blocking fertility in male bonnet monkeys.

Prenatal exposure to dexamethasone alters Leydig cell steroidogenic capacity in immature and adult rats.

This study examines the effects of prenatal exposure to dexamethasone (DEX) on postnatal testosterone production in male rats. Pregnant female rats were treated on gestation days 14-19 with DEX (100 microg/kg body weight per day; n = 9) or vehicle (n = 9). Results show that 35-day-old male offspring from DEX-treated pregnant females (n = 42) had decreased levels of serum testosterone (45.6% lower, P < .05) compared with control offspring (n = 43), although serum luteinizing hormone (LH) levels were not significantly altered. These findings suggest that a direct programming of developing gonadal cells occurs in response to high levels of maternal glucocorticoid. Indeed, testosterone production was significantly reduced in Leydig cells isolated from immature offspring of DEX-treated pregnant females compared with controls (48.3%, P < .001), and LH stimulation of these cells did not compensate for the lowered steroidogenic capacity. The hypothalamic-pituitary-adrenal axis was also affected, because significant reductions in both serum adrenocorticotropic hormone (ACTH; 26.2%, P < .001) and corticosterone (CORT; 32.3%, P < .001) were measured in DEX-exposed immature male offspring. In contrast, adult male offspring from DEX-treated dams had significantly higher levels of serum ACTH (39.2%, P <. 001) and CORT (37.8%, P < .001). These same animals had higher serum testosterone (31.6%, P < or = .05) and a significant reduction in serum LH (30.8%, P < .001). Moreover, Leydig cells isolated from these adult offspring exhibited an increased capacity for testosterone biosynthesis under basal (38.6%, P < .001) and LH-stimulated conditions (33.5%, P < .001). In summary, sustained changes in steroidogenic capacity were observed in male rats exposed to high levels of glucocorticoid during prenatal development. More specifically, DEX exposure in utero perturbed Leydig cell testosterone production in both pubertal and adult rats.

Oestrogenic and antiandrogenic chemicals in the environment: effects on male reproductive health.

Exposures of human populations to pesticides and industrial pollutants, and to synthetic chemicals present in foods, beverages, and plastics, have raised concern that these substances can interfere with endogenous sex hormone function. Interference with sex hormone action can, in turn, result in a variety of developmental and reproductive anomalies. Compounds in this class are thus referred to as endocrine disruptors (EDs). EDs that affect reproductive processes in vertebrates act primarily by altering oestrogenic and antiandrogenic activities. The recent cloning of a second oestrogen receptor (ER) subtype (ERbeta) and its widespread tissue distribution pattern indicates that the first ER to be cloned, ERalpha, may not be the only, or even the primary, mediator of oestrogen action. It is anticipated that this discovery will lead to development of antagonist compounds specific to either ER subtype, and help to determine the function of each receptor subtype in reproductive and other tissues. Growing evidence suggests that EDs interfere with reproductive function at low exposure levels and cause distinct effects at different concentrations within the same organ. Developing organisms have increased susceptibility to the actions of EDs because differentiating tissues are more vulnerable to changes in hormonal milieu. Thus, children are at greater risk of toxicant-related illnesses than adults. However, most data are collected from laboratory studies, and it remains to be determined that the levels of chemicals in the environment can impair human reproductive health. There is also significant genetic variability between human and animal species in their reactions to chemicals. The effects of low-dose, chronic, and multiple chemical exposures warrant further investigation in order to characterize the risk of environmental agents to humans. The aims of this review, which will focus on male reproduction, are to: 1) identify synthetic chemicals in the environment that fall into the ED class; 2) describe their mechanisms of toxicity in reproductive tissues; and, 3) outline the direction of future research efforts with respect to EDs.

Müllerian inhibiting substance inhibits testosterone synthesis in adult rats.

Müllerian inhibiting substance (MIS) is a gonadal hormone that causes regression of the Müllerian ducts during male sexual differentiation. Postnatally, MIS inhibits the proliferation and differentiation of immature Leydig cells, and transgenic mice that overexpress MIS have decreased serum testosterone concentrations. To elucidate the effects of MIS on androgen regulation in the postnatal testis, we examined testosterone synthesis in adult Sprague-Dawley rats following intratesticular and intraperitoneal injections of MIS. Intratesticular MIS injection achieved high local concentrations of MIS (574.0 +/- 60.0 ng/mL) at 4 hours, with a corresponding decline in serum testosterone concentrations to 0.7 +/- 0.1 ng/mL, compared to 1.1 +/- 0.2 ng/mL with intraperitoneal MIS and 1.6 +/- 0.1 ng/mL with intratesticular vehicle (IT-Veh) (P < .001). Intratesticular administration of MIS (IT-MIS) resulted in much higher serum and testicular interstitial fluid MIS concentrations than the intraperitoneal route. To directly examine the testosterone production rate in MIS-treated animals, we isolated Leydig cells from MIS and vehicle-injected testes. Primary Leydig cells exposed to MIS had a lower testosterone production rate and decreased expression of p450c17 (hydroxylase/lyase) and luteinizing hormone (LH) receptor mRNAs than that of vehicle-injected controls or the noninjected contralateral testis. In conclusion, intratesticular administration of MIS caused a decline in serum testosterone concentrations by decreasing the rate of testosterone biosynthesis, confirming that MIS can regulate adult Leydig cell androgen production. The ability of MIS to down-regulate mRNA expression of the p450c17 and LH receptor genes suggests that this effect is mediated transcriptionally. These data indicate that, in addition to its role in embryonic differentiation of the male reproductive tract, MIS has a regulatory function in the postnatal testis. We conclude that one such function is for MIS to directly inhibit adult Leydig cell steroidogenesis.

Modulation of rat Leydig cell steroidogenic function by di(2-ethylhexyl)phthalate.

Exposure of rodents to phthalates is associated with developmental and reproductive anomalies, and there is concern that these compounds may be causing adverse effects on human reproductive health. Testosterone (T), secreted almost exclusively by Leydig cells in the testis, is the primary steroid hormone that maintains male fertility. Leydig cell T biosynthesis is regulated by the pituitary gonadotropin LH. Herein, experiments were conducted to investigate the ability of di(2-ethylhexyl)phthalate (DEHP) to affect Leydig cell androgen biosynthesis. Pregnant dams were gavaged with 100 mg(-1) kg(-1) day(-1) DEHP from Gestation Days 12 to 21. Serum T and LH levels were significantly reduced in male offspring, compared to control, at 21 and 35 days of age. However, these inhibitory effects were no longer apparent at 90 days. In a second set of experiments, prepubertal rats, from 21 or 35 days of age, were gavaged with 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 14 days. This exposure paradigm affected Leydig cell steroidogenesis. For example, exposure of rats to 200 mg(-1) kg(-1) day(-1) DEHP caused a 77% decrease in the activity of the steroidogenic enzyme 17beta-hydroxysteroid dehydrogenase, and reduced Leydig cell T production to 50% of control. Paradoxically, extending the period of DEHP exposure to 28 days (Postnatal Days 21-48) resulted in significant increases in Leydig cell T production capacity and in serum LH levels. The no-observed-effect-level and lowest-observed-effect-level were determined to be 1 mg(-1) kg(-1) day(-1) and 10 mg(-1) kg(-1) day(-1), respectively. In contrast to observations in prepubertal rats, exposure of young adult rats by gavage to 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 28 days (Postnatal Days 62-89) induced no detectable changes in androgen biosynthesis. In conclusion, data from this study show that DEHP effects on Leydig cell steroidogenesis are influenced by the stage of development at exposure and may occur through modulation of T-biosynthetic enzyme activity and serum LH levels.

Purification of rat leydig cells: increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells.

Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure that includes a filtration with nylon mesh (100-micron pore size) to separate interstitial cells from the seminiferous tubules, combining centrifugal elutriation and Percoll density gradient sedimentation, has been used to obtain a 95% enrichment of rat Leydig cells. However, the number of recovered Leydig cells by this procedure represents only a small fraction of the 25 million, on average, that exist in the adult rat testis. The objective of this study was to test whether the yield of purified Leydig cells might be enhanced by substitution of unit-gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters, macrophages, or both would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone production in vitro. The number of purified Leydig cells was 1.9-fold higher for the S method than for the F method, with no differences in purity assessed by 3beta-hydroxysteroid dehydrogenase histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenase dispersion and at the end of the purification. No differences were seen in testosterone production or in the number of macrophages present in the Leydig cells that were prepared by the 2 methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clustered Leydig cells that are systematically eliminated when a filtration step is used.

The soluble murine type I interferon receptor Ifnar-2 is present in serum, is independently regulated, and has both agonistic and antagonistic properties.

The ability to modify responses to type I interferons (IFNs) could alter processes such as hematopoiesis and immunity, which involve endogenous IFNs and responses to exogenous IFNs. The data presented here support a significant role for a recently identified soluble isoform of the murine type I IFN receptor, muIfnar-2a, as an efficient regulator of IFN responses. The messenger RNA (mRNA) transcript encoding muIfnar-2a is generally more abundant than that encoding the transmembrane isoform, muIfnar-2c. Furthermore, the ratio of muIfnar-2a:2c transcripts varied from more than 10:1 in the liver and other organs to less than 1:1 in bone-marrow macrophages, indicating independent regulation of the 2 transcripts encoding receptor isoforms and suggesting that the soluble muIfnar-2a levels are biologically relevant in some organs. Western blot analysis showed that soluble muIfnar-2 was present at high levels in murine serum and other biologic fluids and bound type I IFN. Recombinant muIfnar-2a competitively inhibited the activity of both IFNalpha and beta in reporter assays using the L929 cell line and in antiproliferative and antiviral assays using primary cells. Surprisingly, using primary thymocytes from Ifnar-2(-/-) mice, recombinant muIfnar-2a formed a complex with IFN alpha or beta and muIfnar-1 at the cell surface and transmitted an antiproliferative signal. These data indicate potential dual actions of soluble muIfnar-2 and imply that a signal can be transduced through the Ifnar-1 chain of the receptor complex in the absence of the cytoplasmic domain of Ifnar-2. Therefore, our results suggest that soluble Ifnar-2 is an important regulator of endogenous and systemically administered type I IFN.

Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study.

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0. 01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0. 01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.

Identification of the oxidative 3alpha-hydroxysteroid dehydrogenase activity of rat Leydig cells as type II retinol dehydrogenase.

Dihydrotestosterone (DHT) is the most potent naturally occurring androgen, and its production in the testis may have important consequences in developmental and reproductive processes. In the rat testis, three factors can contribute to intracellular DHT levels: 1) synthesis of DHT from T by 5alpha-reductase, 2) conversion of DHT to 5alpha-androstane-3alpha, 17beta-diol (3alpha-DIOL) by the reductive activity of 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), and 3) conversion of 3alpha-DIOL by an oxidative 3alpha-HSD activity. While the type I 3alpha-HSD enzyme (3alpha-HSD1 or AKR1C9) is an oxidoreductase in vitro and could theoretically be responsible for factors 2 and 3, we have shown previously that rat Leydig cells have two 3alpha-HSD activities: a cytosolic NADP(H)- dependent activity, characteristic of 3alpha-HSD1, and a microsomal NAD(H)-dependent activity. The two activities were separable by both developmental and biochemical criteria, but the identity of the second enzyme was unknown. To identify the microsomal NAD(H)-dependent 3alpha-HSD in rat Leydig cells, degenerate primers were used to amplify a number of short-chain alcohol dehydrogenases. Sequence analysis of cloned PCR products identified retinol dehydrogenase type II (RoDH2) as the prevalent species in purified Leydig cells. RoDH2 cDNA was subcloned into expression vectors and transiently transfected into CHOP and COS-1 cells. Its properties were compared with transiently transfected 3alpha-HSD1. When measured in intact CHOP and COS-1 cells, RoDH2 cDNA produced a protein that catalyzed the conversions of 3alpha-DIOL to DHT and androsterone to androstanedione, but not the reverse reactions. Therefore, the 3alpha-HSD activity of RoDH2 was exclusively oxidative. In contrast, type I 3alpha-HSD cDNA produced a protein that was exclusively a 3alpha-HSD reductase. In cell homogenates and subcellular fractions, RoDH2 catalyzed both 3alpha-HSD oxidation and reduction reactions that were NAD(H) dependent, and the enzyme activities were located in the microsomes. Type I 3alpha-HSD also catalyzed both oxidation and reduction, but was located in the cytosol and was NADP(H) dependent. We conclude that type I 3alpha-HSD and RoDH2 have distinct 3alpha-HSD activities with opposing catalytic directions, thereby controlling the rates of DHT production by Leydig cells.

Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses.

The Kit receptor tyrosine kinase functions in hemato- poiesis, melanogenesis and gametogenesis. Kit receptor-mediated cellular responses include proliferation, survival, adhesion, secretion and differentiation. In mast cells, Kit-mediated recruitment and activation of phosphatidylinositol 3'-kinase (PI 3-kinase) produces phosphatidylinositol 3'-phosphates, plays a critical role in mediating cell adhesion and secretion and has contributory roles in mediating cell survival and proliferation. To investigate the consequences in vivo of blocking Kit-mediated PI 3-kinase activation we have mutated the binding site for the p85 subunit of PI 3-kinase in the Kit gene, using a knock-in strategy. Mutant mice have no pigment deficiency or impairment of steady-state hematopoiesis. However, gametogenesis is affected in several ways and tissue mast cell numbers are affected differentially. While primordial germ cells during embryonic development are not affected, Kit(Y719F)/Kit(Y719F) males are sterile due to a block at the premeiotic stages in spermatogenesis. Furthermore, adult males develop Leydig cell hyperplasia. The Leydig cell hyperplasia implies a role for Kit in Leydig cell differentiation and/or steroidogenesis. In mutant females follicle development is impaired at the cuboidal stages resulting in reduced fertility. Also, adult mutant females develop ovarian cysts and ovarian tubular hyperplasia. Therefore, a block in Kit receptor-mediated PI 3-kinase signaling may be compensated for in hematopoiesis, melanogenesis and primordial germ cell development, but is critical in spermatogenesis and oogenesis.

Initial predominance of the oxidative activity of type I 11beta-hydroxysteroid dehydrogenase in primary rat Leydig cells and transfected cell lines.

Glucocorticoids suppress testosterone production in Leydig cells. The level of glucocorticoid action is set within the Leydig cell by the number of glucocorticoid receptors and by the activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD). This enzyme acts either as an oxidase inactivating glucocorticoid or as a reductase amplifying its action. It is currently unknown whether extracellular conditions might cause 11betaHSD oxidative and reductive activities in Leydig cells to change inversely or independently. The aim of the present study was to determine whether extracellular conditions set in vitro by various culture time and media components, such as glucose and pyruvate, affect the relative rates of 11betaHSD oxidation and reduction. Primary rat Leydig cells and cell lines (COS1 and CHOP cells) transfected with 11betaHSD-I complementary DNA (cDNA), were incubated with 25 nmol/L (physiologic range) or 500 nmol/L (stress range) concentrations of radiolabeled substrates, corticosterone or 11-hydrocorticosterone, for 0 to 24 hours. Oxidative activity predominated over reductive activity under initial conditions when product formation increased linearly with time. For example, in Dulbecco's modified Eagle medium/F12 medium (containing 5.5 mmol/L glucose), the peak ratio of oxidation to reduction (with 1 denoting equivalence of oxidative and reductive activities) was 5.5 in rat Leydig cells, 19.7 in COS1 cells, and 20.8 in CHOP cells. Glucose stimulated reductive activity but did not change the predominant direction of 11betaHSD catalysis at earlier times. In COS1 cells transfected with 11betaHSD-I cDNA, oxidative activity rapidly increased during the first 2 hours of the incubation, then gradually decreased while reductive activity increased steadily. The relative ratio of oxidation to reduction rapidly declined to less than 0.5 at 6 hours, and thus the favored direction of catalysis changed from oxidation to reduction. However, in transfected CHOP cells, 11betaHSD oxidative activity rapidly increased during the first 2 hours and continued to increase for 24 hours. The ratio of oxidative to reductive activity rapidly declined but kept above 1 in CHOP cells for 24 hours, and the favored direction of catalysis remained predominantly oxidative. These results revealed that 11betaHSD-I is a predominant oxidase initially in Leydig cells and 2 cell lines, and that the oxidative activity is gradually lost over time. The data suggest that type I 11betaHSD is a predominant oxidase in Leydig cells in vivo.

A metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane, reduces testosterone biosynthesis in rat leydig cells through suppression of steady-state messenger ribonucleic acid levels of the cholesterol side-chain cleavage enzyme.

Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testosterone (T). T promotes the male phenotype in the prepubertal period and maintains sexual function in adulthood; therefore, disruption of T biosynthesis in Leydig cells can adversely affect male fertility. The present study was designed to evaluate the ability of a xenoestrogen, methoxychlor (the methoxylated isomer of DDT [1,1, 1-trichloro-2,2-bis(p-chlorophenyl)ethane]), to alter Leydig cell steroidogenic function. Purified progenitor, immature, and adult Leydig cells were obtained from, respectively, 21-, 35-, and 90-day-old Sprague-Dawley rats treated with graded concentrations of the biologically active metabolite of methoxychlor, 2, 2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and assessed for T production. HPTE caused a dose-dependent inhibition of basal and LH-stimulated T production by Leydig cells. Compared to the control value, reduced T production by progenitor and immature Leydig cells was apparent after 10 h of HPTE treatment in culture; the equivalent time for adult Leydig cells was 18 h. The reversibility of HPTE-induced inhibition was evaluated by incubating Leydig cells for 3, 6, 10, 14, or 18 h and measuring T production after allowing time for recovery. After treatment with HPTE for 3 h, T production by immature and adult Leydig cells for the 18-h posttreatment period was similar to the control value, but that of progenitor Leydig cells was significantly lower. The onset of HPTE action and the reversibility of its effect showed that Leydig cells are more sensitive to this compound during pubertal differentiation than in adulthood. T production was comparable when control and HPTE-treated immature Leydig cells were incubated with pregnenolone, progesterone, and androstenedione, but HPTE-treated Leydig cells produced significantly reduced amounts of T when incubations were conducted with 22R-hydroxycholesterol (P < 0.01). This finding suggested that HPTE-induced inhibition of T production is related to a decrease in the activity of cytochrome P450 cholesterol side-chain cleavage enzyme (P450(scc)) and cholesterol utilization. The reduced steady-state mRNA level for P450(scc) in HPTE-treated Leydig cells was demonstrated by reverse transcription-polymerase chain reaction and densitometry. In conclusion, this study showed that HPTE causes a direct inhibition of T biosynthesis by Leydig cells at all stages of development. This effect suggests that reduced T production could be a contributory factor in male infertility associated with methoxychlor and, possibly, other DDT-related compounds.