Alternative (backdoor) androgen production and masculinization in the human fetus.

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production, but little is known about the synthesis of backdoor androgens, despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human fetuses using multidimensional and high-resolution mass spectrometry. Results show that androsterone is the principal backdoor androgen in the male fetal circulation and that DHT is undetectable (<1 ng/mL), while in female fetuses, there are significantly lower levels of androsterone and testosterone. In the male, intermediates in the backdoor pathway are found primarily in the placenta and fetal liver, with significant androsterone levels also in the fetal adrenal. Backdoor intermediates, including androsterone, are only present at very low levels in the fetal testes. This is consistent with transcript levels of enzymes involved in the alternate pathway (steroid 5α-reductase type 1 [SRD5A1], aldo-keto reductase type 1C2 [AKR1C2], aldo-keto reductase type 1C4 [AKR1C4], cytochrome P450 17A1 [CYP17A1]), as measured by quantitative PCR (qPCR). These data identify androsterone as the predominant backdoor androgen in the human fetus and show that circulating levels are sex dependent, but also that there is little de novo synthesis in the testis. Instead, the data indicate that placental progesterone acts as substrate for synthesis of backdoor androgens, which occurs across several tissues. Masculinization of the human fetus depends, therefore, on testosterone and androsterone synthesis by both the fetal testes and nongonadal tissues, leading to DHT formation at the genital tubercle. Our findings also provide a solid basis to explain why placental insufficiency is associated with disorders of sex development in humans.

Leydig cell steroidogenesis unexpectedly escapes mitochondrial dysfunction in prematurely aging mice.

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in tissues during aging in animals and humans and are the basis for mitochondrial diseases. Testosterone synthesis occurs in the mitochondria of Leydig cells. Mitochondrial dysfunction (as induced here experimentally in mtDNA mutator mice that carry a proofreading-deficient form of mtDNA polymerase γ, leading to mitochondrial dysfunction in all cells types so far studied) would therefore be expected to lead to low testosterone levels. Although mtDNA mutator mice showed a dramatic reduction in testicle weight (only 15% remaining) and similar decreases in number of spermatozoa, testosterone levels in mtDNA mutator mice were unexpectedly fully unchanged. Leydig cell did not escape mitochondrial damage (only 20% of complex I and complex IV remaining) and did show high levels of reactive oxygen species (ROS) production (>5-fold increased), and permeabilized cells demonstrated absence of normal mitochondrial function. Nevertheless, within intact cells, mitochondrial membrane potential remained high, and testosterone production was maintained. This implies development of a compensatory mechanism. A rescuing mechanism involving electrons from the pentose phosphate pathway transferred via a 3-fold up-regulated cytochrome b5 to cytochrome c, allowing for mitochondrial energization, is suggested. Thus, the Leydig cells escape mitochondrial dysfunction via a unique rescue pathway. Such a pathway, bypassing respiratory chain dysfunction, may be of relevance with regard to mitochondrial disease therapy and to managing ageing in general.

Bortezomib treatment causes long-term testicular dysfunction in young male mice.

BACKGROUND: With increased long-term survivors of childhood cancer patients, therapy-associated infertility has become one of the most common late side-effects and significantly affects their life-quality. Therefore, evaluation of anti-cancer agents on male reproduction and infertility prevention are urgently demanding. The proteasome inhibitor bortezomib has been launched in clinical trials for childhood cancers, however, its potential side effects on reproduction have so far been neither investigated experimentally nor reported in treated children. Thus the present study is designed to explore the impact of bortezomib on male reproductive function and to gain insights into how bortezomib exerts its adverse effects on man gonad, thereby providing pediatric oncologists relevant information. METHODS: 35 day-old male mice were treated with one 11-day cycle of bortezomib and then sacrificed 2 days, 45 days, or 6 months later. A mating study was performed in the group followed for 6 months, and their pups were analyzed on postnatal day 50. Serum follicle-stimulating hormone (FSH) and testicular testosterone levels were measured. Testicular morphology was evaluated by light- and electron microscopy, and the underlying mechanisms and pathways of testis damage were investigated. RESULTS: Testicular damage was visible already 2 days after stopping bortezomib and increased in severity by day 45. Then 80% of seminiferous tubules exhibited hypospermatogenesis with arrest at the levels of spermatogonia, spermatocytes and round spermatids. Germ cells were specifically targeted by bortezomib as evidenced by increased apoptosis mediated through activation of p53 and caspases. Even six months after the bortezomib treatment, testis weight, sperm concentration and seminiferous tubule length remained at a decreased level, indicating that spermatogenesis and tubular outgrowth could not fully recover. Combined with persistently increased serum levels of FSH in these mice, our results demonstrate that bortezomib can have long-term effects on testicular function, although fertility of bortezomib-exposed males remained and their offspring looked healthy. CONCLUSION: Bortezomib treatment causes long-term gonadal dysfunction in male mice. Careful monitoring of gonadal function in male childhood cancer patients treated with bortezomib is thus strongly recommended.

Excessive ovarian production of nerve growth factor elicits granulosa cell apoptosis by setting in motion a tumor necrosis factor α/stathmin-mediated death signaling pathway.

Excessive nerve growth factor (NGF) production by the ovary, achieved via a transgenic approach, results in arrested antral follicle growth, reduced ovulatory capacity, and a predisposition to cyst formation in response to mildly elevated LH levels. Two salient features in these mutant mice (termed 17NF) are an elevated production of 17α-hydroxyprogesterone (17-OHP(4)), testosterone, and estradiol (E(2)) in response to gonadotropins, and an increased frequency of granulosa cell (GC) apoptosis. In this study, we show that the increase in steroidal response is associated with enhanced expression of Cyp17a1, Hsd17b, and Cyp19a1, which encode the enzymes catalyzing the synthesis of 17-OHP(4), testosterone, and E(2) respectively. Using a proteomic approach, we identified stathmin (STMN1), as a protein that is overproduced in 17NF ovaries. In its phosphorylated state, STMN1 mediates a cell death signal initiated by tumor necrosis factor α (TNF). STMN1 is expressed in GCs and excessive NGF increases its abundance as well as that of its forms phosphorylated at serine (Ser) 16, 25, and 38. TNF synthesis is also increased in 17NF ovaries, and this change is abolished by blocking neurotrophic tyrosine kinase receptors. Inhibiting TNF actions in vivo by administering a soluble TNF receptor prevented the increase in total and phosphorylated STMN1 production, as well as GC apoptosis in NGF-overproducing ovaries. These results indicate that an excess of NGF in the ovary promotes steroidogenesis by enhancing the expression of enzyme genes involved in 17-OHP(4), testosterone, and E(2) synthesis, and causes GC apoptosis by activating a TNF/ STMN1-mediated cell death pathway.

The prenylflavonoid phytoestrogens 8-prenylnaringenin and isoxanthohumol diferentially suppress steroidogenesis in rat Leydig cells in ontogenesis.

8-Prenylnaringenin and isoxanthohumol are prenylflavonoids found in the hop plant, Humulus lupulus (Cannabaceae), which is traditionally used to add bitterness and flavor to beer. Flavonoids have previously been reported to exert endocrine disrupting actions. Therefore, we investigated the effects of 8-prenylnaringenin and isoxanthohumol on steroidogenesis activated by human chorionic gonadotropin (hCG) in primary cultures of rat Leydig cells at different stages of their development. The present study is the first to demonstrate that the prenylflavonoids 8-prenylnaringenin and isoxanthohumol exert complex maturation-dependent effects on Leydig cell steroidogenesis. Those compounds inhibited hCG-stimulated androgen production by Leydig cells at all stages of their development, a process that was associated with the reduced ability of the cells to produce cAMP. However, these same compounds up-regulated hCG-activated StAR expression in progenitor (PLC) and immature (ILC) but not adult types of Leydig cells (ALC). Further, 8-prenylnaringenin and isoxanthohumol were not able to suppress androgen production activated by an exogenous analog of cAMP, (Bu)2 cAMP, in ALC and ILC but synergistically stimulated steroidogenesis in PLC. Our data suggest that 8-prenylnaringenin and isoxanthohumol affect cAMP-dependent cellular processes up-stream transport of cholesterol into mitochondria.

Diabetes Type 1 Negatively Influences Leydig Cell Function in Rats, Which is Partially Reversible By Insulin Treatment.

Type 1 diabetes mellitus (T1DM) is associated with impaired spermatogenesis and lower testosterone levels and epididymal weight. However, the underlying processes in the testis are unknown and remain to be elucidated. Therefore, the present study focused on the effects of T1DM on testicular function in a spontaneously diabetic rat model. BB/OKL rats after diabetes manifestation were divided into 3 groups: those without insulin treatment and insulin treatment for a duration of 2 and of 6 weeks. Anthropometrical data, circulating levels of gonadotrophins, testosterone, and inhibin B were measured. Intratesticular testosterone, oxidative stress, inflammation, and apoptosis were analyzed. Key enzymes of steroidogenesis were evaluated in the testis. Untreated diabetic rats had significantly lower serum follicle-stimulating hormone and luteinizing hormone levels. Serum and intratesticular testosterone levels significantly decreased in untreated diabetic rats compared to healthy controls. Key markers of Leydig cell function were significantly downregulated at the RNA level: insulin-like factor 3 (Insl3) by 53% (P = .006), Star by 51% (P = .004), Cyp11A1 by 80% (P = .003), 3Beta-Hsd2 by 61% (P = .005), and Pbr by 52% (P = .002). In the insulin-treated group, only Cyp11A1 and 3Beta-Hsd2 transcripts were significantly lower. Interestingly, the long-term insulin-treated group showed significant upregulation of most steroidogenic enzymes without affecting testosterone levels. Tumor necrosis factor α and apoptosis were significantly increased in the long-term insulin-treated rats. In conclusion T1DM, with a severe lack of insulin, has an adverse action on Leydig cell function. This is partially reversible with well-compensated blood glucose control. Long-term T1DM adversely affects Leydig cell function because of the process of inflammation and apoptosis.

Effects of resveratrol analogs on steroidogenesis and mitochondrial function in rat Leydig cells in vitro.

Resveratrol and its analogs are considered to be a promising drug candidate for treatment of cancer and different age-associated diseases. In the present study we have investigated the effects of resveratrol and its synthetic analogs on steroidogenesis and mitochondrial function in primary cultures of rat Leydig cells. Our findings indicate that resveratrol and its analogs structure-dependently attenuated hCG-activated steroidogenesis in Leydig cells through suppression of the expression of steroidogenic acute regulatory protein and cytochrome P450c17. 3,5-Diacetyl resveratrol was observed to modulate mitochondrial function in Leydig cells, suppressing polarization of inner mitochondrial membrane, and 3,4,4'-trimethoxystilbene stimulated the overall activity of intracellular reductases involved in the reduction of WST-1 to formazan. Thus, the inhibitory actions of resveratrol analogs on steroidogenesis in Leydig cells indicate novel mechanisms of action of these compounds, which may be of potential therapeutic interest, where suppression of androgen action is needed.

Estrogen receptor beta selective ligand 5alpha-Androstane-3beta, 17beta-diol stimulates spermatogonial deoxyribonucleic acid synthesis in rat seminiferous epithelium in vitro.

Gonadotropins and testosterone are important regulators of spermatogenesis, even though gonadotropin receptors and the androgen receptor are not expressed by germ cells. However, a functional role for estrogens in connection with male reproduction has been postulated on the basis of the phenotypes of mice lacking estrogen receptor (ER) and cytochrome P-450 aromatase. This has further support by findings of ER expression in the testis, including that of ERbeta in spermatogonia. 5alpha-Androstane-3beta, 17beta-diol (3betaAdiol), a metabolite of testosterone produced via the intermediate potent androgen 5alpha-dihydrotestosterone (DHT), has been reported to selectively bind ERbeta rather than EpsilonRalpha, but not androgen receptor. Here, we have characterized the influence of 17beta-estradiol (E), the major physiological estrogen, 3betaAdiol, and DHT on DNA synthesis in vitro by segments of the seminiferous epithelium at different stages of the seminiferous epithelial cycle in the rat. E and 3betaAdiol exerted similar stimulatory effects on premitotic DNA synthesis in stage I segments, whereas other stages tested (V, VIIa, and XIII-IX) remained unresponsive. In contrast, DHT had no effect on this process. 5-bromo-2'-deoxyuridine labeling of stage I segments revealed a 30-fold higher labeling index in the presence than in the absence of E, and the labeled cells were identified as spermatogonia. Moreover, high levels of 3betaAdiol were found in the testis of intact rats as well as in primary cultures of rat Leydig cells in response to human chorionic gonadotropin. We suggest that 3betaAdiol may serve as a growth factor for germ cells stimulating premitotic DNA synthesis in connection with spermatogenesis via an ERbeta-dependent pathway.

Ontogeny of gonadal sex steroids.

Sex steroids are crucial hormones for the proper development and function of the body; they regulate sexual differentiation, the secondary sex characteristics, and sexual behaviour patterns. Gonads are the major sources of sex steroids, although adrenal cortex, placenta, and to a lesser extent other tissues contribute to their production in adult life and at various phases of development. Steroidogenesis of gonadal sex hormones is by definition sexually dimorphic, and involves differences not only in hormonal action but also in regulation and temporal patterns of production. This review focuses on the ontogeny and developmental regulation of steroid hormones in the gonads, with an attempt to detail these processes in humans.

Inhibitory effects of mono-ethylhexyl phthalate on steroidogenesis in immature and adult rat Leydig cells in vitro.

Di-2-ethylhexyl (DEHP) phthalate, one of the phthalates most widely distributed in our general environment, causes reproductive toxicity that is attributable to the action of its primary metabolite, mono(2-ethylhexyl) phthalate (MEHP). Here, we have investigated the effects of MEHP on steroidogenesis by primary cultures of immature and adult rat Leydig cells. In both cases MEHP (250muM) was found to inhibit stimulation of androgen production evoked by human chorionic gonadotropin (hCG). This was associated with decreased expression of steroidogenic acute regulatory (StAR) protein and reduced transport of cholesterol into mitochondria but no detectable adverse effect on steroidogenic enzymes. Moreover, upon exposure to MEHP alone, 5alpha-reductase activity was decreased in immature, but not in adult Leydig cells. All together, our findings demonstrate that MEHP exerts suppressive effects on hCG-activated steroidogenesis in primary cultures of immature and adult rat Leydig cells and suppresses 5alpha-reductase activity in immature and not of adult rat cells. This may partly explain the anti-androgenic effects of DEHP in vivo and indicate a higher susceptibility in younger subjects.

Adipose Tissue is a Potential Source of Hyperandrogenism in Obese Female Rats.

OBJECTIVE: Obesity in females is often associated with metabolic complications and hyperandrogenism, but the sources of androgens are not completely understood. Therefore, this study investigated whether adipose tissue could be a source of androgens promoting hyperandrogenism development in obese female rats. METHODS: Gene expression of steroidogenic enzymes and testosterone levels were determined in periovarian and inguinal adipose tissue and in the supernatant of cultured preadipocytes and adipocytes. The conversion of pregnenolone to androgens was analyzed by thin-layer chromatography. RESULTS: Substantial amounts of testosterone in adipose tissue (25-153 ng/g tissue) and in the supernatant of adipocytes (0.33-0.69 ng/ten thousand cells]) were found. StAR and steroidogenic enzymes encoded by genes including Cyp11A1, Cyp17A1, Cyp19, Hsd3b2, Hsd17b3, and Srd5a2 were expressed in adipose tissue and cultured cells. Thin layer chromatography data revealed that preadipocytes and adipocytes were able to convert pregnenolone to testosterone. Higher levels for all steroidogenic enzymes were found in both depots of obese animals compared with lean animals, with significantly higher levels in inguinal tissue. CONCLUSIONS: The whole steroidogenic machinery and capacity for testosterone biosynthesis were found in fat depots of female rats. These findings support the hypothesis that adipose tissue may contribute substantially to the hyperandrogenism in female obesity.

Insulin-like growth factor-I is an important antiapoptotic factor for rat leydig cells during postnatal development.

The present investigation examines the influence of IGF-I and the role of IGF-I receptor (IGF-IR) in the apoptosis/survival of Leydig cells. Immunohistochemical analysis of the rat testis at different ages revealed that the level of the phosphorylated IGF-IR increases from birth to d 20 of postnatal life, remaining high in the adult testis. Western blotting revealed that this level is higher in Leydig cells isolated from 40-d-old than from 10- or 60-d-old rats. Application of the terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay revealed that IGF-I decreases the level of apoptosis in Leydig cells at all stages of development, and the selective inhibitor of IGF-IR, picropodophyllin, blocks this antiapoptotic effect. The mechanism underlying the antiapoptotic action of IGF-I involves the phosphatidylinositol 3-kinase/Akt pathway, and in immature Leydig cells, this growth factor enhances the expression of Bcl-2 and cellular inhibitor of apoptosis proteins 2, while preventing activation of caspase-3 by cleavage. Furthermore, IGF-II and high concentrations of insulin also evoke phosphorylation of IGF-IR and, like IGF-I, enhance the expression of the steroidogenic acute regulatory protein by Leydig cells. Inhibition of IGF-IR by picropodophyllin decreases the survival of Leydig cells, both in the presence and absence of IGF-I, demonstrating that signaling via the IGF-IR plays an important role in Leydig cell survival.

Stimulation of the pituitary-adrenal axis and of adrenocortical steroidogenesis ex vivo by administration of di-2-ethylhexyl phthalate to prepubertal male rats.

Phthalate esters exert deleterious effects on testicular physiology and, consequently, on reproduction and fertility. However, little is presently known concerning potential adverse effects of these environmental pollutants on the hormonal functions of the adrenal gland. Therefore, we have investigated the effects of administering to rats of different developmental ages di-2-ethylhexyl phthalate (DEHP) on the hypothalamic-pituitary-adrenal axis in vivo, as well as on adrenocortical steroidogenesis ex vivo. Oral exposure to DEHP once daily for 4 days elevated the serum levels of ACTH and corticosterone in rats 20 and 40 days of age, but not in adult, 60-day-old animals. Furthermore, primary cultures of adrenocortical cells isolated from 20- and 40-day-old rats treated with DEHP exhibited an enhanced capacity to produce corticosterone in response to ACTH, dibutyryl cAMP, and 22R-hydroxycholesterol, as well as increased ACTH-stimulated transport of endogenous cholesterol into mitochondria. Neither DEHP nor its major metabolite mono-2-ethylhexyl phthalate altered steroidogenesis in cultures of adrenocortical cells isolated from untreated rats. These findings demonstrate that in male rats, DEHP exerts an age-dependent influence on the pituitary-adrenocortical axis in vivo and adrenocortical steroidogenesis ex vivo. Such perturbation may be of pathological significance in connection with disorders of the hormonal stress response, especially in very young human beings.

Resveratrol inhibits steroidogenesis in human fetal adrenocortical cells at the end of first trimester.

SCOPE: Resveratrol has a diverse array of healthful effects on metabolic parameters in different experimental paradigms but has also potential to inhibit steroidogenesis in rodent adrenals. The aim of the present study was to characterize the effects of resveratrol on human fetal adrenal steroidogenesis at gestational weeks (GW) 9-12. METHODS AND RESULTS: Adrenals from aborted fetuses (GW10-12) were used to prepare primary cultures of human fetal adrenocortical cells (HFAC). HFAC were treated in the presence or absence of ACTH (10 ng/mL) with or without resveratrol (10 µM) for 24 h. The production of steroids by HFAC was analyzed by gas and liquid chromatography coupled to tandem/mass spectrometry. The expression of steroidogenic enzymes at GW 9-12 was quantified by automated Western blotting. We observed that resveratrol significantly suppressed synthesis of dehydroepiandrosterone (DHEA), androstenedione and 11-deoxicortisol by ACTH-activated and unstimulated HFAC, which was associated with inhibition of the activities and expression of cytochromes 17α-hydroxylase/17,20 lyase (CYP17) and 21-hydroxylase (CYP21) in these fetal adrenocortical cells. CONCLUSION: Our in vitro findings on the sensitivity of human fetal adrenal steroidogenesis to resveratrol at GW9-12 suggest that intake of this polyphenol at high doses by women who are at early stages of pregnancy is undesirable.

Sesquiterpene lactone helenalin suppresses Leydig and adrenocortical cell steroidogenesis by inhibiting expression of the steroidogenic acute regulatory protein.

Helenalin is a potent anti-inflammatory and anti-neoplastic agent isolated from several plant species of the Asteracea family. Here, we have investigated the effects of helenalin on steroidogenesis activated by adrenocorticotropic hormone (ACTH) and human chorionic gonadotropin (hCG) in primary cultures of rat adrenocortical and Leydig cells. Our findings demonstrate that helenalin inhibits both ACTH- and hCG-activated steroidogenesis in these cells. This effect was already evident after 2-3 h treatment with helenalin. In contrast, steroidogenesis from 22R-OHC, a cell-permeable form of cholesterol, was not inhibited by helenalin, suggesting that the expression of the steroidogenic acute regulartory (StAR) protein might be inhibited by this compound. Indeed, helenalin attenuated StAR protein expression activated by ACTH and hCG in adrenocortical and Leydig cells as assessed by PAGE/Western analyses. This inhibitory action of helenalin on steroidogenic cell functions indicates novel mechanisms of action of this compound which may be of potential therapeutic interest. However, it also poses safety concerns relating to possible negative side-effects on anabolism and systemic stress.

Phthalates Exert Multiple Effects on Leydig Cell Steroidogenesis.

Humans are significantly exposed to phthalates via food packaging, cosmetics and medical devices such as tubings and catheters. Testicular Leydig cells (LCs) are suggested to be among the main targets of phthalate toxicity in the body. However, their sensitivity to phthalates is species-dependent. This paper describes the response of the LCs from different species (mouse, rat and human) to phthalate exposure in different experimental paradigms (in vivo, ex vivo and in vitro), with particular focus on mechanisms of phthalate action on LC steroidogenesis. A comprehensive analysis of the impact of phthalate diesters and phthalate monoesters on LCs in different stages of their development is presented and possible mechanisms of phthalates action are discussed. Finally novel, not yet fully elucidated sites of action of phthalate monoesters on the backdoor pathway of 5α-dihydrotestosterone biosynthesis in immature mouse LCs and their effects on steroidogenesis and redox state in adult mouse LCs are reported.

Leptin Within the Subphysiological to Physiological Range Dose Dependently Improves Male Reproductive Function in an Obesity Mouse Model.

Obesity has recently been linked with reduced fertility, and the mechanisms underpinning this effect are currently unknown. The adipokine leptin is dysregulated in obesity and affects reproductive tracts; therefore, we investigated the dose-dependent effects of leptin on Leydig cell function and spermatogenesis. Eight-week-old leptin-deficient obese (ob/ob) male mice were treated with subphysiological (0.1- or 0.5-mg/kg body weight [BW]/d) or physiological (3.0-mg/kg BW/d) doses of leptin or saline for 12 weeks (chronic treatment) or 72 hours (acute treatment). We then evaluated male reproductive function markers. Mean testis weight increased significantly in the 0.1- and 3.0-mg/kg BW/d groups compared with saline controls (both P < .05). Intratesticular testosterone levels relative to testis weight significantly increased in the 0.5-mg/kg BW/d group compared with saline controls (P < .05). FSH levels increased in a dose-dependent manner with leptin treatment, whereas LH levels did not change. Leptin treatment significantly up-regulated both mRNA and protein expression of the steroidogenic enzyme cytochrome P450 17A1. Spermatogenesis improved in leptin-treated animals. Significantly more seminiferous tubules were observed in stages I-VIII (P < .01), and there were fewer abnormal seminiferous tubule structures (P < .01). Acute treatment with physiological leptin doses partially improved male reproductive markers without changing BW. Administration of subphysiological to physiological doses of leptin improves Leydig cell function and spermatogenesis.

Stimulation of steroidogenesis in immature rat Leydig cells evoked by interleukin-1alpha is potentiated by growth hormone and insulin-like growth factors.

The cytokine IL-1alpha is produced constitutively by the intact testis, but its function in this organ remains largely unknown. In this study we examined cooperation between IL-1alpha and GH and IGFs with regard to stimulation of steroidogenesis by Leydig cells from 40-d-old rats in vitro. IL-1alpha alone stimulated testosterone (T) and dihydrotestosterone (DHT) production. GH, IGF-I, or IGF-II alone was without effect on T production, but they were found to elevate DHT release, albeit without an obvious dose-response effect. Costimulation with IL-1alpha and GH or with IL-1alpha and IGF-I or IGF-II elevated the rate of steroidogenesis (both T and DHT) above that observed with IL-1alpha alone. GH was found to increase the level of IGF-I in the cultured Leydig cells, an effect that was potentiated by IL-1alpha. The costimulatory effect of GH on steroidogenesis was abolished by treatment with picropodophyllin, a specific inhibitor of the IGF-I receptor, indicating that the action of GH is mediated via IGF-I. Moreover, cells costimulated with IL-1alpha and GH exhibited a marked decrease in the level of intact IGF-binding protein-3 in the culture medium due to the induction of proteolytic activity toward this binding protein. In contrast, secretion of IGF-binding protein-2 was increased by such costimulation. These findings suggest that the stimulation of steroidogenesis in Leydig cells evoked by GH and IGFs requires cooperation with IL-1alpha. This cooperation may play an important role in connection with postnatal Leydig cell maturation and steroidogenesis.

The liver X receptor-{beta} is essential for maintaining cholesterol homeostasis in the testis.

The liver X receptor (LXR)alpha and -beta has been found to play a central role in maintaining cellular cholesterol homeostasis. In this study we comprehensively investigated the effect of deleting LXRalpha and -beta on testicular morphology and function. In the absence of LXRbeta, excessive cholesterol accumulated in the Sertoli cells from 2.5 months, resulting in severe cellular disruption and dysregulation of spermatogenesis by 10 months of age. This correlated with gene expression analyses that clearly indicated that LXRbeta was the dominant transcript in the testis Although the LXRalpha(-/-) testis was normal, the LXRalpha(-/-)beta(-/-) testis presented with a more severe phenotype than the LXRbeta(-/-) mice, and males were infertile by 4 months of age, indicating LXRalpha may partially rescue the testicular phenotype. Although Leydig cells did not accumulate excessive cholesterol, declining serum and intratesticular androgen levels with age suggested that these cells were in fact less functional. Treatment of a Sertoli cell line with the LXR agonist T0901317 led to increased expression of known LXR target genes like ATP binding cassette-G1 and sterol regulatory binding protein-1c; similar results were observed in wild-type testis after in vivo administration, suggesting the LXR is functioning in the same way as in other tissues. Ordinarily increased levels of cholesterol activate intracellular sensors to decrease these levels; however, the increasing amount of cholesterol in the Sertoli cells indicates improper control of cholesterol metabolism when LXRbeta is absent. Although the precise molecular mechanism at this time remains unclear, our study highlights the crucial role for LXRbeta in retaining cholesterol homeostasis in Sertoli cells.

Interleukin-18 is expressed in rat testis and may promote germ cell growth.

Although host-defence mechanisms, designed to preserve the integrity of the developing germ cells are operative in the testis, the components of this protective system have yet to be characterised in detail. Here, we report that the cytokine interleukin-18 (IL-18) is expressed in the rat testis and may contribute to these defences. Thus, analysis by RT-PCR and Western blotting revealed pronounced testicular expression of pro-IL-18 from postnatal day 5 and onwards. Expression of both IL-18 mRNA and protein was found to be localised to meiotic and post-meiotic germ cells as evaluated by in situ hybridisation and immunohistochemistry, respectively. The mRNA species coding for the IL-18 receptor and IL-1beta converting enzyme, which activates pro-IL-18, were also shown to be expressed by the seminiferous tubules. Recombinant IL-18 was seen to stimulate spermatogonial DNA synthesis in cultures of staged segments of rat seminiferous tubules, without influencing germ cell apoptosis. These results suggest that IL-18 may have host-protective and growth-promoting functions in the testis, but further investigations need to be done to confirm this.