RESUMEN
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% (Pâ =â .006), Star by 51% (Pâ =â .004), Cyp11A1 by 80% (Pâ =â .003), 3Beta-Hsd2 by 61% (Pâ =â .005), and Pbr by 52% (Pâ =â .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.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Insulina/administración & dosificación , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Animales , Apoptosis/efectos de los fármacos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/fisiopatología , Hormona Folículo Estimulante/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Células Intersticiales del Testículo/citología , Hormona Luteinizante/metabolismo , Masculino , Proteínas/genética , Proteínas/metabolismo , Ratas , Espermatogénesis/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacos , Testículo/metabolismo , Testosterona/metabolismoRESUMEN
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.
Asunto(s)
Andrógenos/biosíntesis , Feto/fisiología , Masculinidad , Dihidrotestosterona/sangre , Dihidrotestosterona/metabolismo , Femenino , Humanos , Masculino , Redes y Vías Metabólicas , Ovario/metabolismo , Embarazo , Segundo Trimestre del Embarazo/sangre , ARN Mensajero/genética , ARN Mensajero/metabolismo , Testículo/metabolismoRESUMEN
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.
Asunto(s)
Tejido Adiposo/fisiología , Hiperandrogenismo/etiología , Obesidad/complicaciones , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Andrógenos/metabolismo , Animales , Células Cultivadas , Femenino , Expresión Génica , Hiperandrogenismo/metabolismo , Lipogénesis/fisiología , Obesidad/metabolismo , Obesidad/patología , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Testosterona/metabolismoRESUMEN
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.
Asunto(s)
Corteza Suprarrenal/efectos de los fármacos , Corteza Suprarrenal/metabolismo , Esteroides/metabolismo , Estilbenos/farmacología , Corteza Suprarrenal/citología , Células Cultivadas , Familia 21 del Citocromo P450/antagonistas & inhibidores , Familia 21 del Citocromo P450/metabolismo , Femenino , Humanos , Embarazo , Primer Trimestre del Embarazo , Resveratrol , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Esteroide 17-alfa-Hidroxilasa/metabolismoRESUMEN
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.
Asunto(s)
Leptina/farmacología , Testículo/efectos de los fármacos , Animales , Ensayo de Inmunoadsorción Enzimática , Hormona Folículo Estimulante/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Ratones , Ratones Obesos , Tamaño de los Órganos/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Túbulos Seminíferos/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismo , Testosterona/metabolismoRESUMEN
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.
Asunto(s)
17-Cetosteroides/metabolismo , Androstanoles/metabolismo , Células Intersticiales del Testículo/metabolismo , Ácidos Ftálicos/toxicidad , Animales , Humanos , Células Intersticiales del Testículo/patología , Masculino , Ratones , Oxidación-Reducción/efectos de los fármacos , RatasRESUMEN
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.
Asunto(s)
Envejecimiento/genética , Células Intersticiales del Testículo/metabolismo , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Envejecimiento/metabolismo , Animales , Citocromos b5/genética , Citocromos b5/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , ADN Mitocondrial/genética , Masculino , Potencial de la Membrana Mitocondrial/genética , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Espermatozoides/metabolismo , Testosterona/genética , Testosterona/metabolismoRESUMEN
Numerous studies have reported on testicular toxicity of phthalates in different experimental paradigms and showed that Leydig cells (LCs) were one of the main targets of phthalate actions. Adverse effects of phthalates on LCs steroidogenesis have been attributed to their metabolites, monophthalates. This study focuses on investigation whether LCs responsiveness to monophthalates action is associated with their potential to produce androgens. We found that of 3 monophthalates investigated [ie, mono-2-ethylhexyl phthalate (MEHP), mono-n-butyl phthalate, and mono-n-benzyl phthalate] only MEHP caused biological effects on the mouse LCs function. This monophthalate stimulated basal steroidogenesis associated with upregulation of StAR protein expression with no effect on hCG-stimulated androgen production by LCs from CBA/Lac and C57BL/6j mouse genotypes were observed. Further, MEHP attenuated ATP production and increased superoxide generation by both phenotypes of mouse LCs that indicated on mitochondrial dysfunction induced by the monophthalate. All together, our data indicate that MEHP-mediated stimulation of steroidogenesis and perturbation in mitochondrial function are not associated with the capacity of the LCs to synthesize androgens. We suggest that this effect of MEHP observed in LCs of rodent origin needs to be taken into consideration in analysis of earlier start of puberty in boys and may highlight a possible influence of phthalates on reproductive health in males.
Asunto(s)
Andrógenos/biosíntesis , Dietilhexil Ftalato/análogos & derivados , Células Intersticiales del Testículo/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Esteroides/biosíntesis , Animales , Dietilhexil Ftalato/toxicidad , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Mitocondrias/metabolismo , Fosfoproteínas/metabolismoRESUMEN
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.
Asunto(s)
Ácidos Borónicos/efectos adversos , Pirazinas/efectos adversos , Espermatogénesis/efectos de los fármacos , Enfermedades Testiculares/patología , Testosterona/biosíntesis , Animales , Ácidos Borónicos/administración & dosificación , Bortezomib , Hormona Folículo Estimulante/sangre , Humanos , Masculino , Ratones , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Inhibidores de Proteasoma/administración & dosificación , Pirazinas/administración & dosificación , Enfermedades Testiculares/inducido químicamente , Testículo/efectos de los fármacos , Testículo/patologíaRESUMEN
AIM: We aim to examine the steroidogenic phenotype and the differentiation potential of human testicular peritubular cells (HTPCs) and to explore their possible relationship to the adult Leydig cell lineage. BACKGROUND: The cells of the adult Leydig cell lineage may reside in the peritubular compartment of the testis. This suggestion is supported by the facts that the rodent peritubular cells can be differentiated toward this lineage and that cAMP enhances their steroidogenic potential. METHODS: Human testicular biopsies, and derived HTPCs, were analyzed by immunohistochemistry, RT-PCR, and Western blotting. After stimulation by forskolin or platelet-derived growth factor-BB, quantitative RT-PCR was used to compare the levels of mRNAs encoding proteins involved in steroidogenesis and steroid production was analyzed by liquid chromatography and tandem mass spectrometry. RESULTS: Immunohistochemical analysis revealed that the peritubular cells that form the outer part of the tubular wall express platelet derived growth factor receptor-α. Furthermore, the pluripotency markers (POU domain class 5 transcription factor 1, GATA-binding protein 4), stem Leydig cell markers (platelet derived growth factor receptor-A, leukemia inhibitory factor receptor), and mRNAs encoding proteins involved in steroidogenesis (nuclear receptor subfamily 5, group A, member 1; steroidogenic acute regulatory protein; CYP11A1; CYP17A1; 3ß-hydroxysteroid dehydrogenase) were expressed by the HTPCs. Stimulation with forskolin increased the expression of the steroidogenic markers, which was accompanied by the production of pregnenolone and progesterone by HTPCs in vitro. Treatment with platelet-derived growth factor-BB induced expression of steroidogenic acute regulatory protein. CONCLUSIONS: Our results indicate that the tubular wall of the human testis is a reservoir for cells of the adult Leydig cell lineage and that the steroidogenic potential of these cells can be activated in culture.
Asunto(s)
Hormonas Esteroides Gonadales/biosíntesis , Células Intersticiales del Testículo/metabolismo , Células Madre/citología , Células Madre/metabolismo , Testículo/citología , Adulto , Células Cultivadas , Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Intersticiales del Testículo/citología , Masculino , Proteína Homeótica Nanog , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Testículo/metabolismo , Testículo/patologíaRESUMEN
Although three-dimensional testicular cell cultures have been demonstrated to mimic the organization of the testis in vivo and support spermatogenesis, the optimal culture conditions and requirements remain unknown. Therefore, utilizing an established three-dimensional cell culture system that promotes differentiation of pre-meiotic murine male germ cells as far as elongated spermatids, the present study was designed to test the influence of different culture media on germ cell differentiation, Leydig cell functionality, and overall cell survival. Single-cell suspensions prepared from 7-day-old rat testes and containing all the different types of testicular cells were cultured for as long as 31 days, with or without stimulation by gonadotropins. Leydig cell functionality was assessed on the basis of testosterone production and the expression of steroidogenic genes. Gonadotropins promoted overall cell survival regardless of the culture medium employed. Of the various media examined, the most pronounced expression of Star and Tspo, genes related to steroidogenesis, as well as the greatest production of testosterone was attained with Dulbecco's modified eagle medium + glutamine. Although direct promotion of germ cell maturation by the cell culture medium could not be observed, morphological evaluation in combination with immunohistochemical staining revealed unfavorable organization of tubules formed de novo in the three-dimensional culture, allowing differentiation to the stage of pachytene spermatocytes. Further differentiation could not be observed, probably due to migration of germ cells out of the cell colonies and the consequent lack of support from Sertoli cells. In conclusion, the observations reported here show that in three-dimensional cultures, containing all types of rat testicular cells, the nature of the medium per se exerts a direct influence on the functionality of the rat Leydig cells, but not on germ cell differentiation, due to the lack of proper organization of the Sertoli cells.
RESUMEN
During the past few decades, scientific evidence has been accumulated concerning the possible adverse effects of the exposure to environmental chemicals on the well-being of wildlife and human populations. One large and growing group of such compounds of anthropogenic or natural origin is referred to as endocrine-disrupting chemicals (EDCs), due to their deleterious action on the endocrine system. This concern was first focused on the control of reproductive function particularly in males, but has later been expanded to include all possible endocrine functions. The present review describes the underlying physiology behind the cascade of developmental events that occur during sexual differentiation of males and the specific role of androgen in the masculinization process and proper organogenesis of the external male genitalia. The impact of the genetic background, environmental exposures and lifestyle factors in the etiology of hypospadias, cryptorchidism and testicular cancer are reviewed and the possible role of EDCs in the development of these reproductive disorders is discussed critically. Finally, the possible direct and programming effects of exposures in utero to widely use therapeutic compounds, environmental estrogens and other chemicals on the incidence of reproductive abnormalities and poor semen quality in humans are also highlighted.
Asunto(s)
Trastornos del Desarrollo Sexual/inducido químicamente , Disruptores Endocrinos/efectos adversos , Efectos Tardíos de la Exposición Prenatal , Andrógenos/fisiología , Animales , Criptorquidismo/etiología , Criptorquidismo/genética , Sistema Endocrino/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Femenino , Genitales Masculinos/embriología , Humanos , Hipospadias/etiología , Hipospadias/genética , Estilo de Vida , Masculino , Ratones , Embarazo , Ratas , Análisis de Semen , Células de Sertoli/efectos de los fármacos , Diferenciación Sexual/efectos de los fármacos , Neoplasias Testiculares/inducido químicamente , Neoplasias Testiculares/genética , Factores de Transcripción/fisiologíaRESUMEN
It is well known that estrogens and estrogen-like endocrine disruptors can suppress steroidogenic gene expression, attenuate androgen production and decrease differentiation of adult Leydig cell lineage. However, there is no information about the possible link between the potency of Leydig cells to produce androgens and their sensitivity to estrogenic stimuli. Thus, the present study explored the relationship between androgen production potential of Leydig cells and their responsiveness to estrogenic compounds. To investigate this relationship we selected mouse genotypes contrasting in sex hormone levels and differing in testosterone/estradiol (T/E2) ratio. We found that two mouse genotypes, CBA/Lac and C57BL/6j have the highest and the lowest serum T/E2 ratio associated with increased serum LH level in C57BL/6j compared to CBA/Lac. Analysis of steroidogenic gene expression demonstrated significant upregulation of Cyp19 gene expression but coordinated suppression of LHR, StAR, 3ßHSDI and Cyp17a1 in Leydig cells from C57BL/6j that was associated with attenuated androgen production in basal and hCG-stimulated conditions compared to CBA/Lac mice. These genotype-dependent differences in steroidogenesis were not linked to changes in the expression of estrogen receptors ERα and Gpr30, while ERß expression was attenuated in Leydig cells from C57BL/6j compared to CBA/Lac. No effects of estrogenic agonists on steroidogenesis in Leydig cells from both genotypes were found. In contrast, xenoestrogen bisphenol A significantly potentiated hCG-activated androgen production by Leydig cells from C57BL/6j and CBA/Lac mice by suppressing conversion of testosterone into corresponding metabolite 5α-androstane-3α,17ß-diol. All together our data indicate that developing mouse Leydig cells with different androgen production potential are resistant to estrogenic stimuli, while xenoestrogen BPA facilitates hCG-induced steroidogenesis in mouse Leydig cells via attenuation of testosterone metabolism. This cellular event can cause premature maturation of Leydig cells that may create abnormal intratesticular paracrine milieu and disturb proper development of germ cells.
Asunto(s)
Andrógenos/biosíntesis , Compuestos de Bencidrilo/farmacología , Estrógenos/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Fenoles/farmacología , Testosterona/metabolismo , Animales , Células Cultivadas , Gonadotropina Coriónica/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genotipo , Humanos , Ligandos , Masculino , Ratones , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Especificidad de la EspecieRESUMEN
Platelet-derived growth factor receptor α (PDGFRα)-positive peritubular cells (PTCs) are suggested to be putative stem Leydig cells. At present little is known about their phenotype and steroidogenic potential. We isolated highly purified PDGFRα-positive neonatal PTCs by magnetic cell sorting (MACS) from 8dpp rat testes and characterized them in vitro. We have demonstrated that PDGFRα-positive PTCs have a mixed phenotype. They expressed PTC-specific genes (αSma, Myh11), pluripotency markers (Pou5f1, nestin, Lifr) and genes encoding steroidogenic enzymes. Treatment with the cAMP-analog (Bu)2cAMP for 7 days upregulated steroidogenic enzyme gene expression and significantly increased their steroidogenic potential. The main end-point steroid was progesterone due to rapid inactivation of CYP17 and 17ßHSD. Long-term culturing of PDGFRα-positive PTCs increased the expression of Myh11, and treatment with (Bu)2cAMP attenuated this process. All together, our findings support the hypothesis that neonatal PDGFRα-positive PTCs are steroidogenic competent progeny of stem Leydig cells (SLCs) which give rise to the adult Leydig cell lineage.
Asunto(s)
Células Intersticiales del Testículo/enzimología , Progesterona/biosíntesis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Testosterona/biosíntesis , Animales , Animales Recién Nacidos , Diferenciación Celular , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , AMP Cíclico/metabolismo , Hormonas Esteroides Gonadales/biosíntesis , Proteínas de Filamentos Intermediarios/metabolismo , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/metabolismo , Células Intersticiales del Testículo/metabolismo , Masculino , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Fenotipo , Cultivo Primario de Células , Progesterona Reductasa/genética , Progesterona Reductasa/metabolismo , Ratas , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario , Túbulos Seminíferos/citología , Células Madre/fisiología , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismo , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismo , TranscriptomaRESUMEN
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.
Asunto(s)
Apoptosis , Células de la Granulosa/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Ovario/metabolismo , Transducción de Señal , Estatmina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Aromatasa/genética , Aromatasa/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hormonas Esteroides Gonadales/metabolismo , Gonadotropinas/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Transgénicos , Factor de Crecimiento Nervioso/genética , Ovario/efectos de los fármacos , Ovario/enzimología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Estatmina/genética , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
We studied steroidogenesis and the regulation of Leydig cell-specific gene expression in primary cultures of highly purified postnatal fetal Leydig cells (PFLCs). PFLCs activated by hCG and (Bu)(2)cAMP demonstrated transient capacity to produce testosterone (T) in vitro. A time dependent decline in T production by (Bu)(2)cAMP-stimulated PFLCs was observed and associated with the accumulation of progesterone in the culture media and complete suppression of P450c17 expression at the translational but not transcriptional level. PFLCs was found to lose their capacity to express Leydig cell-related genes (e.g., 3ßHSD, P450c17, Insl3), which was restored by treatment with (Bu)(2)cAMP. It was also found that PDGFα alone and in combination with (Bu)(2)cAMP significantly stimulated proliferation of the isolated PFLCs in vitro. Our data indicate that cAMP-activated signaling pathway(s) play an important role in the regulation of PFLC differentiation and function.
Asunto(s)
Expresión Génica , Células Intersticiales del Testículo/enzimología , Progesterona/biosíntesis , Testosterona/biosíntesis , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Animales Recién Nacidos , Bucladesina/farmacología , Proliferación Celular , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Gonadotropina Coriónica/farmacología , Pruebas de Enzimas , Insulina/genética , Insulina/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Masculino , Comunicación Paracrina , Fenotipo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Proteínas/genética , Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de HL/genética , Receptores de HL/metabolismo , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismoRESUMEN
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.
Asunto(s)
Flavanonas/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Fitoestrógenos/farmacología , Xantonas/farmacología , Andrógenos/metabolismo , Animales , Colesterol/metabolismo , Gonadotropina Coriónica/metabolismo , AMP Cíclico/metabolismo , Flavonoides/farmacología , Humulus/química , Células Intersticiales del Testículo/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Esteroides/metabolismo , Estilbenos/farmacología , Androstenodiona/metabolismo , Animales , Células Cultivadas , Gonadotropina Coriónica/farmacología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Células Intersticiales del Testículo/enzimología , Células Intersticiales del Testículo/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Ratas , Ratas Sprague-Dawley , Resveratrol , Esteroide 17-alfa-Hidroxilasa/metabolismo , Estilbenos/química , Relación Estructura-Actividad , Sales de Tetrazolio/metabolismoRESUMEN
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.
Asunto(s)
Dietilhexil Ftalato/análogos & derivados , Células Intersticiales del Testículo/efectos de los fármacos , Plastificantes/toxicidad , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Androstano-3,17-diol/metabolismo , Animales , Células Cultivadas , Colesterol/metabolismo , Gonadotropina Coriónica/farmacología , Dietilhexil Ftalato/toxicidad , Células Intersticiales del Testículo/metabolismo , Masculino , Fosfoproteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Testosterona/metabolismoRESUMEN
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.