RESUMEN

Male reproductive function depends on the formation of spermatogonial stem cells from their neonatal precursors, the gonocytes. Previously, we identified several UPS enzymes dynamically altered during gonocyte differentiation. The present work focuses on understanding the role of the RING finger protein 149 (RNF149), an E3 ligase that we found to be strongly expressed in gonocytes and downregulated in spermatogonia. The quantification of RNF149 mRNA from postnatal day (PND) 2 to 35 (puberty) in rat testis, brain, liver, kidney, and heart indicated that its highest levels are found in the testis. RNF149 knock-down in PND3 rat gonocytes was performed to better understand its role in gonocyte development. While a proliferative cocktail of PDGF-BB and 17ß-estradiol (P+E) increased both the expression levels of the cell proliferation marker PCNA and RNF149 in mock cells, the effects of P+E on both genes were reduced in cells treated with RNF149 siRNA, suggesting that RNF149 expression is regulated during gonocyte proliferation and that there might be a functional link between RNF149 and PCNA. To examine RNF149 subcellular localization, EGFP-tagged RNF149 vectors were constructed, after determining the rat testis RNF149 mRNA sequence. Surprisingly, two variant transcripts were expressed in rat tissues, predicting truncated proteins, one containing the PA and the other the RING functional domains. Transfection in mouse F9 embryonal carcinoma cells and C18-4 spermatogonial cell lines showed differential subcellular profiles of the two truncated proteins. Overall, the results of this study support a role for RNF149 in gonocyte proliferation and suggest its transcription to variant mRNAs resulting in two proteins with different functional domains. Future studies will examine the respective roles of these variant proteins in the cell lines and isolated gonocytes.

Asunto(s)

Maduración Sexual , Ubiquitina , Animales , Masculino , Ratones , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Espermatogonias/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética

RESUMEN

BACKGROUND: In infants, fever is often treated with acetaminophen or ibuprofen, two antipyretic and analgesic drugs inhibiting cyclooxygenases (COXs), enzymes catalyzing prostaglandin production. Infancy represents a critical developmental period when neonatal germ cells/gonocytes differentiate to spermatogonial stem cells required for spermatogenesis. OBJECTIVES: (a) Determine the expression of Cox2 and associated genes in postnatal day (PND)3 rat gonocytes compared to spermatogonia. (b) Examine whether acetaminophen or ibuprofen disrupts neonatal gonocyte functions. (c) Determine whether neonatal gonocytes produce prostaglandins and whether this process is altered by acetaminophen and ibuprofen. MATERIALS AND METHODS: The expression of Cox2 and related genes was determined by gene arrays and qPCR. Cox2 protein levels were determined by immunocyto/histochemistry and immunoblots. The effects of acetaminophen and ibuprofen on PND3 gonocyte viability, apoptosis, proliferation, and differentiation were examined alone and with a proliferation cocktail or differentiation factor. Prostaglandins were examined by immunocyto/histochemistry and LC-MS. RESULTS: Cox2 and related genes are highly expressed in gonocytes and spermatogonia. Acetaminophen and ibuprofen did not affect gonocyte survival or apoptosis, but they increased gonocyte proliferation. Ibuprofen significantly reduced RA-induced Stra8 expression, indicating an inhibitory effect on differentiation. Ibuprofen combined with RA decreased Cox2 mRNA and protein expression. PGE2 and PGF2α were produced by neonatal gonocytes and decreased by acetaminophen and ibuprofen. DISCUSSION: The concomitant decrease of Stra8 expression, Cox2 expression, and PGE2 and PGF2a production in gonocytes co-treated with RA suggests that Cox2 plays a role in PND3 gonocyte differentiation. The effects of acetaminophen and ibuprofen on proliferation suggest a negative relationship between Cox2 and proliferation. Treating neonates with acetaminophen or ibuprofen could disrupt gonocyte development, leading to adverse reproductive effects. CONCLUSION: Understanding COX2 role in neonatal gonocytes and the potential risk of acetaminophen and ibuprofen treatment of infants may help prevent male reproductive pathologies.

Asunto(s)

Acetaminofén/toxicidad , Células Madre Germinales Adultas/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/toxicidad , Ibuprofeno/toxicidad , Células Madre Germinales Adultas/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

Peroxiredoxins (PRDXs) are antioxidant enzymes that protect cells from oxidative stress and play a role in reactive oxygen species (ROS)-mediated signaling. We reported that PRDXs are critical for human fertility by maintaining sperm viability and regulating ROS levels during capacitation. Moreover, studies on Prdx6-/- mice revealed the essential role of PRDX6 in the viability, motility, and fertility competence of spermatozoa. Although PRDXs are abundant in the testis and spermatozoa, their potential role at different phases of spermatogenesis and in perinatal germ cells is unknown. Here, we examined the expression and role of PRDXs in isolated rat neonatal gonocytes, the precursors of spermatogonia, including spermatogonial stem cells. Gene array, qPCR analyses showed that PRDX1, 2, 3, 5, and 6 transcripts are among the most abundant antioxidant genes in postnatal day (PND) 3 gonocytes, while immunofluorescence confirmed the expression of PRDX1, 2, and 6 proteins. The role of PRDXs in gonocyte viability was examined using PRDX inhibitors, revealing that the 2-Cys PRDXs and PRDX6 peroxidases activities are critical for gonocytes viability in basal condition, likely preventing an excessive accumulation of endogenous ROS in the cells. In contrast to its crucial role in spermatozoa, PRDX6 independent phospholipase A2 (iPLA2) activity was not critical in gonocytes in basal conditions. However, under conditions of H2O2-induced oxidative stress, all these enzymatic activities were critical to maintain gonocyte viability. The inhibition of PRDXs promoted a two-fold increase in lipid peroxidation and prevented gonocyte differentiation. These results suggest that ROS are produced in neonatal gonocytes, where they are maintained by PRDXs at levels that are non-toxic and permissive for cell differentiation. These findings show that PRDXs play a major role in the antioxidant machinery of gonocytes, to maintain cell viability and allow for differentiation.

RESUMEN

Spermatogenesis consists of a series of highly regulated processes that include mitotic proliferation, meiosis and cellular remodeling. Although alterations in gene expression are well known to modulate spermatogenesis, posttranscriptional mechanisms are less well defined. The ubiquitin proteasome system plays a significant role in protein turnover and may be involved in these posttranscriptional mechanisms. We previously identified ubiquitin ligase Huwe1 in the testis and showed that it can ubiquitinate histones. Since modulation of histones is important at many steps in spermatogenesis, we performed a complete characterization of the functions of Huwe1 in this process by examining the effects of its inactivation in the differentiating spermatogonia, spermatocytes and spermatids. Inactivation of Huwe1 in differentiating spermatogonia led to their depletion and formation of fewer pre-leptotene spermatocytes. The cell degeneration was associated with an accumulation of DNA damage response protein γH2AX, impaired downstream signalling and apoptosis. Inactivation of Huwe1 in spermatocytes indicated that Huwe1 is not essential for meiosis and spermiogenesis, but can result in accumulation of γH2AX. Collectively, these results provide a comprehensive survey of the functions of Huwe1 in spermatogenesis and reveal Huwe1's critical role as a modulator of the DNA damage response pathway in the earliest steps of spermatogonial differentiation.

Asunto(s)

Diferenciación Celular/fisiología , Ligasas/metabolismo , Meiosis/fisiología , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Femenino , Histonas/metabolismo , Masculino , Ratones , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/metabolismo , Espermátides/metabolismo , Espermatocitos/metabolismo , Espermatogonias/fisiología , Testículo/metabolismo , Testículo/fisiología

RESUMEN

Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology.

Asunto(s)

Proteínas Portadoras/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA/metabolismo , Seminoma/metabolismo , Espermatogénesis , Espermatogonias/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Humanos , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Receptores de GABA/genética , Receptores de GABA-A/genética , Espermatogonias/citología , Espermatogonias/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tretinoina/farmacología

RESUMEN

For decades, only few tissues and cell types were defined as steroidogenic, capable of de novo steroid synthesis from cholesterol. However, with the refinement of detection methods, several tissues have now been added to the list of steroidogenic tissues. Besides their critical role as long-range acting hormones, steroids are also playing more discreet roles as local mediators and signaling molecules within the tissues they are produced. In testis, steroidogenesis is carried out by the Leydig cells through a broad network of proteins, mediating cholesterol delivery to CYP11A1, the first cytochrome of the steroidogenic cascade, and the sequential action of enzymes insuring the production of active steroids, the main one being testosterone. The knowledge that male germ cells can be directly regulated by steroids and that they express several steroidogenesis-related proteins led us to hypothesize that germ cells could produce steroids, acting as autocrine, intracrine and juxtacrine modulators, as a way to insure synchronized progression within spermatogenic cycles, and preventing inappropriate cell behaviors between neighboring cells. Gene expression and protein analyses of mouse and rat germ cells from neonatal gonocytes to spermatozoa showed that most steroidogenesis-associated genes are expressed in germ cells, showing cell type-, spermatogenic cycle-, and age-specific expression profiles. Highly expressed genes included genes involved in steroidogenesis and other cell functions, such as Acbd1 and 3, Tspo and Vdac1-3, and genes involved in fatty acids metabolism or synthesis, including Hsb17b4 10 and 12, implying broader roles than steroid synthesis in germ cells. These results support the possibility of an additional level of regulation of spermatogenesis exerted between adjacent germ cells.

Asunto(s)

Biomarcadores/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Esteroides/biosíntesis , Testículo/metabolismo , Animales , Western Blotting , Células Cultivadas , Perfilación de la Expresión Génica , Células Germinativas/citología , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/citología

RESUMEN

The production of spermatozoa relies on a pool of spermatogonial stem cells (SSCs), formed in infancy from the differentiation of their precursor cells, the gonocytes. Throughout adult life, SSCs will either self-renew or differentiate, in order to maintain a stem cell reserve while providing cells to the spermatogenic cycle. By contrast, gonocytes represent a transient and finite phase of development leading to the formation of SSCs or spermatogonia of the first spermatogenic wave. Gonocyte development involves phases of quiescence, cell proliferation, migration, and differentiation. Spermatogonia, on the other hand, remain located at the basement membrane of the seminiferous tubules throughout their successive phases of proliferation and differentiation. Apoptosis is an integral part of both developmental phases, allowing for the removal of defective cells and the maintenance of proper germ-Sertoli cell ratios. While gonocytes and spermatogonia mitosis are regulated by distinct factors, they both undergo differentiation in response to retinoic acid. In contrast to postpubertal spermatogenesis, the early steps of germ cell development have only recently attracted attention, unveiling genes and pathways regulating SSC self-renewal and proliferation. Yet, less is known on the mechanisms regulating differentiation. The processes leading from gonocytes to spermatogonia have been seldom investigated. While the formation of abnormal gonocytes or SSCs could lead to infertility, defective gonocyte differentiation might be at the origin of testicular germ cell tumors. Thus, it is important to better understand the molecular mechanisms regulating these processes. This review summarizes and compares the present knowledge on the mechanisms regulating mammalian gonocyte and spermatogonial differentiation.

Asunto(s)

Diferenciación Celular/genética , Células Germinativas/metabolismo , Células de Sertoli/metabolismo , Espermatogonias/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , Espermatogénesis/genética

RESUMEN

Apoptosis is an integral part of the spermatogenic process, necessary to maintain a proper ratio of Sertoli to germ cell numbers and provide an adequate microenvironment to germ cells. Apoptosis may also represent a protective mechanism mediating the elimination of abnormal germ cells. Extensive apoptosis occurs between the first and second postnatal weeks, at the point when gonocytes, precursors of spermatogonial stem cells, should have migrated toward the basement membrane of the tubules and differentiated into spermatogonia. The mechanisms regulating this process are not well-understood. Gonocytes undergo phases of proliferation, migration, and differentiation which occur in a timely and closely regulated manner. Gonocytes failing to migrate and differentiate properly undergo apoptosis. Inadequate gonocyte differentiation has been suggested to lead to testicular germ cell tumor (TGCT) formation. Here, we examined the expression levels of apoptosis-related genes during gonocyte differentiation by quantitative real-time polymerase chain reaction, identifying 48 pro- and anti-apoptotic genes increased by at least two-fold in rat gonocytes induced to differentiate by retinoic acid, when compared to untreated gonocytes. Further analysis of the most highly expressed genes identified the pro-apoptotic genes Gadd45a and Cycs as upregulated in differentiating gonocytes and in spermatogonia compared with gonocytes. These genes were also significantly downregulated in seminomas, the most common type of TGCT, compared with normal human testicular tissues. These results indicate that apoptosis-related genes are actively regulated during gonocyte differentiation. Moreover, the down-regulation of pro-apoptotic genes in seminomas suggests that they could represent new therapeutic targets in the treatment of TGCTs.

Asunto(s)

Células Madre Adultas/patología , Apoptosis/genética , Diferenciación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Seminoma/genética , Neoplasias Testiculares/genética , Células Madre Adultas/efectos de los fármacos , Animales , Caspasa 9/genética , Diferenciación Celular/efectos de los fármacos , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratas , Ratas Sprague-Dawley , Seminoma/patología , Neoplasias Testiculares/patología , Tretinoina/farmacología , Proteinas GADD45

RESUMEN

Neonatal gonocytes are direct precursors of spermatogonial stem cells, the cell pool that supports spermatogenesis. Although unipotent in vivo, gonocytes express pluripotency genes common with embryonic stem cells. Previously, we found that all-trans retinoic acid (RA) induced the expression of differentiation markers and a truncated form of platelet-derived growth factor receptor (PDGFR)ß in rat gonocytes, as well as in F9 mouse embryonal carcinoma cells, an embryonic stem cell-surrogate that expresses somatic lineage markers in response to RA. The present study is focused on identifying the signaling pathways involved in RA-induced gonocyte and F9 cell differentiation. Mitogen-activated protein kinase kinase (MEK) 1/2 activation was required during F9 cell differentiation towards somatic lineage, whereas its inhibition potentiated RA-induced Stra8 expression, suggesting that MEK1/2 acts as a lineage specification switch in F9 cells. In both cell types, RA increased the expression of the spermatogonial/premeiotic marker Stra8, which is in line with F9 cells being at a stage before somatic-germline lineage specification. Inhibiting PDGFR kinase activity reduced RA-induced Stra8 expression. Interestingly, RA increased the expression of PDGFRα variant forms in both cell types. Together, these results suggest a potential cross talk between RA and PDGFR signaling pathways in cell differentiation. RA receptor-α inhibition partially reduced RA effects on Stra8 in gonocytes, indicating that RA acts in part via RA receptor-α. RA-induced gonocyte differentiation was significantly reduced by inhibiting SRC (v-src avian sarcoma [Schmidt-Ruppin A-2] viral oncogene) and JAK2/STAT5 (Janus kinase 2/signal transducer and activator of transcription 5) activities, implying that these signaling molecules play a role in gonocyte differentiation. These results suggest that gonocyte and F9 cell differentiation is regulated via cross talk between RA and PDGFRs using different downstream pathways.

Asunto(s)

Células Madre de Carcinoma Embrionario/citología , Células Madre de Carcinoma Embrionario/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Tretinoina/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Madre Adultas/citología , Células Madre Adultas/fisiología , Animales , Animales Recién Nacidos , Diferenciación Celular , Línea Celular , Proliferación Celular , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Queratolíticos/farmacología , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Ratones , Factor de Crecimiento Derivado de Plaquetas/genética , Ratas , Ratas Sprague-Dawley , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Testículo/citología , Testículo/crecimiento & desarrollo

RESUMEN

Spermatogenesis represents a complex succession of cell division and differentiation events resulting in the continuous formation of spermatozoa. Such a complex program requires precise expression of enzymes and structural proteins which is effected not only by regulation of gene transcription and translation, but also by targeted protein degradation. In this chapter, we review current knowledge about the role of the ubiquitin-proteasome system in spermatogenesis, describing both proteolytic and non-proteolytic functions of ubiquitination. Ubiquitination plays essential roles in the establishment of both spermatogonial stem cells and differentiating spermatogonia from gonocytes. It also plays critical roles in several key processes during meiosis such as genetic recombination and sex chromosome silencing. Finally, in spermiogenesis, we summarize current knowledge of the role of the ubiquitin-proteasome system in nucleosome removal and establishment of key structures in the mature spermatid. Many mechanisms remain to be precisely defined, but present knowledge indicates that research in this area has significant potential to translate into benefits that will address problems in both human and animal reproduction.

Asunto(s)

Complejo de la Endopetidasa Proteasomal/fisiología , Espermatogénesis/fisiología , Ubiquitina/metabolismo , Acrosoma/fisiología , Animales , Humanos , Masculino , Meiosis , Nucleosomas/fisiología

RESUMEN

The ubiquitin proteasome system (UPS) consists of a cascade of enzymatic reactions leading to the ubiquitination of proteins, with consequent degradation or altered functions of the proteins. Alterations in UPS genes have been associated with male infertility, suggesting the role of UPS in spermatogenesis. In the present study, we questioned whether UPS is involved in extensive remodeling and functional changes occurring during the differentiation of neonatal testicular gonocytes to spermatogonia, a step critical for the establishment of the spermatogonial stem cell population. We found that addition of the proteasome inhibitor lactacystin to isolated gonocytes inhibited their retinoic acid-induced differentiation in a dose-dependent manner, blocking the induction of the spermatogonial gene markers Stra8 and Dazl. We then compared the UPS gene expression profiles of Postnatal Day (PND) 3 gonocytes and PND8 spermatogonia, using gene expression arrays and quantitative real-time PCR analyses. We identified 205 UPS genes, including 91 genes expressed at relatively high levels. From those, 28 genes were differentially expressed between gonocytes and spermatogonia. While ubiquitin-activating enzymes and ligases showed higher expression in gonocytes, most ubiquitin conjugating and deubiquitinating enzymes were expressed at higher levels in spermatogonia. Concomitant with the induction of spermatogonial gene markers, retinoic acid altered the expression of many UPS genes, suggesting that the UPS is remodeled during gonocyte differentiation. In conclusion, these studies identified novel ubiquitin-related genes in gonocytes and spermatogonia and revealed that proteasome function is involved in gonocyte differentiation. Considering the multiple roles of the UPS, it will be important to determine which UPS genes direct substrates to the proteasome and which are involved in proteasome-independent functions in gonocytes and to identify their target proteins.

Asunto(s)

Diferenciación Celular , Complejo de la Endopetidasa Proteasomal/metabolismo , Espermatogonias/metabolismo , Ubiquitina/metabolismo , Animales , Animales Recién Nacidos , Perfilación de la Expresión Génica , Masculino , Complejo de la Endopetidasa Proteasomal/genética , Inhibidores de Proteasoma , Ratas , Ratas Sprague-Dawley , Células de Sertoli/metabolismo , Espermatogonias/citología , Tretinoina

RESUMEN

In recent years, an increasing interest has emerged at understanding how spermatogonial stem cells (SSCs) arise from their precursor cells, the gonocytes. The identification of factors acting directly on gonocytes rather than on the surrounding somatic cells and the study of genes and signaling pathways intrinsic to gonocytes require their isolation from Sertoli and peritubular cells. The present article describes a simple method developed to isolate rat neonatal gonocytes, allowing for the study of their proliferation and differentiation to SSCs. We also present immunocytochemical methods that can be used to study protein expression changes and proliferation in gonocytes.

Asunto(s)

Separación Celular/métodos , Inmunohistoquímica/métodos , Espermatogonias/citología , Espermatogonias/metabolismo , Testículo/citología , Animales , Disección/instrumentación , Disección/métodos , Masculino , Ratas , Ratas Sprague-Dawley , Testículo/anatomía & histología

RESUMEN

We previously found that platelet-derived growth factor (PDGF) and 17beta-estradiol stimulate gonocyte proliferation in a dose-dependent, nonadditive manner. In the present study, we report that gonocytes express RAF1, MAP2K1, and MAPK1/3. Inhibition of RAF1 and MAP2K1/2, but not phosphoinositide-3-kinase, blocked PDGF-induced proliferation. AG-370, an inhibitor of PDGF receptor kinase activity, suppressed not only PDGF-induced proliferation but also that induced by 17beta-estradiol. In addition, RAF1 and MAP2K1/2 inhibitors blocked 17beta-estradiol-activated proliferation. The estrogen receptor antagonist ICI 182780 inhibited both the effects of 17beta-estradiol and PDGF. PDGF lost its stimulatory effect when steroid-depleted serum or no serum was used. Similarly, 17beta-estradiol did not induce gonocyte proliferation in the absence of PDGF. The xenoestrogens genistein, bisphenol A, and DES, but not coumestrol, stimulated gonocyte proliferation in a dose-dependent and PDGF-dependent manner similarly to 17beta-estradiol. Their effects were blocked by ICI 182780, suggesting that they act via the estrogen receptor. AG-370 blocked genistein and bisphenol A effects, demonstrating their requirement of PDGF receptor activation in a manner similar to 17beta-estradiol. These results demonstrate the interdependence of PDGF and estrogen pathways in stimulating in vitro gonocyte proliferation, suggesting that this critical step in gonocyte development might be regulated in vivo by the coordinated action of PDGF and estrogen. Thus, the inappropriate exposure of gonocytes to xenoestrogens might disrupt the crosstalk between the two pathways and potentially interfere with gonocyte development.

Asunto(s)

Proliferación Celular , Estradiol/fisiología , Células Germinativas/fisiología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Sistemas de Mensajero Secundario/fisiología , Espermatogénesis/fisiología , Animales , Inmunohistoquímica , MAP Quinasa Quinasa 1/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-raf , Ratas , Ratas Sprague-Dawley , Receptor Cross-Talk/fisiología , Transducción de Señal/fisiología , Espermatogonias/fisiología , Testículo/citología , Testículo/metabolismo

RESUMEN

Concerns have been raised about the possible role of the phytoestrogen genistein and the xenoestrogen Bisphenol A (BPA) as endocrine disruptors. In the present study, we examined the effects of fetal exposure to genistein and BPA on the Mitogen-activated protein kinase (MAPK) pathway and on testicular cell populations in neonatal and adult rat testes. At postnatal day (PND) 3, genistein (0.1-10 mg/kg/day) and BPA (1-200 mg/kg/day) induced Raf1 and Erk1/2 mRNA and protein increases in testes, mainly in Sertoli cells. No changes were seen for Mek1. At PND60, Erk1/2 protein expression remained robust in Sertoli cells and in some spermatogonia. Raf1 was predominant in Leydig cells while Mek1 was expressed strongly in spermatogonia, and they were both expressed in pachytene spermatocytes. No consistent change was seen in these proteins at PND60. Transient effects were observed on germ cell populations, while the only remaining effect on adult testicular cells was an increase in Leydig cell number. Rats exposed in utero to the two compounds did not present significant changes in circulating testosterone levels, suggesting normally functioning adult Leydig cells. Furthermore, Sertoli cell numbers were not affected by exposure to genistein and BPA. Finally, around 10% of genistein and BPA exposed rats were sterile, whereas all control rats were fertile. These data suggest that fetal exposure to genistein and BPA exerts transient effects in rat testes and that the changes observed at PND3 did not correlate with relevant changes in germ cell populations, Leydig cell function, or fertility in the adult.

Asunto(s)

Estrógenos/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células de Sertoli/efectos de los fármacos , Espermatocitos/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Testículo/efectos de los fármacos , Animales , Animales Recién Nacidos , Compuestos de Bencidrilo , Recuento de Células , Perfilación de la Expresión Génica , Genisteína/farmacología , Infertilidad/inducido químicamente , Masculino , Fenoles/farmacología , Ratas , Transducción de Señal/efectos de los fármacos , Testículo/crecimiento & desarrollo