RESUMO
BACKGROUND: Cadmium (Cd) is a common environmental pollutant and a major constituent of tobacco smoke. Adverse effects of this heavy metal on reproductive function have been identified in adults; however, no studies have examined its effects on human reproductive organs during development. OBJECTIVES: Using our previously developed organ culture system, we investigated the effects of cadmium chloride on human gonads at the beginning of fetal life, a critical stage in the development of reproductive function. METHODS: Human fetal gonads were recovered during the first trimester (711 weeks postconception) and cultured with or without Cd. We used different concentrations of Cd and compared results with those obtained with mouse fetal gonads at similar stages. RESULTS: Cd, at concentrations as low as 1 microM, significantly decreased the germ cell density in human fetal ovaries. This correlated with an increase in germ cell apoptosis, but there was no effect on proliferation. Similarly, in the human fetal testis, Cd (1 microM) reduced germ cell number without affecting testosterone secretion. In mouse fetal gonads, Cd increased only female germ cell apoptosis. CONCLUSIONS: This is the first experimental demonstration that Cd, at low concentrations, alters the survival of male and female germ cells in humans. Considering data demonstrating extensive human exposure, we believe that current environmental levels of Cd could be deleterious to early gametogenesis.
Assuntos
Poluentes Atmosféricos/toxicidade , Apoptose/efeitos dos fármacos , Cádmio/toxicidade , Feto/citologia , Células Germinativas/efeitos dos fármacos , Exposição Materna/efeitos adversos , Adulto , Animais , Feminino , Feto/anatomia & histologia , Células Germinativas/citologia , Gônadas/citologia , Gônadas/efeitos dos fármacos , Gônadas/embriologia , Humanos , Masculino , Camundongos , Técnicas de Cultura de Órgãos , Gravidez , Primeiro Trimestre da GravidezRESUMO
There are great concerns about the increasing incidence of abnormalities in male reproductive function. Human sperm counts have markedly dropped and the rate of testicular cancer has clearly augmented over the past four decades. Moreover, the prevalence rates of cryptorchidism and hypospadias are also probably increasing. It has been hypothesized that all these adverse trends in male reproduction result from abnormalities in the development of the testis during foetal and neonatal life. Furthermore, many recent epidemiological, clinical and experimental data suggest that these male reproductive disorders could be due to the effects of xenobiotics termed endocrine disruptors, which are becoming more and more concentrated and prevalent in our environment. Among these endocrine disruptors, we chose to focus this review on the phthalates for different reasons: 1) they are widespread in the environment; 2) their concentrations in many human biological fluids have been measured; 3) the experimental data using rodent models suggesting a reprotoxicity are numerous and are the most convincing; 4) their deleterious effects on the in vivo and in vitro development and function of the rat foetal testis have been largely studied; 5) some epidemiological data in humans suggest a reprotoxic effect at environmental concentrations at least during neonatal life. However, the direct effects of phthalates on human foetal testis have never been explored. Thus, as we did for the rat in the 1990s, we recently developed and validated an organ culture system which allows maintenance of the development of the different cell types of human foetal testis. In this system, addition of 10-4 M MEHP (mono-2-ethylhexyl phthalate), the most produced phthalate, had no effect on basal or LH-stimulated production of testosterone, but it reduced the number of germ cells by increasing their apoptosis, without modification of their proliferation. This is the first experimental demonstration that phthalates alter the development of the foetal testis in humans. Using our organotypic culture system, we and others are currently investigating the effect of MEHP in the mouse and the rat, and it will be interesting to compare the results between these species to analyse the relevance of toxicological tests based on rodent models.
Assuntos
Disruptores Endócrinos , Testículo , Animais , Humanos , Masculino , Neoplasias Testiculares , Testículo/metabolismo , TestosteronaRESUMO
CONTEXT: Germ cells formed during human fetal life are essential for fertility of the adult, and several studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. However, factors inducing a genotoxic stress may also be implicated. OBJECTIVES: We investigated the effect of gamma-irradiation on the functions of human fetal testis during the first trimester of gestation by using an organ culture system. Then we focused on the role of the p53 pathway in the observed effects. RESULTS: Germ cells were highly sensitive to irradiation even at doses as low as 0.1 and 0.2 Gy. Indeed, for these doses, one third of germ cells died by apoptosis. Other germ cells were blocked in their cycle, but no repair seemed to occur, and longer culture with the highest dose used showed that they were destined to die. Sertoli cells were less affected, although their proliferation and the level of anti-Müllerian hormone were reduced. Irradiation had no effect on testosterone secretion or on the expression of steroidogenic enzymes by Leydig cells. After irradiation, p53 phosphorylated on serine 15 was detected from 1-24 h in all cell types. This activation of p53 was accompanied by an increase in mRNA levels of proapoptotic factors Bax and Puma, whereas that of antiapoptotic Bcl-2 remained unchanged. P21, which is responsible for cell cycle arrest, was also up-regulated 6, 30, and 72 h after irradiation. Finally, when we added pifithrin-alpha, a specific inhibitor of p53 functions, a significant decrease in irradiation-induced apoptosis in both germ and Sertoli cells was observed, indicating the involvement of the p53 pathway in irradiation-induced apoptosis. CONCLUSIONS: This study demonstrated here for the first time the great sensitivity of human fetal germ cells to genotoxic stress caused by ionizing radiation.
Assuntos
Apoptose/efeitos da radiação , Tolerância a Radiação/fisiologia , Testículo/embriologia , Testículo/efeitos da radiação , Caspase 3/metabolismo , Relação Dose-Resposta à Radiação , Idade Gestacional , Humanos , Antígeno Ki-67/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/efeitos da radiação , Masculino , Técnicas de Cultura de Órgãos , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Células de Sertoli/efeitos da radiação , Espermatozoides/citologia , Espermatozoides/metabolismo , Espermatozoides/efeitos da radiação , Testículo/citologia , Testículo/metabolismo , Testosterona/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The key role of the fetal testis in the masculinization of genital organs has been known for a long time. More recently, the observed increases in male reproductive disorders has been postulated to be the result of changes in fetal and neonatal testis development in response to increasing environmental pollution. However, few tools are available for studying fetal testis development and the effects of physiological or toxic substances. We have developed an organ culture system in which rat fetal testis is grown on a filter floating on a synthetic medium containing no serum, hormones or biological factors. In this study, we have compared the long-term morpho-functional development of the various testicular cell types in this system with that observed in vivo and have extended this system to the mouse. Rat Leydig, Sertoli and germ cells and macrophages develop normally over a period of 1-2 weeks in this system. Fewer cells are produced than in vivo but the level of differentiated function is similar. Germ cells, which are difficult to culture in vitro, resume mitosis after a quiescent period, at the same time as in vivo. Similar results have been obtained with mouse fetuses, except that Leydig cells dedifferentiate in vitro if the testis is explanted after 13.5 days post conception. Testicular architecture and intercellular communication are sufficiently preserved for the development of the main fetal and neonatal testicular cell types in vitro with no added factors. Our floating-filter organotypic culture system in synthetic medium therefore allows the morpho-functional development of somatic and germ cells in fetal testis explants taken at all developmental stages in rat and at early stages in mouse. This method is potentially useful for studies of the effects of various factors, and of xenobiotics, in particular.
Assuntos
Testículo/citologia , Animais , Diferenciação Celular , Células Cultivadas , Desenvolvimento Fetal , Células Intersticiais do Testículo/citologia , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Células de Sertoli/citologia , Espermatozoides/citologia , Testosterona/metabolismoRESUMO
Epidemiological, clinical, and experimental studies have suggested that excessive exposure to estrogens during fetal/neonatal life can lead to reproductive disorders and sperm abnormalities in adulthood. However, it is unknown whether endogenous concentrations of estrogens affect the establishment of the male fetal germ cell lineage. We addressed this question by studying the testicular development of mice in which the estrogen receptor (ER) beta or the ERalpha gene was inactivated. The homozygous inactivation of ERbeta (ERbeta-/-) increased the number of gonocytes by 50% in 2- and 6-d-old neonates. The numbers of Sertoli and Leydig cells and the level of testicular testosterone production were unaffected, suggesting that estrogens act directly on the gonocytes. The increase in the number of gonocytes did not occur during fetal life but instead occurred just after birth, when gonocytes resumed mitosis and apoptosis. It seems to result from a decrease in the apoptosis rate evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and cleaved caspase-3 immunohistochemical detection. Last, mice heterozygous for the ERbeta gene inactivation behaved similarly to their ERbeta-/- littermates in terms of the number of gonocytes, apoptosis, and mitosis, suggesting that these cells are highly sensitive to the binding of estrogens to ERbeta. ERalpha inactivation had no effect on the number of neonatal gonocytes and Sertoli cells. In conclusion, this study provides the first demonstration that endogenous estrogens can physiologically inhibit germ cell growth in the male. This finding may have important implications concerning the potential action of environmental estrogens.