In vitro effects of Uranium on human fetal germ cells.

Uranium (U) is found in the environment and its use in industrial or military activities has led to concerns about its potential toxicity. The reprotoxicity of this heavy metal has been established in adult animals; however, no studies have examined its effect on human fetal gonads. Using an organ culture system, we investigated the effects of uranyl acetate on human gonads during the first trimester of gestation (7-12 weeks), which is a critical step in the development of a functional reproductive system. In human fetal ovaries, 0.05 mM U significantly decreased germ cell density by increasing their apoptosis rate. In human fetal testes, 0.1mM U similarly reduced the number of germ cells. The human fetal germ cells are more sensitive to U than mouse germ cells in the same experimental conditions. This is the first evidence that U may impair the development of the human gonads.

Conclusions of the French Food Safety Agency on the toxicity of bisphenol A.

Since more than 10 years, risk assessment of bisphenol A (BPA) is debated at the international level. In 2008, the U.S. National Toxicology Program (NTP) expressed some concern for adverse effects, at current level of exposure to BPA, on developmental toxicity. In this context, the French Food Safety Agency (AFSSA) decided to review the toxicity data on BPA with a special focus on this endpoint at doses below 5mg/kg bw/day (the no observed adverse effect level set by different regulatory bodies). This paper summarizes the conclusions of a collective assessment conducted by an expert Working Group from AFSSA. Studies were classified into 3 groups: (i) finding no toxicity, (ii) reporting results not considered to be of concern and (iii) indicating warning signals. The term "warning signal" means that no formal conclusion can be drawn regarding the establishment of a health based guidance value but the study raises some questions about the toxicity of BPA at low doses. It was concluded that studies are needed to ascertain the significance for human health of these warning signals and to be able to propose new methodologies for assessing the risks associated with low doses of BPA and more generally of endocrine disruptors.

TGFbeta signaling in male germ cells regulates gonocyte quiescence and fertility in mice.

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence. These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.

Cadmium increases human fetal germ cell apoptosis.

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.

Determination of hydroxyl rate constants by a high-throughput fluorimetric assay: towards a unified reactivity scale for antioxidants.

We describe in this article the development of a new method for the determination of rate constants of reaction of the hydroxyl radical, generated by radiolysis of water, with almost any possible molecule. It has been designed to provide a fast and reliable screening of antioxidant banks using microplates. Our particular approach is based on the use of the coumarin molecule as a competitor against the tested molecules: after a fast pulse of low dose irradiation, the fluorescence of 7-hydroxycoumarin produced by the oxidation of coumarin is measured and is inversely proportional to the scavenging ability of the tested antioxidant. We have validated our protocol using 32 molecules whose rate constants with HO had already been evaluated and found a good agreement between our rate constants and the latter ones. The scopes and limitations of our method, as well as those of other rate constant determination methods, are discussed.

Phthalates impair germ cell development in the human fetal testis in vitro without change in testosterone production.

BACKGROUND: 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. Phthalate esters represent a class of environmental endocrine-active chemicals known to disrupt development of the male reproductive tract by decreasing testosterone production in the fetal rat. OBJECTIVES: Using the organ culture system we developed previously, we investigated the effects on the development of human fetal testis of one phthalate--mono-2-ethylhexyl phthalate (MEHP)--an industrial chemical found in many products, which has been incriminated as a disruptor of male reproductive function. METHODS: Human fetal testes were recovered during the first trimester (7-12 weeks) of gestation, a critical period for testicular differentiation, and cultured for 3 days with or without MEHP in basal conditions or stimulated with luteinizing hormone (LH). RESULTS: Whatever the dose, MEHP treatment had no effect on basal or LH-stimulated testosterone produced by the human fetal testis in vitro, although testosterone production can be modulated in our culture system. MEHP (10(-4) M) did not affect proliferation or apoptosis of Sertoli cells, but it reduced the mRNA expression of anti-Müllerian hormone. MEHP (10(-4) M) reduced the number of germ cells by increasing their apoptosis, measured by the detection of caspase-3-positive germ cells, without modification of their proliferation. CONCLUSIONS: This is the first experimental demonstration that phthalates alter the development of the germ cell lineage in humans. However, in contrast to results observed in the rat, phthalates did not affect steroidogenesis.

Sex-specific differences in fetal germ cell apoptosis induced by ionizing radiation.

BACKGROUND: We have previously shown that male human fetal germ cells are highly radiosensitive and that their death depends on p53 activation. Male germ cell apoptosis was initiated with doses as low as 0.1 Gy and was prevented by pifithrin alpha, a p53 inhibitor. In this study, we investigated the radiosensitivity of early female and male fetal proliferating germ cells. METHODS AND RESULTS: Both male and female fetal germ cells displayed a similar number of gamma H2AX foci in response to ionizing radiation (IR). In organ culture of human fetal ovaries, the germ cells underwent apoptosis only when exposed to high doses of IR (1.5 Gy and above). Accumulation of p53 was detected in irradiated male human fetal germ cells but not in female ones. Inhibition of p53 with pifithrin alpha did not affect oogonia apoptosis following irradiation. IR induced apoptosis similarly in mouse fetal ovaries in organ culture and in vivo during oogonial proliferation. Germ cell survival in testes from p53 knockout or p63 knockout mice exposed to IR was better than wild-type, whereas female germ cell survival was unaffected by p53 or p63 knockout. CONCLUSIONS: These findings show that pre-meiotic male and female fetal germ cells behave differently in response to a genotoxic stress--irradiation--with oogonia being less sensitive and undergoing p53-independent apoptosis.

Ontogenesis of testicular function in humans.

The two major functions of the testis, steroidogenesis and gametogenesis, take place during fetal life. These two functions have been extensively studied in rodents and adult humans. However, their onset during fetal life is poorly documented in humans. In the first part of this work we presented both our experimental data and some data of literature concerning the development of the human fetal testis. In the second part of this article, using the organ culture system we previously developed, we have investigated the regulations or perturbations of fetal testis development both in rodent and human models. Our findings provide important insight into the potential role of exposure to environmental pollutants (physical factors, in particular ionizing radiation, cadmium and endocrine disruptors such as phthalates) during fetal testicular development and their potential deleterious effects on male fertility in adulthood. Our results highlight the specificity of the human model compared with rodent models.

Adverse effects of endocrine disruptors on the foetal testis development: focus on the phthalates.

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.

p63 null mutation protects mouse oocytes from radio-induced apoptosis.

Female fertility in mammals is determined by the pool of primordial follicles and low doses of radiation induce a major loss of primordial follicles in the ovary. We investigated the expression of p53 and its homologues, p63 and p73, in the normal and irradiated neonatal ovary. p63 was the only member of the p53 family detected in oocyte nucleus. No p63 transcripts or protein were detected in the early foetal ovary. p63 production began in late pachytene-stage oocytes and peaked in diplotene oocytes in mice and humans. The production of p63 was correlated with meiotic DNA double-strand break repair. Only transactivation (TA) isoforms were present in the ovary, with TAp63 alpha by far the most abundant in terms of mRNA and protein levels. Complete p63 null mutation did not affect normal ovary development. Irradiation rapidly triggered p63 phosphorylation. p63 null mutation prevented the cleavage of caspases-9 and -3 and the follicle loss induced by ionising radiation. Thus, our results evidence that irradiation-induced depletion of the primordial follicle pool results from the activation of p63 in quiescent oocytes.

High radiosensitivity of germ cells in human male fetus.

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.

The role of p63 in germ cell apoptosis in the developing testis.

The fetal and neonatal development of male germ cells (gonocytes) is a poorly understood but crucial process for establishing fertility. In rodents, gonocytes go through two phases of proliferation accompanied by apoptosis and separated by a quiescent period during the end of fetal development. P63 is a member of the P53 gene family that yields six isoforms. We detected only the p63 protein and no p53 and p73 in the nucleus of the gonocytes of mouse testes. We report for the first time the ontogeny of each p63 mRNA isoform during testis development. We observed a strong expression of p63gamma mRNA and protein when gonocytes are in the quiescent period. In vitro treatment with retinoic acid prevented gonocytes from entering the quiescent period and was correlated with a reduced production of p63gamma isoform mRNA. We investigated the function of p63 by studying the testicular phenotype of P63-null mice. P63 invalidation slightly, but significantly increased the number of gonocytes counted during the quiescent period. As P63-null animals die at birth we used an original organ culture that mimicked neonatal in vivo development to study further the testicular development. P63 invalidation resulted in a sharply increased number of gonocytes during the culture period due to a decrease in spontaneous apoptosis with no change in proliferation. P63 invalidation also caused abnormal morphologies in the germ cells that were also found in P63(+/-) adult male mice. Thus, p63 appears as an important regulator of germ cell development.

Use of organ culture to study the human fetal testis development: effect of retinoic acid.

CONTEXT: In human, the chronology of the testicular development has been extensively studied, but the factors implicated in the onset and the regulation of gametogenesis and steroidogenesis remain hardly known. OBJECTIVES: To identify these factors, we developed an organ culture system for human fetal testes recovered during the first trimester (6-12 wk) of gestation. We first aimed at investigating the characteristics of this system by comparing the in vivo and in vitro gametogenesis and steroidogenesis. Second, we used organ culture to investigate the effect on the human testicular functions of retinoic acid (RA), previously described as a regulator of gonadal development in rodents. RESULTS: Organ culture proved to be an efficient tool for studying the early development of the testicular functions. Indeed, this system was able to maintain satisfactory development of the germ cells and Leydig cells in the absence of any added factor. For older fetuses, the number of germ cells decreased in culture and the LH was necessary to maintain the steroidogenic activity. The addition of 10(-6) m RA decreased the total number of germ cells in the fetal testis at all studied stages. This resulted from an increase in apoptosis, which slightly exceeded the increase of proliferation. However, RA had a stimulatory effect on the steroidogenic function for the youngest fetuses over a short period of time by increasing the expression of P450 cholesterol side-chain cleavage, 17 alpha-hydroxylase/C17-20 lyase, and steroidogenic acute regulatory protein. CONCLUSIONS: Thus, RA appears as a potential regulator of both gametogenesis and steroidogenesis in human fetal testis. Our organ culture is an interesting tool for studying the effects of various factors on the development of human fetal testis, in particular the effect of hormone-disrupting chemicals.

Effects of prenatal irradiation with an accelerated heavy-ion beam on postnatal development in rats: I. Neurophysiological alterations.

Effects on postnatal neurophysiological development in offspring were studied after exposure of pregnant Wistar rats to accelerated carbon-ion beams with an LET of about 13 keV/ mum at doses ranging from 0.1 Gy to 2.5 Gy on the 15th day of gestation. The age at which four physiological markers appeared and five reflexes were acquired was examined prior to weaning. Gain in body weight was monitored until the offspring were 3 months old. Male offspring were evaluated as young adults using two behavioral tests. The effects of X rays estimated for the same biological end points were studied for comparison. For most of the end points at early age, no significant alterations were observed in offspring that received prenatal irradiation with 0.1 Gy of either accelerated carbon ions or X rays compared to the offspring of sham-irradiated dams. However, all offspring whose dams received 2.5 Gy died prior to weaning. Offspring from dams irradiated with accelerated carbon ions generally showed higher incidences of prenatal death and preweaning mortality, markedly delayed accomplishment in their physiological markers and reflexes, and gain in body weight compared to those exposed to X rays at doses of 0.5 to 2 Gy. Significantly reduced ratios of main organ weight to body weight at the postnatal ages of 30, 60 and 90 days were also observed within this dose range. The results indicate that irradiation with 0.5 to 2 Gy on day 15 of gestation caused permanent alterations in offspring that were dependent on dose. The alterations include permanent growth retardation, morphological malformations in main organs, including microcephaly, diminished reflex attainment, delayed appearance of physiological markers, and changes in adult behavior. Exposure to 1 to 2 Gy of radiation resulted in growth retardation and behavioral alterations that persisted throughout life. Accelerated carbon ions generally induced more detrimental effects than X rays.

Establishment of the reproductive function and transient fertility of female rats lacking primordial follicle stock after fetal gamma-irradiation.

In mammals, the primordial follicle stock is not renewable, and its size, therefore, limits the reproductive life span of the female. In this study we have investigated the morphological and functional differentiation of dysgenesic ovaries in female rats exposed in utero to 1.5 Gy gamma-irradiation. As a consequence of the severe depletion in oocytes, females evidenced premature ovarian failure from 6 months on. Nevertheless, puberty onset and fertility at the beginning of reproductive life were similar to those of controls. The differentiation and evolution of the entire follicular population were followed during the immature period, using follicle counts, in situ hybridization of follicular maturation markers, and analysis of atresia. Primordial follicles were much more affected by irradiation (1.4-1.9% of controls) than growing follicles (30-45% of controls). As the very low number of primordial follicles remained constant throughout this period, it may be considered that the growing follicle pool plays the role of follicular reserve, permitting the transient normal fertility of irradiated females. Within the neonatal period, primary and secondary follicles, as revealed by proliferating cell nuclear antigen immunostaining, remain quiescent longer in irradiated than in control ovaries. Consequently, the majority of the most mature follicles (i.e. the first follicular wave) characterized by a high expression of aromatase transcripts during the infantile period, are missing in irradiated ovaries. Concomitantly, the 17beta-estradiol plasma peak is absent, and plasma FSH levels are higher than those in control females. In conclusion, these observations emphasize that the female reproductive life span depends not merely on the size of the primordial follicle stock, but also on the entire follicle complement as well as follicular dynamics during the immature period.