Foetal exposure to the bisphenols BADGE and BPAF impairs meiosis through DNA oxidation in mouse ovaries.

Many endocrine disruptors have been proven to impair the meiotic process which is required for the production of healthy gametes. Bisphenol A is emblematic of such disruptors, as it impairs meiotic prophase I and causes oocyte aneuploidy following in utero exposure. However, the mechanisms underlying these deleterious effects remain poorly understood. Furthermore, the increasing use of BPA alternatives raises concerns for public health. Here, we investigated the effects of foetal exposure to two BPA alternatives, bisphenol A Diglycidyl Ether (BADGE) and bisphenol AF (BPAF), on oogenesis in mice. These compounds delay meiosis initiation, increase the number of MLH1 foci per cell and induce oocyte aneuploidy. We further demonstrate that these defects are accompanied by changes in gene expression in foetal premeiotic germ cells and aberrant mRNA splicing of meiotic genes. We observed an increase in DNA oxidation after exposure to BPA alternatives. Specific induction of oxidative DNA damage during foetal germ cell differentiation causes similar defects during oogenesis, as observed in 8-oxoguanine DNA Glycosylase (OGG1)-deficient mice or after in utero exposure to potassium bromate (KBrO3), an inducer of oxidative DNA damage. The supplementation of BPA alternatives with N-acetylcysteine (NAC) counteracts the effects of bisphenols on meiosis. Together, our results propose oxidative DNA lesion as an event that negatively impacts female meiosis with major consequences on oocyte quality. This could be a common mechanism of action for numerous environmental pro-oxidant pollutants, and its discovery, could lead to reconsider the adverse effect of bisphenol mixtures that are simultaneously present in our environment.

Unexpected Interacting Effects of Physical (Radiation) and Chemical (Bisphenol A) Treatments on Male Reproductive Functions in Mice.

For decades, numerous chemical pollutants have been described to interfere with endogenous hormone metabolism/signaling altering reproductive functions. Among these endocrine disrupting substances, Bisphenol A (BPA), a widely used compound, is known to negatively impact germ and somatic cells in the testis. Physical agents, such as ionizing radiation, were also described to perturb spermatogenesis. Despite the fact that we are constantly exposed to numerous environmental chemical and physical compounds, very few studies explore the impact of combined exposure to chemical and physical pollutants on reproductive health. The aim of this study was to describe the impact of fetal co-exposure to BPA and IR on testicular function in mice. We exposed pregnant mice to 10 µM BPA (corresponding to 0.5 mg/kg/day) in drinking water from 10.5 dpc until birth, and we irradiated mice with 0.2 Gy (γ-ray, RAD) at 12.5 days post-conception. Co-exposure to BPA and γ-ray induces DNA damage in fetal germ cells in an additive manner, leading to a long-lasting decrease in germ cell abundance. We also observed significant alteration of adult steroidogenesis by RAD exposure independently of the BPA exposure. This is illustrated by the downregulation of steroidogenic genes and the decrease of the number of adult Leydig cells. As a consequence, courtship behavior is modified, and male ultrasonic vocalizations associated with courtship decreased. In conclusion, this study provides evidence for the importance of broadening the concept of endocrine disruptors to include physical agents, leading to a reevaluation of risk management and regulatory decisions.

Mouse model of radiation-induced premature ovarian insufficiency reveals compromised oocyte quality: implications for fertility preservation.

RESEARCH QUESTION: What is the impact of radiation exposure on oocyte quality and female fertility? DESIGN: Prepubertal mice underwent whole-body irradiation with a single dose (0.02, 0.1, 0.5, 2, 8 Gy) of gamma- or X-rays. Oocytes were quantified in irradiated (n = 36) and sham-treated (n = 8) mice. After a single exposure to 2 Gy, formation of DNA double-strand breaks (n = 10), activation of checkpoint kinase (Chk2) (n = 10) and dynamics of follicular growth (n = 18) were analysed. Fertility assessment was performed in adult irradiated mice and controls from the number of pups per mouse (n = 28) and the fetal abortion rate (n = 24). Ploidy of mature oocytes (n = 20) was analysed after CREST immunostaining, and uterine sections were examined. RESULTS: Radiation exposure induced a massive loss of primordial follicles with LD50 below 50 mGy for both gamma and X-rays. Growing follicles survived doses up to 8 Gy. This difference in radiosensitivity was not due to a different amount of radio-induced DNA damage, and Chk2 was activated in all oocytes. Exposure to a 2 Gy dose abolished the long-term fertility of females due to depletion of the ovarian reserve. Detailed analysis indicates that surviving oocytes were able to complete folliculogenesis and could be fertilized. This transient fertility allowed irradiated females to produce a single litter albeit with a high rate of fetal abortion (23%, P = 0.0096), related to altered ploidy in the surviving oocytes (25.5%, P = 0.0035). CONCLUSIONS: The effects of radiation on surviving oocyte quality question natural conception as a first-line approach in cancer survivors. Together, the data emphasize the need for fertility preservation before radiation exposure and call for reassessment of the use of cryopreserved oocytes.

Two populations of self-maintaining monocyte-independent macrophages exist in adult epididymis and testis.

Macrophages are the principal immune cells of the epididymis and testis, but their origins, heterogeneity, development, and maintenance are not well understood. Here, we describe distinct populations of epididymal and testicular macrophages that display an organ-specific cellular identity. Combining in vivo fate-mapping, chimeric and parabiotic mouse models with in-depth cellular analyses, we found that CD64hiMHCIIlo and CD64loMHCIIhi macrophage populations of epididymis and testis arise sequentially from yolk sac erythro-myeloid progenitors, embryonic hematopoiesis, and nascent neonatal monocytes. While monocytes were the major developmental source of both epididymal and testicular macrophages, both populations self-maintain in the steady-state independent of bone marrow hematopoietic precursors. However, after radiation-induced macrophage ablation or during infection, bone marrow-derived circulating monocytes are recruited to the epididymis and testis, giving rise to inflammatory macrophages that promote tissue damage. These results define the layered ontogeny, maintenance and inflammatory response of macrophage populations in the male reproductive organs.

Nuclear receptors and endocrine disruptors in fetal and neonatal testes: a gapped landscape.

During the last decades, many studies reported that male reproductive disorders are increasing among humans. It is currently acknowledged that these abnormalities can result from fetal exposure to environmental chemicals that are progressively becoming more concentrated and widespread in our environment. Among the chemicals present in the environment (air, water, food, and many consumer products), several can act as endocrine disrupting compounds (EDCs), thus interfering with the endocrine system. Phthalates, bisphenol A (BPA), and diethylstilbestrol (DES) have been largely incriminated, particularly during the fetal and neonatal period, due to their estrogenic and/or anti-androgenic properties. Indeed, many epidemiological and experimental studies have highlighted their deleterious impact on fetal and neonatal testis development. As EDCs can affect many different genomic and non-genomic pathways, the mechanisms underlying the adverse effects of EDC exposure are difficult to elucidate. Using literature data and results from our laboratory, in the present review, we discuss the role of classical nuclear receptors (genomic pathway) in the fetal and neonatal testis response to EDC exposure, particularly to phthalates, BPA, and DES. Among the nuclear receptors, we focused on some of the most likely candidates, such as peroxisome-proliferator activated receptor (PPAR), androgen receptor (AR), estrogen receptors (ERα and ß), liver X receptors (LXR), and small heterodimer partner (SHP). First, we describe the expression and potential functions (based on data from studies using receptor agonists and mouse knockout models) of these nuclear receptors in the developing testis. Then, for each EDC studied, we summarize the main evidences indicating that the reprotoxic effect of each EDC under study is mediated through a specific nuclear receptor(s). We also point-out the involvement of other receptors and nuclear receptor-independent pathways.

Differential effects of bisphenol A and diethylstilbestrol on human, rat and mouse fetal leydig cell function.

Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10(-12) to 10(-5) M BPA concentrations on fetal Leydig cell function, as fetal life is a critical period of sensitivity to ED effects on male reproductive function. To this aim, fetal testes from human at 6.5-10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1-3 days. BPA concentrations as low as 10(-8) M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10(-5) M BPA were required. Similarly, 10(-8) M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10(-5) and 10(-6) M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10(-8) M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.

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.