Chronic exposure to low concentrations of chlorpyrifos affects normal cyclicity and histology of the uterus in female rats.

Chlorpyrifos (CPF), the most used insecticide in Argentina, can act as an endocrine disruptor at low doses. We previously demonstrated that chronic exposure to CPF induces hormonal imbalance in vivo. The aim of this work was to study the effects of low concentrations of CPF (0.01 and 1 mg/kg/day) on the reproductive system of virgin adult rats. In the ovary, we studied the effects of CPF on steroidogenesis by determining steroid hormone content by RIA and CYP11 and CYP19 enzyme expression by qRT-PCR. The estrous cycle was evaluated by microscopic observation of vaginal smear, as well as by changes in uterine histology. In endometrium, we determined the fractal dimension and expression of PCNA, ERα and PR by IHC. Our results showed that chronic exposure to CPF affects ovarian steroid synthesis, causing alterations in the normal cyclicity of animals. In addition, CPF induced proliferative changes in the uterus, suggesting that it could affect reproduction or act as a risk factor in the development of uterine proliferative pathologies.

Early-Pregnancy Dydrogesterone Supplementation Mimicking Luteal-Phase Support in ART Patients Did Not Provoke Major Reproductive Disorders in Pregnant Mice and Their Progeny.

Progestogens are frequently administered during early pregnancy to patients undergoing assisted reproductive techniques (ART) to overcome progesterone deficits following ART procedures. Orally administered dydrogesterone (DG) shows equal efficacy to other progestogens with a higher level of patient compliance. However, potential harmful effects of DG on critical pregnancy processes and on the health of the progeny are not yet completely ruled out. We treated pregnant mice with DG in the mode, duration, and doses comparable to ART patients. Subsequently, we studied DG effects on embryo implantation, placental and fetal growth, fetal-maternal circulation, fetal survival, and the uterine immune status. After birth of in utero DG-exposed progeny, we assessed their sex ratios, weight gain, and reproductive performance. Early-pregnancy DG administration did not interfere with placental and fetal development, fetal-maternal circulation, or fetal survival, and provoked only minor changes in the uterine immune compartment. DG-exposed offspring grew normally, were fertile, and showed no reproductive abnormalities with the exception of an altered spermiogram in male progeny. Notably, DG shifted the sex ratio in favor of female progeny. Even though our data may be reassuring for the use of DG in ART patients, the detrimental effects on spermatogenesis in mice warrants further investigations and may be a reason for caution for routine DG supplementation in early pregnancy.

Perinatal exposure to Bisphenol A disturbs the early differentiation of male germ cells.

Understanding the effects of Bisphenol A (BPA) on early germ cell differentiation and their consequences in adult life is an area of growing interest in the field of endocrine disruption. Herein, we investigate whether perinatal exposure to BPA affects the differentiation of male germ cells in early life using a transgenic mouse expressing the GFP reporter protein under the Oct4 promoter. In this model, the expression of GFP reflects the expression of the Oct4 gene. This pluripotency gene is required to maintain the spermatogonial stem cells in an undifferentiated stage. Thus, GFP expression was used as a parameter to evaluate the effect of BPA on early germ cell development. Female pregnant transgenic mice were exposed to BPA by oral gavage, from embryonic day 5.5 to postnatal day 7 (PND7). The effects of BPA on male germ cell differentiation were evaluated at PND7, while sperm quality, testicular morphology, and protein expression of androgen receptor and proliferating cell nuclear antigen were studied at PND130. We found that perinatal/lactational exposure to BPA up-regulates the expression of Oct4-driven GFP in testicular cells at PND7. This finding suggests a higher proportion of undifferentiated spermatogonia in BPA-treated animals compared with non-exposed mice. Moreover, in adulthood, the number of spermatozoa per epididymis was reduced in those animals perinatally exposed to BPA. This work shows that developmental exposure to BPA disturbed the normal differentiation of male germ cells early in life, mainly by altering the expression of Oct4 and exerted long-lasting sequelae at the adult stage, affecting sperm count and testis.

Dermal exposure to the UV filter benzophenone-3 during early pregnancy affects fetal growth and sex ratio of the progeny in mice.

The aim of this study was to analyze whether dermal exposure to benzophenone 3 (BP-3) during pregnancy affects critical parameters of pregnancy, and whether this exposure may affect the outcome of a second pregnancy in mice. Pregnant mice were exposed to 50-mg BP-3/kg body weight/day or olive oil (vehicle) from gestation day (gd) 0 to gd6 by dermal exposure. High-frequency ultrasound imaging was used to follow up fetal and placental growth in vivo. Blood flow parameters in uterine and umbilical arteries were analyzed by Doppler measurements. Mice were killed at gd5, gd10, and gd14 on the first pregnancy, and at gd10 and 14 on the second pregnancy. The weight of the first and second progenies was recorded, and sex ratio was analyzed. BP-3 levels were analyzed in serum and amniotic fluid. BP-3 reduced the fetal weight at gd14 and feto-placenta index of first pregnancy, with 16.13% of fetuses under the 5th percentile; arteria uterina parameters showed altered pattern at gd10. BP-3 was detected in serum 4 h after the exposure at gd6, and in amniotic fluid at gd14. Offspring weight of first progeny was lower in BP-3 group. Placenta weights of BP-3 group were decreased in second pregnancy. First and second progenies of mothers exposed to BP-3 showed a higher percentage of females (female sex ratio). Dermal exposure to low dose of BP-3 during early pregnancy resulted in an intrauterine growth restriction (IUGR) phenotype, disturbed sex ratio and alterations in the growth curve of the offspring in mouse model.

Culturing Rat Whole Ovary for UV Filter Benzophenone-3 Treatment.

We describe a detailed protocol to establish a newborn rat whole ovary culture, which enables the study of direct effects (independent of hypothalamic-pituitary-gonadal axis) of endocrine disrupting chemicals (EDCs), such as benzophenone-3 (BP-3). This method is useful to understand changes in follicle formation, primordial to primary transition, and expression of regulatory molecules linked to these processes and also provides an alternative to animal models. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Rat ovarian surgery Basic Protocol 2: Whole organ/ovarian culture Basic Protocol 3: RNA isolation and quantitative real-time PCR Basic Protocol 4: Histological processing and staining.

Exposure to 17α-ethinyl estradiol during early pregnancy affects fetal growth and survival in mice.

17α-ethinyl estradiol (EE2) is a synthetic compound widely used in the generation of contraceptive pills. EE2 is present in the urine of women taking contraceptives and its presence has been confirmed at increasing concentrations contaminating rivers all over the world. Because of this cycle, it can entry the human food chain when watering plants. A negative influence of EE2 on fertility and reproductive capacity of wildlife was already suggested. The short-term impact of exposure to contaminating EE2 on pregnancy outcome has not been addressed. Pregnant mice were exposed to either 0.005 µg (concentrations found in water) or 5 µg EE2/kg (contraceptive dose) body weight/day from gestation day 1-7 by oral gavage. Control mice received a 0.1% ethanol solution. High frequency ultrasound imaging was used to follow-up fetal and placental growth in vivo. Doppler measurements were utilized to analyze blood flow parameters in uterine and umbilical arteries. Mice were sacrificed at gd5, 10, and 14. We show that most fetuses of mothers exposed to the high EE2 dose die intrauterine at gd10, with implantation sizes beginning to be smaller already at gd8. Mothers exposed to the low EE2 dose show an impaired remodeling of the spiral arteries, a higher placental weight and pups that are large for gestational age. The insulin-like growth factor system that regulates fetal and placental growth and development is affected by the EE2 treatment. Our results show that a short-term exposure to EE2 during early pregnancy has severe consequences for fetal growth and survival depending on the dose. Exposition to synthetic estrogens affects placenta growth and angiogenesis. These findings urge to the study of mechanisms dysregulated upon environmental exposition to estrogens.

Bisphenol A exposure during early pregnancy impairs uterine spiral artery remodeling and provokes intrauterine growth restriction in mice.

Endocrine disrupting chemicals are long suspected to impair reproductive health. Bisphenol A (BPA) has estrogenic activity and therefore the capacity of interfering with endocrine pathways. No studies dissected its short-term effects on pregnancy and possible underlying mechanisms. Here, we studied how BPA exposure around implantation affects pregnancy, particularly concentrating on placentation and uterine remodeling. We exposed pregnant female mice to 50 µg/kg BPA/day or 0.1% ethanol by oral gavage from day 1 to 7 of gestation. High frequency ultrasound was employed to document the presence and size of implantations, placentas and fetuses throughout pregnancy. Blood velocity in the arteria uterina was analyzed by Doppler measurements. The progeny of mothers exposed to BPA was growth-restricted compared to the controls; this was evident in vivo as early as at day 12 as analyzed by ultrasound and confirmed by diminished fetal and placenta weights observed after sacrificing the animals at day 14 of gestation. The remodeling of uterine spiral arteries (SAs) was considerably impaired. We show that short-term exposure to a so-called "safe" BPA dose around implantation has severe consequences. The intrauterine growth restriction observed in more than half of the fetuses from BPA-treated mothers may owe to the direct negative effect of BPA on the remodeling of uterine SAs that limits the blood supply to the fetus. Our work reveals unsuspected short-term effects of BPA on pregnancy and urges to more studies dissecting the mechanisms behind the negative actions of BPA during early pregnancy.

Ovarian dysfunctions in adult female rat offspring born to mothers perinatally exposed to low doses of bisphenol A.

The study of oral exposure to the environmental estrogen bisphenol A (BPA) during the perinatal period and its effects on ovarian functionality in adulthood has generated special interest. Thus, our objective was to investigate ovarian folliculogenesis and steroidogenesis in adult female rat offspring born to mothers exposed to low doses of BPA (BPA50: 50µg/kgday; BPA0.5: 0.5µg/kgday) by the oral route during gestation and breastfeeding. Ovaries from both BPA-treated groups showed reduced primordial follicle recruitment and a greater number of corpora lutea, indicating an increased number of ovulated oocytes, coupled with higher levels of mRNA expression of 3ß-hydroxysteroid dehydrogenase and serum progesterone. BPA50-treated animals had lower expression of androgen receptor (AR) at different stages of the growing follicle population. BPA0.5-treated rats evidenced an imbalance of AR expression between primordial/primary follicles, with higher mRNA-follicle-stimulating hormone receptor expression. These results add to the growing evidence that folliculogenesis and steroidogenesis are targets of BPA within the ovary.

Characterization of Oct4-GFP transgenic mice as a model to study the effect of environmental estrogens on the maturation of male germ cells by using flow cytometry.

Oct4 is involved in regulation of pluripotency during normal development and is down-regulated during formation of postnatal reservoir of germ cells. We propose thatOct4/GFP transgenic mouse, which mimics the endogenous expression pattern of Oct4, could be used as a mammalian model to study the effects of environmental estrogens on the development of male germ cells. Oct4/GFP maturation profile was assessed during postnatal days -PND- 3, 5, 7, 10, 14 and 80, using flow cytometry. Then, we exposed pregnant mothers to 17α-ethinylestradiol (EE2) from day post coitum (dpc) 5 to PND7. Percentage of Oct4/GFP-expressing cells and levels of expression of Oct4/GPF were increased in PND7 after EE2 exposure. These observations were confirmed by analysis of GFP and endogenous Oct4 protein in the seminiferous tubules and by a reduction in epididymal sperm count in adult mice. We introduced Oct4/GFP mouse together with flow cytometry as a tool to evaluate changes in male germ cells development.

Neonatal exposure to xenoestrogens impairs the ovarian response to gonadotropin treatment in lambs.

Bisphenol A (BPA) and diethylstilbestrol (DES) are xenoestrogens, which have been associated with altered effects on reproduction. We hypothesized that neonatal xenoestrogen exposure affects the ovarian functionality in lambs. Thus, we evaluated the ovarian response to exogenous ovine FSH (oFSH) administered from postnatal day 30 (PND30) to PND32 in female lambs previously exposed to low doses of DES or BPA (BPA50: 50 µg/kg per day, BPA0.5: 0.5 µg/kg per day) from PND1 to PND14. We determined: i) follicular growth, ii) circulating levels of 17ß-estradiol (E2), iii) steroid receptors (estrogen receptor alpha, estrogen receptor beta, and androgen receptor (AR)) and atresia, and iv) mRNA expression levels of the ovarian bone morphogenetic protein (BMPs) system (BMP6, BMP15, BMPR1B, and GDF9) and FSH receptor (FSHR). Lambs neonatally exposed to DES or BPA showed an impaired ovarian response to oFSH with a lower number of follicles ≥2 mm in diameter together with a lower number of atretic follicles and no increase in E2 serum levels in response to oFSH treatment. In addition, AR induction by oFSH was disrupted in granulosa and theca cells of lambs exposed to DES or BPA. An increase in GDF9 mRNA expression levels was observed in oFSH-primed lambs previously treated with DES or BPA50. In contrast, a decrease in BMPR1B was observed in BPA0.5-postnatally exposed lambs. The modifications in AR, GDF9, and BMPR1B may be associated with the altered ovarian function due to neonatal xenoestrogen exposure in response to an exogenous gonadotropin stimulus. These alterations may be the pathophysiological basis of subfertility syndrome in adulthood.

Neonatal exposure to bisphenol A reduces the pool of primordial follicles in the rat ovary.

We evaluated whether exposure to bisphenol A (BPA) disrupts neonatal follicle development in rats. From postnatal day 1 (PND1) to PND7, pups received corn oil (control), diethylstilbestrol (DES20: 20 µg/kg-d, DES0.2: 0.2 µg/kg-d), or BPA (BPA20: 20mg/kg-d, BPA0.05: 0.05 mg/kg-d). We examined follicular dynamics, multioocyte follicles (MOFs) incidence, proliferation and apoptosis rates, expression of steroid receptors (ERα, ERß, PR, AR) and cyclin-dependent kinase inhibitor 1B (p27) in PND8 ovaries. DES20, DES0.2 and BPA20-ovaries showed fewer primordial follicles and increased growing follicles. DES20-ovaries exhibited increased incidence of MOFs. Oocyte survival, AR, PR and apoptosis were not changed. Primordial and recruited follicles from BPA20-ovaries showed higher p27, whereas ERß and proliferation were both increased in recruited follicles. ERα positive primary follicles increased in BPA 20-ovaries. Results show that BPA reduces the primordial follicle pool by stimulating the neonatal initial recruitment, associated with an increased proliferation rate likely mediated by an estrogenic pathway.