Chemical mixture that targets the epidermal growth factor pathway impairs human trophoblast cell functions.

Pregnant women are exposed to complex chemical mixtures, many of which reach the placenta. Some of these chemicals interfere with epidermal growth factor receptor (EGFR) activation, a receptor tyrosine kinase that modulates several placenta cell functions. We hypothesized that a mixture of chemicals (Chem-Mix) known to reduce EGFR activation (polychlorinated biphenyl (PCB)-126, PCB-153, atrazine, trans-nonachlor, niclosamide, and bisphenol S) would interfere with EGFR-mediated trophoblast cell functions. To test this, we determined the chemicals' EGFR binding ability, EGFR and downstream effectors activation, and trophoblast functions (proliferation, invasion, and endovascular differentiation) known to be regulated by EGFR in extravillous trophoblasts (EVTs). The Chem-Mix competed with EGF for EGFR binding, however only PCB-153, niclosamide, trans-nonachlor, and BPS competed for binding as single chemicals. The effects of the Chem-Mix on EGFR phosphorylation were tested by exposing the placental EVT cell line, HTR-8/SVneo to control (0.1% DMSO), Chem-Mix (1, 10, or 100 ng/ml), EGF (30 ng/ml), or Chem-Mix + EGF. The Chem-Mix - but not the individual chemicals - reduced EGF-mediated EGFR phosphorylation in a dose dependent manner, while no effect was observed in its downstream effectors (AKT and STAT3). None of the individual chemicals affected EVT cell invasion, but the Chem-Mix reduced EVT cell invasion independent of EGF. In support of previous studies that have explored chemicals targeting a specific pathway (estrogen/androgen receptor), current findings indicate that exposure to a chemical mixture that targets the EGFR pathway can result in a greater impact compared to individual chemicals in the context of placental cell functions.

Organotin mixtures reveal interactions that modulate adipogenic differentiation in 3T3-L1 preadipocytes.

Organotin chemicals (butyltins and phenyltins) are the most widely used organometallic chemicals worldwide and are used in industrial applications, such as biocides and anti-fouling paints. Tributyltin (TBT) and more recently, dibutyltin (DBT) and triphenyltin (TPT) have been reported to stimulate adipogenic differentiation. Although these chemicals co-exist in the environment, their effect in combination remains unknown. We first investigated the adipogenic effect of eight organotin chemicals (monobutyltin (MBT), DBT, TBT, tetrabutyltin (TeBT), monophenyltin (MPT), diphenyltin (DPT), TPT, and tin chloride (SnCl4)) in the 3T3-L1 preadipocyte cell line in single exposures at two doses (10 and 50 ng/ml). Only three out of the eight organotins induced adipogenic differentiation with TBT eliciting the strongest adipogenic differentiation (in a dose-dependent manner) followed by TPT and DBT, as demonstrated by lipid accumulation and gene expression. We then hypothesized that, in combination (TBT, DBT, and TPT), adipogenic effects will be exacerbated compared to single exposures. However, at the higher dose (50 ng/ml), TBT-induced differentiation was reduced by TPT and DBT when in dual or triple combination. We tested whether TPT or DBT would interfere with adipogenic differentiation stimulated by a peroxisome proliferator-activated receptor (PPARγ) agonist (rosiglitazone) or a glucocorticoid receptor agonist (dexamethasone). Both DBT50 and TPT50 reduced rosiglitazone-, but not dexamethasone-stimulated adipogenic differentiation. In conclusion, DBT and TPT interfere with TBT's adipogenic differentiation possibly via PPARγ signaling. These findings highlight the antagonistic effects among organotins and the need to understand the effects and mechanism of action of complex organotin mixtures on adipogenic outcomes.

The organotin triphenyltin disrupts cholesterol signaling in mammalian ovarian steroidogenic cells through a combination of LXR and RXR modulation.

Organotins, a chemical family with over 30 congeners to which humans are directly exposed to through food consumption, are a chemical class widely used as stabilizers in polyvinyl chloride, and biocides in antifouling products. Aside from tributyltin (TBT), toxicological information on other organotin congeners, such as triphenyltin (TPT), remains scarce. Our previous work has demonstrated that TBT can interfere with cholesterol trafficking in steroidogenic cells. Given their structural similarities, we hypothesized that TPT, similar to TBT, disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. To test this, human and ovine primary ovarian theca cells were isolated, purified and exposed to TPT at environmentally relevant doses (1 or 10 ng/ml) in pre-luteinized (48 h exposure) or luteinizing cells (72 h exposure). Intracellular cholesterol levels, progesterone, and testosterone secretion and gene expression of nuclear receptors, cholesterol transporters, and steroidogenic enzymes were evaluated. In ovine cells, TPT upregulated StAR, ABCA1, and SREBF1 mRNA and ABCA1 protein in both pre-luteinized and luteinized stages. TPT did not alter intracellular cholesterol or testosterone synthesis, but upregulated progesterone production. Inhibitor and shRNA knockdown approaches were then used to evaluate the role of retinoid X receptor (RXR) and liver X receptor (LXR) on TPT's effects. TPT upregulated ABCA1 and StAR expression was blocked by both LXR and RXR antagonists. TPT's effect on ABCA1 expression was reduced in LXRß and RXRß knockdown theca cells. Similar findings were obtained with primary human theca cells. No synergistic effect of TBT and TPT was observed. In conclusion, at an environmentally relevant dose, TPT upregulates theca cell cholesterol transporter ABCA1 expression via RXR and LXR pathways. Similar effects of TPT on human and sheep theca cells supports its conserved mechanism across mammalian theca cells.

Bisphenol S Impairs Invasion and Proliferation of Extravillous Trophoblasts Cells by Interfering with Epidermal Growth Factor Receptor Signaling.

The placenta supports fetal growth and is vulnerable to exogenous chemical exposures. We have previously demonstrated that exposure to the emerging chemical bisphenol S (BPS) can alter placental endocrine function. Mechanistically, we have demonstrated that BPS interferes with epidermal growth factor receptor (EGFR) signaling, reducing placenta cell fusion. Extravillous trophoblasts (EVTs), a placenta cell type that aids with vascular remodeling, require EGF to invade into the maternal endometrium. We hypothesized that BPS would impair EGF-mediated invasion and proliferation in EVTs. Using human EVTs (HTR-8/SVneo cells), we tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional endpoints of EGFR signaling, including EGF endocytosis, cell invasion and proliferation, and endovascular differentiation. We demonstrated that BPS blocked EGF-induced phosphorylation of EGFR by acting as a competitive antagonist to EGFR. Transwell assay and a three-dimensional microfluidic chip invasion assay revealed that BPS exposure can block EGF-mediated cell invasion. BPS also blocked EGF-mediated proliferation and endovascular differentiation. In conclusion, BPS can prevent EGF-mediated EVT proliferation and invasion through EGFR antagonism. Given the role of EGFR in trophoblast proliferation and differentiation during placental development, our findings suggest that maternal exposure to BPS may contribute to placental dysfunction via EGFR-mediated mechanisms.

A modified parachute assay for assessment of gap junction intercellular communication in placental trophoblast cells.

Gap junction intercellular communication (GJIC) is a necessary process for placental development. GJIC can be assessed with a parachute assay, where fluorescent dye-loaded donor cells are 'parachuted' onto acceptor cells and dye diffuses to adjacent cells with active GJIC. During co-culture, donor cells can attach, but the assay does not allow their distinction from acceptor cells, which presents as a major limitation. We have developed a modified parachute assay that permits distinction between donor and acceptor cells, using the extravillous trophoblast cell line HTR-8/SVneo and a lentiviral transduction technique. Using PKA activator CW008 as a positive control and 12-o-tetradecanoylphorbol-13-acetate as a negative control, this modified parachute assay reliably detects both enhanced and attenuated GJIC. Importantly, the ease and accuracy of quantification over currently available methods makes this modified assay optimal for automation and represents a useful tool for in vitro placental toxicological testing.

Automated lipid droplet quantification system for phenotypic analysis of adipocytes using CellProfiler.

Adipogenic differentiation is the process by which preadipocytes become mature adipocytes, cells that store energy and regulate metabolic homeostasis. During differentiation, neutral lipids that accumulate in adipocytes can be detected using stains and used as an index of cell differentiation. However, imaging tools for evaluating intracellular lipid droplets remain at their infancy. Nutrition, stress, or chemical exposure can dysregulate adipogenic differentiation and lipid metabolism. Therefore, the aims of this study were to develop an accurate, standardized approach to quantify lipid droplet size of mature adipocytes and a clustering approach to analyze the total lipid content per adipocyte. For the lipid droplet analysis, we used two approaches, the free online computer software of reference, ImageJ, and another free online computer software, CellProfiler. For ImageJ, we used an already developed macro designed to identify particles and quantify their area, and for CellProfiler, we developed a new analysis pipeline. Our results show that CellProfiler is able to accurately identify a greater number of lipid droplets compared to ImageJ. A clustering analysis is also possible using CellProfiler which allows for the quantification of total lipid content per individual adipocyte to provide insight into single-cell responsiveness to adipogenic stimuli. CellProfiler streamlines the lipid droplet phenotypic analysis of adipocytes compared to more traditional analysis methods. In conclusion, this novel image analysis tool can provide a more precise evaluation of lipid droplet and adipogenesis dysregulation, a critical need in the understanding of metabolic disorders.

Undernutrition and hyperandrogenism during pregnancy: Role in programming of cardiovascular disease and infertility.

Maternal nutritional status programs the development of several systems in female offspring, with effects that depend on the severity, duration, and window of development when the nutritional perturbation is imposed. On the basis of the developmental origins of health and disease concept, we hypothesize that gestational low caloric intake may induce maternal subclinical hyperandrogenism during early pregnancy and compromise cardiovascular health and fertility in the female offspring. To examine this possibility, a literature search for human and animal studies was conducted using two electronic databases, PubMed and Cochrane until April 2019 to address the following questions: (a) Do androgens have a developmental role in cardiovascular and ovarian development? (b) Is excess maternal testosterone linked to cardiovascular disease and infertility? and (c) Could early pregnancy undernutrition enhance maternal androgen production and compromise health and fertility in female offspring? The observations reviewed, establish a potential causative link between maternal undernutrition and subclinical hyperandrogenism with hypertension and reduced ovarian reserve in the progeny. Further studies in appropriate models are needed to better understand whether low energy intake and subclinical maternal hyperandrogenism during early pregnancy can negatively affect the health of the female offspring.

Multispecies study: low-dose tributyltin impairs ovarian theca cell cholesterol homeostasis through the RXR pathway in five mammalian species including humans.

Tributyltin (TBT), an organotin chemical used as a catalyst and biocide, can stimulate cholesterol efflux in non-steroidogenic cells. Since cholesterol is the first limiting step for sex hormone production, we hypothesized that TBT disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. We investigated TBT's effect on cholesterol trafficking, luteinization, and steroidogenesis in theca cells of five species (human, sheep, cow, pig, and mice). Primary theca cells were exposed to an environmentally relevant dose of TBT (1 or 10 ng/ml) and/or retinoid X receptor (RXR) antagonist. The expression of RXRα in sheep theca cells was knocked down using shRNA. Steroidogenic enzymes, cholesterol transport factors, and nuclear receptors were measured by RT-qPCR and Western blotting, and intracellular cholesterol, progesterone, and testosterone secretion by ELISA. TBT upregulated StAR and ABCA1 in ovine cells, and SREBF1 mRNA in theca cells. TBT also reduced intracellular cholesterol and upregulated ABCA1 protein expression but did not alter testosterone or progesterone production. RXR antagonist and RXRα knockdown demonstrates that TBT's effect is partially through RXR. TBT's effect on ABCA1 and StAR expression was recapitulated in all five species. TBT, at an environmentally relevant dose, stimulates theca cell cholesterol extracellular efflux via the RXR pathway, triggers a compensatory upregulation of StAR that regulates cholesterol transfer into the mitochondria and SREBF1 for de novo cholesterol synthesis. Similar results were obtained in all five species evaluated (human, sheep, cow, pig, and mice) and are supportive of TBT's conserved mechanism of action across mammalian species.

Developmental programming: Sex-specific programming of growth upon prenatal bisphenol A exposure.

In both human and animals, in utero exposure to bisphenol A (BPA), an endocrine-disrupting chemical used in the production of plastics and epoxy resins, has been shown to affect offspring reproductive and metabolic health during adult life. We hypothesized that the effect of prenatal exposure to environmentally relevant doses of BPA will be evident during fetal organogenesis and fetal/postnatal growth trajectory. Pregnant ewes were administered BPA subcutaneously from 30 to 90 days of gestation (term 147 days). Fetal organ weight, anthropometric measures, maternal/fetal hormones and postnatal growth trajectory were measured in both sexes. Gestational BPA administration resulted in higher accumulation in male than female fetuses only at fetal day 65, with minimal impact on fetal/maternal steroid milieu in both sexes at both time points. BPA-treated male fetuses were heavier than BPA-treated female fetuses at fetal day 90 whereas this sex difference was not evident in the control group. At the organ level, liver weight was reduced in prenatal BPA-treated female fetuses, while heart and thyroid gland weights were increased in BPA-treated male fetuses relative to their sex-matched control groups. Prenatal BPA treatment also altered the postnatal growth trajectory in a sex-specific manner. Males grew slower during the early postnatal period and caught up later. Females, in contrast, demonstrated the opposite growth trend. Prenatal BPA-induced changes in fetal organ differentiation and early life growth strongly implicate translational relevance of in utero contributions to reproductive and metabolic defects previously reported in adult female offspring.

Gestational bisphenol S impairs placental endocrine function and the fusogenic trophoblast signaling pathway.

Exposure to bisphenolic chemicals during pregnancy occurs in > 90% of pregnancies. Bisphenolic compounds can cross the placental barrier reaching fetal circulation. However, the effects of emerging bisphenolic compounds, such as bisphenol S (BPS), on placental function remain untested. The aim was to determine if bisphenol A (BPA) or BPS, at an environmentally relevant dose, impairs placental function. Pregnant sheep were randomly distributed into three treatment groups (n = 7-8/group): control, BPA, and BPS. All animals received daily injections of corn oil (control), BPA, or BPS (0.5 mg/kg; s.c.; internal fetal doses were ~ 2.6 ng/mL unconjugated BPA and ~ 7.7 ng/mL of BPS) from gestational day 30-100. After a 20-day washout period, placentas were weighed and placentomes collected. Placental endocrine function was assessed on biweekly maternal blood samples. Gestational exposure to BPS, but not BPA, reduced maternal circulating pregnancy-associated glycoproteins without change in placental weight or placental stereology. BPS-exposed placentas had 50% lower e-cadherin protein expression, ~ 20% fewer binucleate cells, and ~ threefold higher glial cell missing-1 protein expression. BPA placentas were not affected highlighting the intrinsic differences among bisphenolic chemicals. This is the first study to demonstrate that gestational BPS can result in placental endocrine dysfunction and points to a dysregulation in the fusogenic trophoblast signaling pathway.

PPARγ agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes.

BACKGROUND: Although the 3T3-L1 preadipocyte cell line represents an informative model for in vitro adipogenesis research, primary cultured cells are often needed to understand particular human or animal metabolic phenotypes. As demonstrated by in vitro cultured preadipocytes from large mammalian species, primary cultured cells require specific adipogenic differentiation conditions different to that of the 3T3-L1 cell line. These conditions are also species-specific and require optimization steps. However, efficient protocols to differentiate primary preadipocytes using alternative species to rodents are scarce. Sheep represent an amenable animal model for fetal biology and developmental origins of health and disease studies. In this work, we present with the first detailed procedure to efficiently differentiate primary fetal and adult ovine preadipocytes. METHODS: Fetal and adult ovine adipose and skin tissue harvest, preadipocyte and fibroblast isolation, proliferation, and standardization and optimization of a new adipogenic differentiation protocol. Use of commercial cell lines (3T3-L1 and NIH-3T3) for validation purposes. Oil red O stain and gene expression were used to validate adipogenic differentiation. ANOVA and Fisher's exact test were used to determine statistical significance. RESULTS: Our optimized adipogenic differentiation method included a prolonged adipogenic cocktail exposure time from 2 to 8 days, higher insulin concentration, and supplementation with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone. This protocol was optimized for both, fetal and adult preadipocytes. CONCLUSIONS: Our protocol enables successful adipogenic differentiation of fetal and adult ovine preadipocytes. This work demonstrates that compared to the 3T3-L1 cell line, fetal ovine preadipocytes require a longer exposure to the differentiation cocktail, and the need for IMBX, dexamethasone, and/or the PPARγ agonist rosiglitazone through the terminal differentiation phase. They also require higher insulin concentration during differentiation to enhance lipid accumulation and similar to human primary preadipocytes, PPARγ agonist supplementation is also required for ovine adipogenic differentiation. This work highlights species-specific differences requirements for adipogenic differentiation and the need to develop standardized methods to investigate comparative adipocyte biology.

Developmental programming: interaction between prenatal BPA exposure and postnatal adiposity on metabolic variables in female sheep.

Among potential contributors for the increased incidence of metabolic diseases is the developmental exposure to endocrine-disrupting chemicals such as bisphenol A (BPA). BPA is an estrogenic chemical used in a variety of consumer products. Evidence points to interactions of BPA with the prevailing environment. The aim of this study was to assess the effects of prenatal exposure to BPA on postnatal metabolic outcomes, including insulin resistance, adipose tissue distribution, adipocyte morphometry, and expression of inflammatory markers in adipose tissue as well as to assess whether postnatal overfeeding would exacerbate these effects. Findings indicate that prenatal BPA exposure leads to insulin resistance in adulthood in the first breeder cohort (study 1), but not in the second cohort (study 2), which is suggestive of potential differences in genetic susceptibility. BPA exposure induced adipocyte hypertrophy in the visceral fat depot without an accompanying increase in visceral fat mass or increased CD68, a marker of macrophage infiltration, in the subcutaneous fat depot. Cohens effect size analysis found the ratio of visceral to subcutaneous fat depot in the prenatal BPA-treated overfed group to be higher compared with the control-overfed group. Altogether, these results suggest that exposure to BPA during fetal life at levels found in humans can program metabolic outcomes that lead to insulin resistance, a forerunner of type 2 diabetes, with postnatal obesity failing to manifest any interaction with prenatal BPA relative to insulin resistance and adipocyte hypertrophy.

Developmental programming: postnatal estradiol modulation of prenatally organized reproductive neuroendocrine function in sheep.

Gestational testosterone (TS) excess, acting via both the androgenic and estrogenic pathways, advances puberty and disrupts the neuroendocrine estradiol (E2) feedback and periovulatory hormonal dynamics in female sheep. These prenatally programmed defects may be subject to postnatal modifications by continued organizational and/or activational effects of steroids. This study investigated (1) the organizational contribution of prenatal estrogen excess and (2) the impact of postnatal exposure to E2 in modulating the effects of prenatal androgen excess (TS and dihydrotestosterone (DHT)) on puberty, neuroendocrine feedback mechanisms, and periovulatory hormonal dynamics in sheep. Pregnant Suffolk sheep were treated with TS, DHT, E2, or E2 plus DHT (ED) from days 30 to 90 of gestation. A subset of the control (C), TS, and DHT female offspring received a constant-release E2 implant postnatally. Findings revealed that (1) prenatal E2-treatment failed to reproduce the neuroendocrine disruptions predicted to be programmed by the estrogenic pathway and (2) prenatal E2D-treatment did not adequately replicate the reproductive neuroendocrine defects induced by prenatal TS excess. More importantly, continuous postnatal E2-treatment, while delaying the onset of puberty and reducing the inhibitory effects of E2 on tonic luteinizing hormone (LH) release, failed to amplify the E2-positive feedback and periovulatory defects induced by prenatal TS-treatment. Our results indicate that disruptions in E2-positive feedback mechanisms and periovulatory gonadotropin secretion induced by prenatal TS-treatment are programmed predominantly during the prenatal life with postnatal exposure to E2 excess not contributing further to these disruptions.

Developmental Programming: Does Prenatal Steroid Excess Disrupt the Ovarian VEGF System in Sheep?

Prenatal testosterone (T), but not dihydrotestosterone (DHT), excess disrupts ovarian cyclicity and increases follicular recruitment and persistence. We hypothesized that the disruption in the vascular endothelial growth factor (VEGF) system contributes to the enhancement of follicular recruitment and persistence in prenatal T-treated sheep. The impact of T/DHT treatments from Days 30 to 90 of gestation on VEGFA, VEGFB, and their receptor (VEGFR-1 [FLT1], VEGFR-2 [KDR], and VEGFR-3 [FLT4]) protein expression was examined by immunohistochemistry on Fetal Days 90 and 140, 22 wk, 10 mo (postpubertal), and 21 mo (adult) of age. Arterial morphometry was performed in Fetal Day 140 and postpubertal ovaries. VEGFA and VEGFB expression were found in granulosa cells at all stages of follicular development with increased expression in antral follicles. VEGFA was present in theca interna, while VEGFB was present in theca interna/externa and stromal cells. All three receptors were expressed in the granulosa, theca, and stromal cells during all stages of follicular development. VEGFR-3 increased with follicular differentiation with the highest level seen in the granulosa cells of antral follicles. None of the members of the VEGF family or their receptor expression were altered by age or prenatal T/DHT treatments. At Fetal Day 140, area, wall thickness, and wall area of arteries from the ovarian hilum were larger in prenatal T- and DHT-treated females, suggestive of early androgenic programming of arterial differentiation. This may facilitate increased delivery of endocrine factors and thus indirectly contribute to the development of the multifollicular phenotype.

A Three-Dimensional Trophoblast Invasion Microfluidic Platform for Toxicological Screening.

To improve our understanding of human placental function and placental cell responses to pregnancy stressors, the development of in vitro models that better recapitulate the in vivo placental microenvironment is needed. Here, we describe a three-dimensional (3D) silicone polymer polydimethylsiloxane (PDMS) microfluidic platform for modeling human trophoblast invasion recreating a placental heterocellular microenvironment. This platform allows the formation of a cellular barrier establishing a chemical gradient and real-time evaluation of trophoblast cell invasion and heterocellular cell-to-cell interactions.

Evaluation of Lipids and Lipid-Related Transcripts in Human and Ovine Theca Cells and an in Vitro Mouse Model Exposed to the Obesogen Chemical Tributyltin.

BACKGROUND: Exposure to obesogenic chemicals has been reported to result in enhanced adipogenesis, higher adipose tissue accumulation, and reduced ovarian hormonal synthesis and follicular function. We have reported that organotins [tributyltin (TBT) and triphenyltin (TPT)] dysregulate cholesterol trafficking in ovarian theca cells, but, whether organotins also exert lipogenic effects on ovarian cells remains unexplored. OBJECTIVE: We investigated if environmentally relevant exposures to organotins [TBT, TPT, or dibutyltin (DBT)] induce lipid dysregulation in ovarian theca cells and the role of the liver X receptor (LXR) in this effect. We also tested the effect of TBT on oocyte maturation and neutral lipid accumulation, and lipid-related transcript expression in cumulus cells and preimplantation embryos. METHODS: Primary theca cell cultures derived from human and ovine ovaries were exposed to TBT, TPT, or DBT (1, 10, or 50 ng/ml). The effect of these chemical exposures on neutral lipid accumulation, lipid abundance and composition, lipid homeostasis-related gene expression, and cytokine secretion was evaluated using liquid chromatography-mass spectrometry (LC-MS), inhibitor-based methods, cytokine secretion, and lipid ontology analyses. We also exposed murine cumulus-oocyte complexes to TBT and evaluated oocyte maturation, embryo development, and lipid homeostasis-related mRNA expression in cumulus cells and blastocysts. RESULTS: Exposure to TBT resulted in higher intracellular neutral lipids in human and ovine primary theca cells. In ovine theca cells, this effect was dose-dependent, independent of cell stage, and partially mediated by LXR. DBT and TPT resulted in higher intracellular neutral lipids but to a lesser extent in comparison with TBT. More than 140 lipids and 9 cytokines were dysregulated in TBT-exposed human theca cells. Expression of genes involved in lipogenesis and fatty acid synthesis were higher in theca cells, as well as in cumulus cells and blastocysts exposed to TBT. However, TBT did not impact the rates of oocyte maturation or blastocyst development. DISCUSSION: TBT induced dyslipidemia in primary human and ovine theca cells, which may be responsible for some of the TBT-induced fertility dysregulations reported in rodent models of TBT exposure. https://doi.org/10.1289/EHP13955.

Birth weight, growth indices, and seminal parameters in male offspring are resilient features to maternal pre-conceptional dietary manipulation in sheep.

Gestational diet manipulation can lead to inadequate fetal nutrient supply resulting in low birth weight, limited postnatal growth, and consequently, reduced reproductive performance in the progeny. However, effects of short-term maternal pre-conceptional dietary manipulation on postnatal growth and reproductive parameters of male offspring in large animals remains unexplored. To determine these consequences, female crossbred (Polypay x Dorset) sheep were allocated to three groups (n = 33/group) of dietary manipulation for 21 days prior to mating under the following conditions: (1) control at 100 % of maintenance energy requirements (40 Kcal of metabolizable energy/kg body weight [BW]), (2) undernutrition (UN) at 50 % of Control intake, and (3) overnutrition (ON) at 200 % of maintenance energy. Singleton ram lambs (UN:9; C:12; ON:6) were monitored from birth until 8 months of age, including birth weight, weekly weights, weight gain, body mass index (BMI), and circulating testosterone. After weaning, monthly scrotal circumference and subcutaneous fat depth were measured. Semen morphology and motility were evaluated at 7 and 8 months of age. Birth weight, weight gain, and BMI at birth and weaning were not significantly different among nutritional treatments. None of the pre-conceptional diets affected body weight change from weaning until 36 weeks of age, BMI, fat depth, or scrotal circumference across the experiment. A sustained rise in plasma testosterone concentrations was detected when ram lambs were, on average, 82 days old and 37 kg. Both testosterone concentrations and scrotal circumference were positively correlated to body weight regardless of treatment group. In addition, seminal parameters did not differ among treatments, but a transient increase in plasma testosterone at 18 weeks of age was observed in ON ram lambs compared to control rams. In conclusion, birth weight, growth indices, and seminal parameters in singleton rams are resilient features in the progeny upon maternal pre-conceptional dietary manipulation in sheep.

Utilising male stimulus to improve the reproductive efficiency of 8-month-old nulliparous ewes and adult parous ewes.

We tested whether utilising the male effect to stimulate ewes before the mating period can reduce the time to conception following the introduction of entire rams, and increase fertility, prolificacy, and reproductive rate (number of fetuses per 100 ewes exposed to fertile rams). A retrospective analysis was used to analyse records from 59,716 ewes collected over 34 years (1986-2020) from seven genotypes: Border Leicester, Composite (crossbred), Dorset, Merino, Dorset x Polypay, Rambouillet, White Suffolk. The dataset also included nulliparous young ewes (mated at age 8 months) and adult parous ewes. Vasectomized rams were used to stimulate 20,632 ewes before a mating period that lasted 2 or 3 estrous cycles, and the outcomes were compared with those from 39,084 ewes that had not been stimulated. Independently of genotype, utilising the male stimulus advanced the average conception date by 8 days for young ewes (P < 0.0001) and by 1 day for adult ewes (P < 0.0001). The male stimulus also increased the proportion of ewes that conceived in their first cycle by 33 % for young ewes and by 6 % for adult ewes (P < 0.0001). For the cycle of conception, there were significant (P < 0.0001) effects of two interactions: male stimulus x age at mating and male stimulus x live weight at mating. The male stimulus improved fertility in both adult ewes (99.8 % vs 89 %; P < 0.001) and young ewes (77.7 % vs 81.3 %; P < 0.001). The male stimulus increased the number of young ewes (41.9 % vs 11.1 %; P < 0.001) and adult ewes (16.6 % vs 2.7 %; P < 0.001) that conceived multiple fetuses in the first 17 days of the mating period. The reproductive rate was improved by the male stimulus in young ewes (129 % vs 135 %; P < 0.001) but not in adult ewes (120 % vs 122 %; P = 0.12). When all animals for all breeds were included in the analyses, there were improvements in fertility, prolificacy, and reproductive rate as age and live weight increased at mating (P < 0.0001). We conclude that, independently of genotype, utilising the male stimulus before the mating period reduces the time to conception and improves reproductive performance in both young and adult ewes.

Binding sites in the epidermal growth factor receptor are responsible for bisphenol S effects on trophoblast cell invasion.

Bisphenol S (BPS) is an endocrine disrupting chemical and the second most abundant bisphenol detected in humans. We have recently demonstrated that in utero exposure to BPS reduces human placenta cell fusion by interfering with epidermal growth factor (EGF)-dependent EGF receptor (EGFR) activation. Our previous work suggests that this occurs via binding of BPS to the extracellular domain of EGFR. However, whether BPS directly binds to EGFR has not been confirmed. We evaluated the binding ability of BPA, BPF and BPS to EGFR to determine whether EGFR binding is a unique attribute of BPS. To test these hypotheses, we first exposed HTR-8/SVneo cells to BPS, BPA, or BPF, with or without EGF. When co-exposed to EGF, BPS, but not BPA nor BPF, reduced EGFR phosphorylation by ∼60%, demonstrating that only BPS can interfere with EGF-dependent EGFR activation. As this indicates that BPS binding to the extracellular domain is responsible for its effect, we performed a computational search for putative binding sites on the EGFR extracellular domain, and performed ligand docking of BPS, BPA, and BPF at these sites. We identified three sites where polar interactions between positively charged residues and the sulfonyl group of BPS could lead binding selectivity over BPA and BPF. To test whether EGFR mutations at the predicted BPS binding sites (Arg255, Lys454, and Arg297) could prevent BPS's interference on EGFR activation, mutations for each EGFR target amino acids (R255A, R297A, and K454A) were introduced. For variants with R297A or K454A mutations, BPS did not affect EGF-mediated EGFR phosphorylation or EGFR-mediated cell invasion, suggesting that these residues are needed for the BPS antagonism effect on EGFR. In conclusion, BPS, but not BPA or BPF, interferes with EGFR-mediated trophoblast cell functions through binding at Arg297 and Lys454 amino acid residues in the extracellular domain of EGFR.

Developmental programming: impact of prenatal testosterone excess on ovarian cell proliferation and apoptotic factors in sheep.

Prenatal testosterone (T) excess leads to reproductive dysfunctions in sheep, which include increased ovarian follicular recruitment and persistence. To test the hypothesis that follicular disruptions in T sheep stem from changes in the developmental ontogeny of ovarian proliferation and apoptotic factors, pregnant Suffolk sheep were injected twice weekly with T propionate or dihydrotestosterone propionate (DHT; a nonaromatizable androgen) from Days 30 to 90 of gestation. Changes in developmental expression of proliferating cell nuclear antigen (PCNA), BCL2, BAX, activated CASP3, and FAS/FASLG were determined at Fetal Days 90 and 140, 22 wk, 10 mo, and 21 mo of age by immunocytochemisty. Prenatal T treatment induced changes in expression of proliferative and apoptotic markers in a follicle-, age-, and steroid-specific manner. Changes in BAX were evident only during fetal life and PCNA, BCL2, and CASP3 only postnatally. Prenatal T and not DHT increased PCNA and decreased BCL2 in granulosa/theca cells of antral follicles at 10 and 21 mo but decreased CASP3 in granulosa/theca cells of antral follicles at 22 wk (prepubertal) and 10 and 21 mo. Both treatments decreased BAX immunostaining in granulosa cells of Fetal Day 90 primordial/primary follicles. Neither treatment affected FAS expression at any developmental time point in any follicular compartment. Effects on BAX appear to be programmed by androgenic actions and PCNA, BCL2, and CASP3 by estrogenic actions of T. Overall, the findings demonstrate that fetal exposure to excess T disrupts the ovarian proliferation/apoptosis balance, thus providing a basis for the follicular disruptions evidenced in these females.