Male Rat Fetuses Research Articles

Autophagy activated by GR/miR-421-3p/mTOR pathway as a compensatory mechanism participates in chondrodysplasia induced by prenatal caffeine exposure in male fetal rats

Autophagy has been implicated in the developmental toxicity of multiple organs in offspring caused by adverse environmental conditions during pregnancy. We have previously found that prenatal caffeine exposure (PCE) can cause fetal overexposure to maternal glucocorticoids, leading to chondrodysplasia. However, whether autophagy is involved and what role it plays has not been reported. In this study, a PCE rat model was established by gavage of caffeine (120mg/kg.d) on gestational day 9-20. The results showed that reduced cartilage matrix synthesis in male fetal rats in the PCE group was accompanied by increased autophagy compared to the control group. Furthermore, the expression of mTOR, miR-421-3p, and glucocorticoid receptor (GR) in male fetal rat cartilage of PCE group was increased. At the cellular level, we confirmed that corticosterone inhibited matrix synthesis in fetal chondrocytes while increasing autophagic flux. However, administration of autophagy enhancer (rapamycin) or inhibitor (bafilomycin A1 or 3-methyladenine) partially increased or further decreased aggrecan expression respectively. At the same time, we found that corticosterone could increase the expression of miR-421-3p through GR and target to inhibit the expression of mTOR, thereby enhancing autophagy. In conclusion, PCE can cause chondrodysplasia and autophagy enhancement in male fetal rats. Intrauterine high corticosterone activates GR/miR-421-3p signaling and down-regulates mTOR signaling in fetal chondrocytes, resulting in enhanced autophagy, which can partially compensate for corticosterone-induced fetal chondrodysplasia. This study confirmed the compensatory protective effect of autophagy on the developmental toxicity of fetal cartilage induced by PCE and its epigenetic mechanism, providing novel insights for exploring the early intervention and therapeutic target of fetal-originated osteoarthritis.

Maternal organophosphate flame retardant exposure alters the developing mesencephalic dopamine system in fetal rat.

Organophosphate flame retardants (OPFRs) have become the predominant substitution for legacy brominated flame retardants but there is concern about their potential developmental neurotoxicity (DNT). OPFRs readily dissociate from the fireproofed substrate to the environment, and they (or their metabolites) have been detected in diverse matrices including air, water, soil, and biota, including human urine and breastmilk. Given this ubiquitous contamination, it becomes increasingly important to understand the potential effects of OPFRs on the developing nervous system. We have previously shown that maternal exposure to OPFRs results in neuroendocrine disruption, alterations to developmental metabolism of serotonin (5-HT) and axonal extension in male fetal rats, and potentiates adult anxiety-like behaviors. The development of the serotonin and dopamine systems occur in parallel and interact, therefore, we first sought to enhance our prior 5-HT work by first examining the ascending 5-HT system on embryonic day 14 using whole mount clearing of fetal heads and 3-dimensional (3D) brain imaging. We also investigated the effects of maternal OPFR exposure on the development of the mesocortical dopamine system in the same animals through 2-dimensional and 3D analysis following immunohistochemistry for tyrosine hydroxylase (TH). Maternal OPFR exposure induced morphological changes to the putative ventral tegmental area and substantia nigra in both sexes and reduced the overall volume of this structure in males, whereas 5-HT nuclei were unchanged. Additionally, dopaminergic axogenesis was disrupted in OPFR exposed animals, as the dorsoventral spread of ventral telencephalic TH afferents were greater at embryonic day 14, while sparing 5-HT fibers. These results indicate maternal exposure to OPFRs alters the development trajectory of the embryonic dopaminergic system and adds to growing evidence of OPFR DNT.

The anti-androgenic fungicide triticonazole induces region-specific transcriptional changes in the developing rat perineum and phallus

Intrauterine exposure to endocrine disrupting chemicals can interfere with male reproductive development. This can lead to male reproductive disorders such as hypospadias, cryptorchidism and reduced fertility, as well as shorter anogenital distance (AGD) – a biomarker for incomplete androgen-dependent fetal masculinization. However, it remains challenging to predict adverse in vivo outcomes based on in vitro effect patterns for many chemicals. This is a challenge for modern toxicology, which aims to reduce animal testing for chemical safety assessments. To enable the transition towards higher reliance on alternative test methods, we need to better map underlying mechanisms leading to adverse effects. Herein, we have analyzed the transcriptome of the perineum and phallus of male fetal rats and defined the impacts of exposure to an anti-androgenic fungicide, triticonazole. Previously we have shown that developmental exposure to triticonazole can induce short male AGD, but without a marked effect on the transcriptome of the fetal testes. In contrast, we report here significant changes to the transcriptional landscape of the perineum and phallus, including regional differences between these adjacent tissues. This highlights the importance of analyzing the correct tissue when characterizing mechanisms of complex in vivo effect outcomes. Our results provide a rich resource for the spatiotemporal gene networks that are involved in the development of male external genitalia, and that can be disrupted upon exposure to chemicals that prevent normal masculinization of the perineum and phallus. Such data will be critical in the development of novel alternative test methods to determine the endocrine disrupting potential of existing and emerging chemicals.

The upregulation of glutamate decarboxylase 67 against hippocampal excitability damage in male fetal rats by prenatal caffeine exposure.

As a kind of xanthine alkaloid, caffeine is widely present in beverages, food, and analgesic drugs. Our previous studies have shown that prenatal caffeine exposure (PCE) can induce programmed hypersensitivity of the hypothalamic-pituitary-adrenal (HPA) axis in offspring rats, which is involved in developing many chronic adult diseases. The present study further examined the potential molecular mechanism and toxicity targets of hippocampal dysfunction, which might mediate the programmed hypersensitivity of the HPA axis in offspring. Pregnant rats were intragastrically administered with 0, 30, and 120 mg/kg/day caffeine from gestational days (GD) 9-20, and the fetal rats were extracted at GD20. Rat fetal hippocampal H19-7/IGF1R cell line was treated with caffeine, adenosine A2A receptor (A2AR) agonist (CGS-21680) or adenylate cyclase agonist (forskolin) plus caffeine. Compared with the control group, hippocampal neurons of male fetal rats by PCE displayed increased apoptosis and reduced synaptic plasticity, whereas glutamate decarboxylase 67 (GAD67) expression was increased. Moreover, the expression of A2AR was down-regulated, PCE inhibited the cAMP/PKA/CREB/BDNF/TrkB pathway. Furthermore, the results in vitro were consistent with the in vivo study. Both CGS21680 and forskolin could reverse the above alteration caused by caffeine. These results indicated that PCE inhibits the BDNF pathway and mediates the hippocampus's glutamate (Glu) excitotoxicity. The compensatory up-regulation of GAD67 unbalanced the Glu/gamma-aminobutyric acid (GABA)ergic output, leading to the impaired negative feedback to the hypothalamus and hypersensitivity of the HPA axis.

Altered hippocampal GR/KCC2 signaling mediates susceptibility to convulsion in male offspring following dexamethasone exposure during pregnancy in rats

Epidemiological research suggests that convulsions may have an intrauterine developmental origin related to the application of dexamethasone, an artificially synthesized glucocorticoid. Here, using a rat animal model of prenatal dexamethasone exposure (PDE) we confirm that PDE can cause susceptibility to convulsions in male offspring and explore the epigenetic programming mechanism underlying this effect related to intrauterine type 2 K+-Cl- cotransporter (KCC2). Wistar rats were injected with dexamethasone (0.2 mg/kg/d) subcutaneously during the gestational days (GD) 9–20 and part of the offspring was given lithium pilocarpine (LiPC) at postnatal week 10. Our results showed that male offspring of the PDE+LiPC group exhibited convulsions susceptibility, as well as increased hippocampal gamma-aminobutyric acid (GABA) and intracellular chloride ions level and decreased GABA receptor expression. The offspring also showed a decrease of hippocampal KCC2 H3K14ac levels and KCC2 expression. PDE male fetal rats (GD20) showed similar changes to male offspring after birth and exhibited an increased expression of glucocorticoid receptor (GR) and histone deacetylase type 2 (HDAC2). We observed effects consistent with those observed in PDE fetal rats following in vitro dexamethasone treatment of the fetal rat hippocampal neuron H19–7 cell line, and the effects could be reversed by treatment with a GR inhibitor (RU486) or HDAC2 inhibitor (romidepsin). Taken together, this study confirmed that PDE causes a reduction of H3K14ac levels in the KCC2 promoter region caused by activation of fetal hippocampal GR-HDAC2-KCC2 signaling. We proposed that this abnormal epigenetic modification is the mechanism underlying offspring convulsions susceptibility. CategoriesMechanism of toxicity.

Maternal supplementation of \u03b1-lipoic acid attenuates prenatal cytarabine exposure-induced oxidative stress, steroidogenesis suppression and testicular damage in F1 male rat fetus

BackgroundCytarabine (Ara-C) is an anticancer drug, which is considered as the mainstay in the treatment of hematological malignancies, known to cause various teratogenic effects. Alpha-lipoic acid (ALA) is a natural antioxidant and its supplementation proved to improve pregnancy outcomes in several pathological conditions. We aimed at exploring the benefits of maternal supplementation of ALA against in-utero Ara-C exposure-induced testicular toxicity in rat fetuses.MethodsPregnant rats (dams) received normal saline (control group), ALA 200 mg/kg (ALA group), Ara-C 12.5 mg/kg (Ara-C 12.5 group), Ara-C 25 mg/kg (Ara-C 25 group), and Ara-C 25 mg/kg + ALA 200 mg/kg (protection group) from gestational day (GD)8 to GD21. Ara-C and ALA were administered via the intraperitoneal and oral routes, respectively. The day of parturition was considered as postnatal day (PND)1. On PND1, all the live male pups were collected. The maternal parameters evaluated include (a) food intake, (b) bodyweight, and (c) oxidative stress (OS) markers. The fetal parameters evaluated include (a) bodyweight, (b) anogenital distances (AGD), (c) testicular weight (d) testicular testosterone levels (e) testicular histopathology, and (f) morphometrical parameters.ResultsA significant and dose-dependent decrease in maternal food intake, weight gain, and an increase in oxidative stress (OS) were observed in the pregnant rats of the Ara-C groups as compared to pregnant rats of the control group. Further, a significant and dose-dependent (a) reduction in bodyweight, AGD, testicular weight, and testosterone levels, (b) increase in OS, and (c) structural and morphometrical anomalies in fetal testes were observed in fetuses of Ara-C groups as compared to fetuses of the control rats. These deleterious effects observed in the Ara-C groups were found to be diminished in the pregnant rats and fetuses of the Protection group as compared to the pregnant rats and fetuses of the Ara-C 25 group.ConclusionsFrom the results of this study, we conclude that the maternal supplementation of ALA may ameliorate the Ara-C exposure-induced impairment in prenatal development and function of the testes in the rat fetuses. However, future experimental and clinical studies are warranted to explore the possible mechanisms involved in the protection offered by maternal supplementation of ALA against Ara-C induced testicular toxicity.Graphical

Low H3K27 acetylation of SF1 in PBMC: a biomarker for prenatal dexamethasone exposure-caused adrenal insufficiency of steroid synthesis in male offspring.

Dexamethasone is widely used to treat pregnancy disorders related to premature delivery. However, lots of researches have confirmed that prenatal dexamethasone exposure (PDE) could increase the risk of offspring multiple diseases. This study was designed to elucidate the epigenetic mechanism of adrenal developmental programming and explore its early warning marker in peripheral blood mononuclear cells (PBMC). We found the adrenal morphological and functional changes of PDE male offspring rats before and after birth, which were mainly performed as the decreased serum corticosterone concentration, steroidogenic acute regulatory (StAR) protein expression, and histone 3 lysine 27 acetylation (H3K27ac) level of steroidogenic factor 1 (SF1) promoter region and its expression. Simultaneously, the expressions of glucocorticoid receptor (GR) and histone acetylation enzyme 5 (HDAC5) in the PDE male fetal rats were increased. In vitro, dexamethasone reduced the expression of SF1, StAR, and cortisol production and still increased the expression of GR and HDAC5, the binding between GR and SF1 promoter region, and protein interaction between GR and HDAC5. GR siRNA or HDAC5 siRNA was able to reverse the above roles of dexamethasone. Furthermore, in vivo, we confirmed that H3K27ac levels of SF1 promoter region and its expression in PBMC of the PDE group were decreased before and after birth, showing a positive correlation with the same indexes in adrenal. Meanwhile, in clinical trials, we confirmed that prenatal dexamethasone application decreased H3K27ac of SF1 promoter region and its expression in neonatal PBMC. In conclusion, PDE-caused adrenal insufficiency of male offspring rats was related to adrenal GR activated by dexamethasone in uterus. The activated GR, on the one hand, increased its direct binding to SF1 promoter region to inhibit its expression, on the other hand, upregulated and recruited HDAC5 to decrease H3K27ac level of SF1 promoter region, and strengthened the inhibition of SF1 and subsequent StAR expression.

Identification and validation of reference genes for RT-qPCR analysis in fetal rat pancreas

The choice of reference gene is crucial for quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) assay. To screen and determine the suitable reference genes in fetal rat pancreas, we selected eight candidate reference genes (Gapdh, Actb, Rn18 s, B2m, Rpl13a, Tbp, Ywhaz and Ubc), and evaluated the constancy of gene expression from fetal rat pancreases in non-pathological situation and prenatal dexamethasone exposure (PDE) model, using four algorithms: GeNorm, NormFinder, Bestkeeper and Comparative ΔCt method. In addition, the alteration of mRNA levels of pancreatic insulin was compared between control and PDE groups to validate the reliability of selected reference genes for data normalization of RT-qPCR. The comprehensive ranking of reference genes under physiological condition was as follow: Gapdh >Actb >Ywhaz >Ubc >Rn18s >Rpl13a >B2m >Tbp (female); Actb >Ywhaz >Gapdh >Ubc >B2m >Rpl13a >Rn18 s | Tbp (male). The top ranking reference genes were also stably expressed in PDE fetal pancreas. The best reference gene combinations are: Ywhaz+Actb for female and Ywhaz+Gapdh for male fetal rat pancreas, respectively. Compared with low ranking or single reference gene, the change trend of insulin mRNA normalized by the best reference gene combination between control and PDE groups was more significant and consistent with that of serum insulin level. In conclusion, our results provided the optimal combination of stable reference genes for RT-qPCR assay in pancreatic developmental toxicity study.

Maternal exposure to trace cadmium affects gonadal differentiation and development in male offspring rats though a star pathway

To investigate effect and mechanism of maternal exposure to trace cadmium on the differentiation and development in testis of male offspring rats. Thirty-two pregnant female SD rats were randomly divided into 4 groups: CdCl2 treated group (group A) was intraperitoneally injected with CdCl2 (1.5 mg/kg.d); DES group(group B) was subcutaneously injected with DES (100 μg/kg.d); CdCl2 +TMX group(group C) was treated with CdCl2 (same as above)+TMX (50 mg/kg.d); control group (group D) was treated with the same dose normal saline instead of CdCl2. On day 30th after parturition, the weight and sex ratio of off-spring male rats were recorded; Offspring testes were used for ultrastructural observation. Ultrastructural changes including swollen mitochondria and expanded endoplasmic reticulum in Sertoli cells, gonocyte and Leydig cells, moreover, lipid droplets cumulated in Leydig cells were observed in group A and group B, and so changes in group C were slightly more than those in group A or group B. The expression of StAR and inhibin-B in Leydig cells decreased significantly in group A and group B (p < 0.05), but there were no difference between group C and group D (P > 0.05). Cadmium has similar effect as estrogen which would affect gonadal differentiation and development in male fetal rats.

Prenatal Exposure to BPA: The Effects on Hepatic Lipid Metabolism in Male and Female Rat Fetuses.

Bisphenol A (BPA) is an organic chemical compound widely used for manufacturing plastics. BPA exposure originates principally from the diet, but it can also originate from dermal contact. In over 90% of individuals, including pregnant women, BPA is detectable in several body fluids. The effects of this exposure on the fetus are under active investigation in several research laboratories. The aim of our work was to study the impact of prenatal exposure to BPA in the liver of rat fetuses from a sex-dependent point of view. We particularly investigated the effects of prenatal BPA exposure on hepatic lipids because of their crucial role, not only for the liver, but also for the whole-body functions. Our results demonstrate that the liver of rat fetuses, in utero exposed to a very low dose of BPA (2.5 µg/kg/day), displays significant modulations with regard to proteins involved in cholesterol and fatty acid biosynthesis and trafficking. Moreover, an impact on inflammatory process has been observed. All these effects are dependent on sex, being observable only in female rat fetuses. In conclusion, this work demonstrates that maternal exposure to BPA compromises hepatic lipid metabolism in female offspring, and it also reveals the perspective impact of BPA on human health at doses currently considered safe.

Exposure to 4-bromodiphenyl ether during pregnancy blocks testis development in male rat fetuses

4-Bromodiphenyl ether (BDE3) is a photodegradation product of higher polybrominated diphenyl ether flame retardants and is known as an endocrine disruptor. However, it is unclear whether and how BDE3 affects the development of fetal testes. This study aimed to investigate the effect of in utero exposure to BDE3 on fetal testicular development in rats. From gestational day (GD) 12–21, BDE3 (0, 50, 100, and 200 mg/kg) was daily gavaged to female pregnant Sprague Dawley rats. BDE3 significantly reduced serum testosterone levels of male pups starting at 50 mg/kg. BDE3 reduced fetal Leydig cell number at a dose of 200 mg/kg without affecting fetal Leydig cell cluster frequency and Sertoli cell number. In addition, BDE3 down-regulated the expression of fetal Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and their proteins at 100 and/or 200 mg/kg. RNA-seq analysis showed that genes responsive to cAMP (Ass1, Gpd1, Rpl13a) were down-regulated and hypoxia-related genes (Egln3 and P4ha1) were up-regulated at 200 mg/kg. In utero exposure to BDE3 can promote autophagy and apoptosis of fetal Leydig cells via increasing the levels of Beclin1, LC3-II, BAX, and by decreasing the levels of p62 and BCL2. In conclusion, in utero exposure to BDE3 blocks the development of fetal rat testes.

Prenatal dexamethasone exposure induces nonalcoholic fatty liver disease in male rat offspring via the miR-122/YY1/ACE2-MAS1 pathway

Epidemiological studies have shown that nonalcoholic fatty liver disease (NAFLD) has an intrauterine developmental origin. We aimed to demonstrate that NAFLD is caused by prenatal dexamethasone exposure (PDE) in adult male rat offspring and to investigate the intrauterine programming mechanism. Liver samples were obtained on gestational day (GD) 21 and postnatal week (PW) 28. The effects and epigenetic mechanism of dexamethasone were studied with bone marrow mesenchymal stem cells (BMSCs) hepatoid differentiated cells and other cell models. In the PDE group, lipid accumulation increased, triglyceride synthesis-related gene expression increased, and oxidation-related gene expression decreased in livers of adult male rat offspring. In utero, hepatic triglyceride synthesis increased and oxidative function decreased in PDE fetal male rats. Moreover, low hepatic miR-122 expression, high Yin Yang-1 (YY1) expression and angiotensin-converting enzyme 2 (ACE2)-Mas receptor (MAS1) signaling pathway inhibition were observed before and after birth. At the cellular level, dexamethasone (100–2500 nM) elevated the intracellular triglyceride content, increased triglyceride synthesis-related gene expression and decreased oxidation-related gene expression. Dexamethasone treatment also decreased miR-122 expression, increased YY1 expression and inhibited the ACE2-MAS1 signaling pathway. Interference or overexpression of glucocorticoid receptor (GR), miR-122, YY1 and ACE2 could reverse the changes in downstream gene expression. In summary, PDE could induce NAFLD in adult male rat offspring. The programming mechanism included inhibition of miR-122 expression after GR activation, and dexamethasone increased hepatocyte YY1 expression; these effects resulted in ACE2-MAS1 signaling pathway inhibition, which led to increased hepatic triglyceride synthesis and decreased oxidative function. The increased triglyceride synthesis and decreased oxidative function of hepatocytes caused by low miR-122 expression due to dexamethasone could continue postnatally, eventually leading to NAFLD in adult rat offspring.

The effects of NONRATT008453.2 on autophagy in genital tubercle fibroblasts of rats with hypospadias induced by dibutyl phthalate.

BackgroundHypospadias is a common birth defect that might be caused by inadequate fusion of the urethral folds in the process of male external genital development. We intended to discover the crucial long noncoding RNAs (lncRNAs) regulating autophagy from the gene expression profile of the genital tubercle (GT) of dibutyl phthalate (DBP) induced hypospadiac rats.MethodsWhole transcriptome resequencing was used to determine the expression of the total RNA in GTs and cultured fibroblasts obtained from GTs of DBP‐induced hypospadiac male rat fetuses. Autophagosomes and autolysosomes were examined under a transmission electron microscope after overexpression of lncRNA NONRATT008453.2 in the fibroblasts by adenovirus transfection. Finally, the protein expression levels of Atg5, Beclin‐1, Atg7, and the LC3A/B‐II:LC3A/B‐I ratio were detected in the fibroblasts by western blotting.ResultsNONRATT008453.2 suppressed autophagy by promoting the expression of Atg7, but inhibited the expressions of Atg5, Beclin‐1, and the LC3A/B‐II:LC3A/B‐I ratio in the GT fibroblasts.ConclusionsNONRATT008453.2 may have an influence on autophagy in the fibroblasts of the GT in DBP‐induced hypospadiac rats.

Transcriptome analysis of fetal rat testis following intrauterine exposure to the azole fungicides triticonazole and flusilazole reveals subtle changes despite adverse endocrine effects

Azoles are used in agriculture and medicine to combat fungal infections. We have previously examined the endocrine disrupting properties of the agricultural azole fungicides triticonazole and flusilazole. Triticonazole displayed strong androgen receptor (AR) antagonism in vitro, whereas in utero exposure resulted in anti-androgenic effects in vivo evidenced by shorter anogenital distance (AGD) in fetal male rats. Flusilazole displayed strong AR antagonism, but less potent than triticonazole, and disrupted steroidogenesis in vitro, whereas in utero exposure disrupted fetal male plasma hormone levels. To elaborate on how these azole fungicides can disrupt male reproductive development by different mechanisms, and to investigate whether feminization effects such as short AGD in males can also be detected at the transcript level in fetal testes, we profiled fetal testis transcriptomes after in utero exposure to triticonazole and flusilazole by 3′Digital Gene Expression (3′DGE). The analysis revealed few transcriptional changes after exposure to either compound at gestation day 17 and 21. This suggests that the observed influence of flusilazole on hormone production may be by directly targeting steroidogenic enzyme activity in the testis at the protein level, whereas observations of shorter AGD by triticonazole may primarily be due to disturbed androgen signaling in androgen-sensitive tissues. Expression of Calb2 and Gsta2 was altered by flusilazole but not triticonazole and may pinpoint novel pathways of disrupted testicular steroid synthesis. Our findings have wider implication for how we integrate omics data in chemical testing frameworks, including selection of non-animal test methods and building of Adverse Outcome Pathways for regulatory purposes.

Gestational vinclozolin exposure suppresses fetal testis development in rats

Vinclozolin is a common dicarboximide fungicide used to protect crops from diseases. It is also an endocrine disruptor and is thought to be related to abnormalities of the reproductive tract. However, its mechanism of inducing abnormalities of the male reproductive tract is still unclear. The purpose of this study was to study the effect of gestational vinclozolin exposure on the development of rat fetal Leydig cells. Female pregnant Sprague-Dawley rats were exposed to vinclozolin (0, 25, 50, and 100 mg/kg body weight/day) by gavage from gestational day 14–21. Vinclozolin dose-dependently reduced serum testosterone levels at doses of 50 and 100 mg/kg and the anogenital distance at 100 mg/kg. RNA-seq, qPCR, and Western blotting showed that vinclozolin down-regulated the expression of Nr5a1, Sox9, Lhcgr, Cyp11a1, Hsd3b1, Hsd17b3, Amh, Pdgfa, and Dhh and their encoded proteins. Vinclozolin reduced the number of NR2F2-positive stem Leydig cells at a dose of 100 mg/kg and enhanced autophagy in the testes. In conclusion, vinclozolin disrupts reproductive tract development and testis development in male fetal rats via several pathways.

Taxifolin attenuates the developmental testicular toxicity induced by di-n-butyl phthalate in fetal male rats

Di-n-butyl phthalate (DBP) is widely used in consumer products as a plasticizer. Here, we report a natural product taxifolin that can attenuate developmental and reproductive toxicity of DBP. Pregnant rats were daily gavaged with 500 mg/kg DBP alone or together with taxifolin (10 and 20 mg/kg) from gestational day (GD) 12–21. At GD21, sera and testes of male fetus were collected. DBP significantly lowered serum testosterone level at 500 mg/kg and taxifolin can completely reverse its action. DBP caused abnormal aggregation of fetal Leydig cells and taxifolin can reverse it. DBP down-regulated the expression of the genes of cholesterol side-chain cleavage enzyme (Cyp11a1), 17β-hydroxysteroid dehydrogenase 3 (Hsd17b3), and insulin-like 3 (Insl3) and taxifolin can reverse its action. DBP increased malondialdehyde levels and decreased superoxide dismutase and glutathione peroxidase expression and taxifolin can reverse it. DBP increased incidence of multinucleated gonocytes and taxifolin can prevent it. Moreover, DBP lowered sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and phosphorylated AMP-activated protein kinase (pAMPK) signalling and taxifolin antagonized DBP. In conclusion, in utero exposure to DBP caused developmental/reproductive toxicity of male offspring via increasing reactive oxygen species and taxifolin is an effective food component that completely reverses DBP-mediated action.

Programming of a developmental imbalance in hypothalamic glutamatergic/GABAergic afferents mediates low basal activity of the hypothalamic-pituitary-adrenal axis induced by prenatal dexamethasone exposure in male offspring rats

This study was intended to demonstrate that prenatal dexamethasone exposure (PDE) can induce low basal activity of the hypothalamic-pituitary-adrenal axis (HPAA) in male offspring rats and explore the underlying mechanism. Pregnant rats were subcutaneously administered 0.2 mg/kg/d dexamethasone from gestational day (GD) 9 to GD20. Male GD20 fetuses and postnatal day 85 adult male offspring rats were sacrificed under anesthesia. Hypothalamic cells were from GD20∼postnatal day (PD) 7 fetal male rats, treated with different concentrations of dexamethasone and the glucocorticoid receptor (GR) antagonist mifepristone for 5 days. The results suggested that dexamethasone enhanced the expression of hypothalamic L-glutamic acid decarboxylase (GAD) 67 by activating GR, further stimulating the conversion of glutamate to gamma-aminobutyric acid (GABA) and inducing an imbalance in glutamatergic/GABAergic afferents in the hypothalamic paraventricular nucleus (PVN). This imbalance change was maintained postnatally, leading to the inhibition of parvocellular neurons, and mediating the low basal activity of the HPAA in PDE offspring rats, which was manifested by decreased levels of blood adrenocorticotropic hormone and corticosterone as well as reduced expression levels of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) in the hypothalamus. Programming of a developmental imbalance in glutamatergic/GABAergic afferents in the PVN is a potential mechanism responsible for low basal activity of the HPAA in male PDE rats.

Maternal plasma concentrations of perfluoroalkyl and polyfluoroalkyl substances during pregnancy and anogenital distance in male infants.

Are maternal plasma concentrations of perfluoroalkyl and polyfluoroalkyl substances (PFASs) during pregnancy associated with anogenital distance (AGD) in male infants at birth, 6, and 12 months of age? Higher maternal plasma concentrations of some PFASs were associated with shorter AGD in male infants at birth and 6months of age. Two animal studies have found that exposure to PFASs was associated with shorter AGD in male rat fetuses and wild male minks. There is only one human study on the topic that did not identify consistent patterns between maternal serum concentrations of PFASs during pregnancy and AGD in male infants. In the prospective cohort study, a total of 1292 eligible pregnant women were recruited at 12-16weeks of gestation between April and December 2012 at the Maternal and Child Health Hospital of Minhang district in Shanghai, China. At delivery, 667 male singletons were born. They were then followed up at birth (n = 439) and at 6 (n = 411) and 12months (n = 376) of age when anopenile distance (AGDAP) and anoscrotal distance (AGDAS) were measured. A total of 500 male infants who had both maternal plasma concentrations of PFASs and at least one AGD measurement of at three time points were included in the present study. Multiple linear regression models were used to evaluate the potential linear associations between maternal concentrations of PFASs and AGD. Maternal plasma concentrations (ln-transformed) of perfluorooctane sulfonate (PFOS), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUdA) were inversely associated with AGDAS or AGDAP at birth (AGDAS: per ln unit increase in PFAS concentrations: β (95% CI): -0.65 (-1.27 to -0.02)mm for PFOS; -0.58 (-1.11 to -0.06)mm for PFDA; and -0.57 (-1.09 to -0.06)mm for PFUdA; AGDAP: per ln unit increase in PFAS concentrations: β (95% CI): -0.63 (-1.24 to -0.01)mm for PFDA and - 0.76 (-1.36 to -0.16)mm for PFUdA). At 6months of age, per unit increase in maternal ln concentrations of PFOS and perfluorotridecanoic acid (PFTrDA), AGDAS decreased on average by -2.21 (95% CI: -4.28 to -0.14) and -1.11 (95% CI: -2.17 to -0.06)mm, respectively. Additionally, ln-transformed perfluorooctanoic acid (PFOA) showed nonsignificant but inverse associations with both AGDAS and AGDAP at 6months of age. We found no significant associations between ln-transformed maternal concentrations of PFASs and either AGDAS or AGDAP at 12months of age. However, significantly inverse association of ln-transformed PFOA with AGDAP was observed in male infants who never or shortly breastfed (<3months) at 12months of age. AGD measurements were performed by different examiners at each follow-up visit, and the intra-examiner variation was not assessed, which might cause intra-rater and inter-rater measurement errors. Additionally, our study may have selection bias since a considerable number of participants withdrew from the cohort although the differences in demographic characteristics were not statistically significant between included mother-infant pairs and those excluded. No statistical correction was made for multiple comparisons. Our findings may have important implications for the early development of genital health in male infants since PFASs can be detected in almost all pregnant women and infants worldwide. This study was supported by grants from the National Key Research and Development program of China (2018YFC1002801 and 2016YFC1000505), the Science and Technology Commission of Shanghai Municipality (16ZR1430100), the National Natural Science Foundation of China (81428011), and the Innovation-Oriented Science and Technology Grant from National Health Commission Key Laboratory of Reproduction Regulation (CX2017-06). The authors have no conflicts of interest to declare. N/A.

Distinct Transcriptional Profiles of the Female, Male, and Finasteride-Induced Feminized Male Anogenital Region in Rat Fetuses.

A short anogenital distance (AGD) in males is a marker for incomplete masculinization and a predictor of adverse effects on male reproductive health. For this reason, AGD is used to assess the endocrine disrupting potential of chemicals for risk assessment purposes. The molecular mechanisms underpinning this chemically induced shortening of the AGD, however, remains unclear. Although it is clear that androgen receptor-mediated signaling is essential, evidence also suggest the involvement of other signaling pathways. This study presents the first global transcriptional profile of the anogenital tissue in male rat fetuses with chemically induced short AGD, also including comparison to normal male and female control animals. The antiandrogenic drug finasteride (10 mg/kg bw/day) was used to induce short AGD by exposing time-mated Sprague Dawley rats at gestation days 7-21. The AGD was 37% shorter in exposed male fetuses compared with control males at gestation day 21. Transcriptomics analysis on anogenital tissues revealed a sexually dimorphic transcriptional profile. More than 350 genes were found to be differentially expressed between the 3 groups. The expression pattern of 4 genes of particular interest (Esr1, Padi2, Wnt2, and Sfrp4) was also tested by RT-qPCR analyses, indicating that estrogen and Wnt2 signaling play a role in the sexually dimorphic development of the anogenital region. Our transcriptomics profiles provide a stepping-stone for future studies aimed at characterizing the molecular events governing development of the anogenital tissues, as well as describing the detailed Adverse Outcome Pathways for short AGD; an accepted biomarker of endocrine effects for chemical risk assessment.

In utero exposure to simvastatin reduces postnatal survival and permanently alters reproductive tract development in the Crl:CD(SD) male rat

We showed previously that in utero exposure to the cholesterol-lowering drug simvastatin (SMV) during sex differentiation lowers fetal lipids and testicular testosterone production (T Prod) in Hsd:SD rats. Here, the effects of SMV on fetal lipids and T Prod in Crl:CD(SD) rats were correlated with postnatal alterations in F1 males. The current study was conducted in two parts: 1) a prenatal assessment to confirm and further characterize the dose response relationship among previously reported alterations of SMV on fetal T Prod and the fetal lipid profile and 2) a postnatal assessment to determine the effects of SMV exposure during the periods of major organogenesis and/or sexual differentiation on F1 offspring growth and development. We hypothesized that SMV would have adverse effects on postnatal development and sexual differentiation as a consequence of the disruptions of fetal lipid levels and testicular T Prod since fetal cholesterol is essential for normal intrauterine growth and development and steroid synthesis.In the prenatal assessment, SMV was administered orally at 0, 15.6, 31.25, 62.5, 80, 90, 100, and 110 mg SMV/kg/d from GD 14-18, the period that cover the critical window of sex differentiation in the male rat fetus. T Prod was maximally reduced by ~40% at 62.5 mg/kg/d, and higher doses induced overt maternal and toxicity.In the postnatal assessment, SMV was administered at 0, 15.6, 31.25, and 62.5 mg/kg/d from GD 8–18 to determine if it altered postnatal development. We found that exposure during this time frame to 62.5 mg SMV/kg/d reduced pup viability by 92%, decreased neonatal anogenital distance, and altered testis histology and morphology in 17% of the F1 males. In another group, SMV was administered only during the masculinizing window (GD14-18) at 62.5 mg/kg/d to determine if male rat sexual differentiation and postnatal reproductive development were altered. SMV-exposed F1 males displayed female-like areolae/nipples, delayed puberty, and reduced seminal vesicle and levator ani-bulbocavernosus weights.Together, these results demonstrate that in utero exposure to SMV reduces offspring viability and permanently disrupts reproductive tract development in the male offspring. While the effects of high dose, short term in utero exposure to SMV in the adult male are likely androgen-dependent and consistent with the 40% reduction in T Prod in the fetal testes, long-term, lower dose administration induced some effects that were likely not mediated by decreased T Prod.