SAT-201 Diet-Induced Obesity and Prenatal Androgen Exposure Increase Transgenerational Susceptibility to Metabolic Dysfunction in Male Adult Offspring

We know relatively little of the consequences of male obesity for reproductive success compared to female obesity. Conflicting evidence exists in both humans and rodents regarding whether paternal obesity alters sperm motility and concentration. However, we have described impaired embryo and fetal development, and implantation, in rodents as a consequence of paternal obesity. This study investigated whether founder male obesity influenced the reproductive and metabolic fitness of males of the subsequent F1 generation. C57BL/6 founder male mice were fed a standard chow (CD) or a high-fat diet (HF) for 8wks. This increases adiposity in the absence of changes in fasting glucose levels. Males were mated to female C57BL/6 mice, and subsequent m ale F1 offspring from HF (HF-F1) or CD (CD-F1) founders were weighed weekly and maintained on standard chow. At 8 weeks and 14 weeks glucose tolerance tests were performed and following euthanasia, tissues and sperm collected. Sperm reactive oxygen species (ROS) and DNA damage levels were determined, and various organs weighed. HF-F1 male pups were significantly heavier relative to CD-F1 males (P < 0.05) although adult bodyweight did not differ significantly. Despite this, liver, pancreas, testes and epididymis weight was significantly elevated for HF-F1 males at 17wks of age (P < 0.05). At both 8wks and 14wks of age HF-F1 males were hypoglycaemic and had impaired glucose metabolism. Sperm analysis of HF-F1 males indicated a significant increase in ROS levels (P < 0.05), DNA damage (P < 0.05) and a decrease in fertilization rates in vitro (P < 0.05). This data indicates significant physiological changes and perturbed sperm parameters in F1 males as a consequence of founder male obesity. It supports further interrogation of male and female F1 offspring, and warrants examination of potential effects for a subsequent F2 population.

There is a paucity of studies on the multigenerational reproductive toxicity of fine particle matter (PM2.5) exposure during pregnancy on male offspring and the underlying mechanisms. This study explored the effects of PM2.5 exposure during pregnancy on the spermatogenesis of three consecutive generations of male mouse offspring. We randomized pregnant C57BL/6 mice into the control group, the Quartz Fiber Membrane control group, and two experimental groups exposed to different concentrations of PM2.5 (4.8 and 43.2 mg/kg B.Wt.). Pregnant mice from experimental groups received intratracheal instillation of PM2.5 of different doses on a three-day basis until birth. F1 mature male offspring from PM2.5-exposed pregnant mice were mated with normal female C57BL/6 mice. Likewise, their F2 mature male followed the same to produce the F3 generation. The results showed that PM2.5 exposure during pregnancy led to decreased body and tail length, body weight, and survival rates, decreased sperm concentration and sperm motility, and increased sperm abnormality rates significantly in F1 male offspring. We barely observed significant impacts of PM2.5 on the birth number, survival rates, and index of testes in the F2 and F3 offspring. Further exploration showed that PM2.5 exposure during pregnancy caused the morphological abnormality of Sertoli cells, downregulated androgen receptor (AR) and connexin43, upregulated anti-Müllerian hormone (AMH), cytokeratin-18 (CK-18), caspase-3, and cleaved caspase-3, decreased thyroid-stimulating hormone (TSH) and testosterone (T), and increased triiodothyronine (T3) in F1 male mouse offspring. Overall, we hypothesize that PM2.5 exposure during pregnancy mainly negatively impacts spermatogenesis in the F1 offspring. The possible mechanism could be that PM2.5 exposure during pregnancy disrupts endocrine hormone release in the F1 generation, thereby influencing the maturation and proliferation of their Sertoli cells and hindering spermatogenesis. This study for the first time investigates the role of Sertoli cells in the reproductive toxicity of PM2.5 on offspring.

Transgenerational effects of social stress on social behavior, corticosterone, oxytocin, and prolactin in rats

Grandmaternal exercise improves the metabolic health of second-generation offspring generated from F1 females

Obesity is predominant in women of reproductive age. Roux-en-Y gastric bypass (RYGB) is the most common bariatric procedure that is performed in obese women for weight loss and metabolic improvement. However, some studies suggest that this procedure negatively affects offspring. Herein, using Western diet (WD)-obese female rats, we investigated the effects of maternal RYGB on postnatal body development, glucose tolerance, insulin secretion and action in their adult male F1 offspring. Female Wistar rats consumed a Western diet (WD) for 18weeks, before being submitted to RYGB (WD-RYGB) or SHAM (WD-SHAM) operations. After 5weeks, WD-RYGB and WD-SHAM females were mated with control male breeders, and the F1 offspring were identified as: WD-RYGB-F1 and WD-SHAM-F1. The male F1 offspring of WD-RYGB dams exhibited decreased BW, but enhanced total nasoanal length gain. At 120days of age, WD-RYGB-F1 rats displayed normal fasting glycemia and glucose tolerance but demonstrated reduced insulinemia and higher glucose disappearance after insulin stimulus. In addition, these rodents presented insulin resistance in the gastrocnemius muscle and retroperitoneal fat, as judged by lower Akt phosphorylation after insulin administration, but an increase in this protein in the liver. Finally, the islets from WD-RYGB-F1 rats secreted less insulin in response to glucose and displayed increased β-cell area and mass. RYGB in WD dams negatively affected their F1 offspring, leading to catch-up growth, insulin resistance in skeletal muscle and white fat, and β-cell dysfunction. Therefore, our data are the first to demonstrate that the RYGB in female rats may aggravate the metabolic imprinting induced by maternal WD consumption, in their male F1 descendants. However, since we only used male F1 rats, further studies are necessary to demonstrate if such effect may also occur in female F1 offspring from dams that underwent RYGB operation.

Transgenerational effects on intestinal inflammation status in mice perinatally exposed to bisphenol S

Zearalenone affects reproductive functions of male offspring via transgenerational cytotoxicity on spermatogonia in mouse

BPA is a monomer that is used in the manufacture of polycarbonate plastics, epoxy resins and a multitude of consumer products. It easily leaches from the inner lining of tin cans and microwave containers into food during heating, from dental sealant into saliva and into beverages from repeated usage or contact with any acidic/alkaline content in polycarbonate bottles. The detection of BPA in biological fluids like maternal plasma, fetal plasma, placental tissue, amniotic fluid and umbilical cord blood have indicated that it can easily transverse the placental barrier. Perinatal period (gestation day (GD) 12-post natal day (PND) 21) encompasses the critical period of gestation and lactation wherein development of the hypothalamus-pituitary-testicular (HPT) axis occurs, especially development of testes that occurs on GD12. Studies on perinatal exposure of BPA have documented its effect on expression of steroid receptors (SRs) in target organs like the mammary gland and brain respectively, resulting in changes in morphogenesis and disturbances in sexual and mating behavior. However, no information is currently available on the effects of BPA in the male offspring of rats exposed to environmentally relevant doses during the critical perinatal period of development and growth of the reproductive tract. The present study aimed at investigating the effects of BPA exposure during this critical window of development (GD12-PND21) on fertility of F1 offspring and understanding its mechanism. Pregnant Holtzman rats (F0) were gavaged with BPA (1.2 and 2.4 μg/Kg /day ) from GD 12-PND 22. Pregnant female treated similarly with DES served as positive control. ON PND 75, fertility assessment of F1 males was performed by cohabitating them with normal cycling adult females. Copulated females were distributed in two sets, one was allowed to deliver the pups and the other was sacrificed on GD 20. The copulated females were examined for various fertility parameters. After fertility assessment male rats (F1) were sacrificed and serum hormonal profile, epididymal sperm count and motility was determined. Histological examination of testis and staging of seminiferous tubules Immuno histochemistry for steroid receptors and their co regulators was performed in testis and quantitative image analysis was performed to assess their levels of expression. A significant increase in the number of copulated females exhibiting resorption was observed at both dose groups of BPA. The adult F1 male offspring showed significant decrease in LH, FSH T and E2. A significant reduction in epididymal sperm count and motility was observed. At the testicular level majority of the seminiferous tubules exhibited sloughing of germ cells. Immunohistchemical localization studies demonstrated significant increase in ER with no effect on AR where as a significant reduction in the expression of SRC-1 and NCoR with a parallel increase in the expression of p/CIP and GRIP -1 was observed. Perinatal exposure to environmentally relevant dose of BPA affects the male gem line leading to impairment in the fertility of F1 male offspring. (platform)

The fetal life is a critical window of developmental patterning affecting growth trajectories before birth and can influence the risk of reproductive and metabolic disease in adulthood. Maternal obesity and elevated prenatal androgens are two prominent potential exposures which may affect the development of the embryo and its postnatal life. If in utero androgen exposure with or without maternal obesity increase female offspring susceptibility for transgenerational inheritance of reproductive and metabolic disease has not been investigated. Therefore we fed female mice high-fat high sucrose (HFHS) or control diet (CD) diet for 6 weeks prior mating to induce obesity and exposed pregnant female mice to prenatal androgen exposure with dihydrotestosterone (DHT) or vehicle during the embryonic day (E) 16.5-E18.5. At weaning, first, second and third generation (F1, F2, and F3) female offspring from DHT exposed mothers with diet-induced obesity had longer anogenital distance (AGD), a marker of in utero androgen exposure, and disturbed estrous cycle compared with offspring from vehicle treated mothers fed CD. F1 and F3 female offspring from DHT exposed mothers and great-grand mothers with diet-induced obesity weighed more compared to offspring from vehicle-treated obese mothers, and F3 female offspring from great-grand DHT exposed mothers had more fat mass assessed by EchoMRI. Additionally, F1 and F3 female offspring from DHT exposed mothers and great-grand mothers with or without diet-induced obesity displayed a lower respiration exchange ratio and reduced energy expenditure than offspring from vehicle-treated mothers fed CD assessed by the metabolic cages. Finally, mice were superovulated to collect MII oocytes for single-cell RNA sequencing. Our preliminary analysis reveal that genes related to preimplantation embryonic imprinting are differentially expressed in F1, F2, and F3 female offspring from DHT exposed mothers, grandmothers, and great-grandmothers fed CD, and in mice from mothers with diet-induced obesity. These results demonstrate that in utero androgen exposure and diet-induced obesity contribute to transgenerational effects on reproductive and metabolic phenotypes in adult female offspring, involving altered expression of preimplantation imprinting genes in germ cells.

If and how obesity and elevated androgens in women with polycystic ovary syndrome (PCOS) affect their offspring’s psychiatric health is unclear. Using data from Swedish population health registers, we showed that daughters of mothers with PCOS have a 78% increased risk of being diagnosed with anxiety disorders. We next generated a PCOS-like mouse (F0) model induced by androgen exposure during late gestation, with or without diet-induced maternal obesity, and showed that the first generation (F1) female offspring develop anxiety-like behavior, which is transgenerationally transmitted through the female germline into the third generation of female offspring (F3) in the androgenized lineage. In contrast, following the male germline, F3 male offspring (mF3) displayed anxiety-like behavior in the androgenized and the obese lineages. Using a targeted approach to search for molecular targets within the amygdala, we identified five differentially expressed genes involved in anxiety-like behavior in F3 females in the androgenized lineage and eight genes in the obese lineage. In mF3 male offspring, three genes were dysregulated in the obese lineage but none in the androgenized lineage. Finally, we performed in vitro fertilization (IVF) using a PCOS mouse model of continuous androgen exposure. We showed that the IVF generated F1 and F2 offspring in the female germline did not develop anxiety-like behavior, while the F2 male offspring (mF2) in the male germline did. Our findings provide evidence that elevated maternal androgens in PCOS and maternal obesity may underlie the risk of a transgenerational transmission of anxiety disorders in children of women with PCOS.

Background:Exposure to the environmental endocrine disruptor bisphenol A (BPA) is ubiquitous and associated with the increased risk of diabetes and obesity. However, the underlying mechanisms remain unknown. We recently demonstrated that perinatal BPA exposure is associated with higher body fat, impaired glucose tolerance, and reduced insulin secretion in first- (F1) and second-generation (F2) C57BL/6J male mice offspring.Objective:We sought to determine the multigenerational effects of maternal bisphenol A exposure on mouse pancreatic islets.Methods:Cellular and molecular mechanisms underlying these persistent changes were determined in F1 and F2 adult offspring of F0 mothers exposed to two relevant human exposure levels of BPA ( and ).Results:Both doses of BPA significantly impaired insulin secretion in male but not female F1 and F2 offspring. Surprisingly, LowerB and UpperB induced islet inflammation in male F1 offspring that persisted into the next generation. We also observed dose-specific effects of BPA on islets in males. UpperB exposure impaired mitochondrial function, whereas LowerB exposure significantly reduced mass and increased death that persisted in the F2 generation. Transcriptome analyses supported these physiologic findings and there were significant dose-specific changes in the expression of genes regulating inflammation and mitochondrial function. Previously we observed increased expression of the critically important gene, Igf2 in whole F1 embryos. Surprisingly, increased Igf2 expression persisted in the islets of male F1 and F2 offspring and was associated with altered DNA methylation.Conclusion:These findings demonstrate that maternal BPA exposure has dose- and sex-specific effects on pancreatic islets of adult F1 and F2 mice offspring. The transmission of these changes across multiple generations may involve either mitochondrial dysfunction and/or epigenetic modifications. https://doi.org/10.1289/EHP1674

Maternal obesity and diet are critical risk factors for development of obesity and diabetes in both first (F1) and second (F2) generation offspring. Maternal exercise in mice improves F1 metabolic health and prevents the detrimental effects of maternal high-fat-diet (HFD) on F1 offspring. Whether the beneficial effects of maternal exercise can be transmitted to the F2 generation is unknown. Here, C57BL/6 females were fed a chow or HFD and subdivided into cages with (Exercise) or without (Sedentary) running wheels for 2wks before breeding and during ∼3wks gestation. Male F1 offspring were sedentary and chow-fed, and at 8 wks of age were bred with age-matched females from untreated parents. F2 offspring were studied based on grand-maternal treatment: chow sedentary; chow trained; HFD sedentary; HFD trained (n= ∼10 litters/group; 348 total mice). F2 offspring were sedentary and chow-fed and studied up to 52 wks of age. For F2 males, grand-maternal exercise significantly improved glucose tolerance at 24, 36 and 52 wks and decreased insulin concentrations at 52 wks. Grand-maternal exercise also resulted in F2 males with lower body weights, decreased fat mass and increased lean mass at 52wks. Grand-maternal HFD decreased glucose uptake in skeletal muscles and adipose tissue in F2 males, but there was no effect of grand-maternal exercise, suggesting that the mechanism for improved glucose tolerance in F2 males does not involve glucose uptake in these tissues. For female F2, grand-maternal exercise had no effect on insulin concentrations, body weights or tissue glucose uptake. However, grand-maternal exercise significantly improved glucose tolerance in female F2 at 36 and 52 wks, and decreased fat mass and increased lean mass. Maternal exercise has beneficial effects on the metabolic health of adult F2 male and female offspring. These novel findings suggest that maternal exercise can prevent obesity and diabetes in multiple generations and may help reduce the incidence of these diseases worldwide. Disclosure A.B.A. Wagner: None. J. Kusuyama: None. P. Nigro: None. N.S. Makarewicz: None. M.F. Hirshman: Stock/Shareholder; Self; Abbott Laboratories, AbbVie Inc., Amgen, Colgate-Palmolive, Eli Lilly and Company, Medtronic. L.J. Goodyear: None. Funding American Diabetes Association (1-17-PMF-009 to A.B.A.W.); National Institutes of Health (R01DK101043)

Vinclozolin—The lack of a transgenerational effect after oral maternal exposure during organogenesis

This study determined the effects of the paternal dietary ratio of n-6: n-3 polyunsaturated fatty acids (PUFAs) on leptin expression in the offspring and associated gene imprinting in a mouse model. Three- to four-week-old male C57BL/6J mice (F0) were fed an n-3 PUFA-deficient (n-3 D) diet, a diet with normal n-3 PUFA content (n-3 N; n-6: n-3 = 4.3:1), or a diet with a high n-3 PUFA content (n-3 H; n-6: n-3 = 1.5:1) for 8 weeks. Two subsequent generations were generated by mating F0 and F1 male mice with 10-week-old virgin female C57 BL/6J mice, to produce F1 and F2 offspring. Compared to the paternal n-3 D diet, paternal n-3 N and n-3 H diets reduced adipose mRNA expression of leptin (Lep) and its plasma concentrations in juvenile F1 male and female offspring, and adult F1 male and F2 female offspring, with upregulated Lep receptor mRNA expression in the hypothalamus. Meanwhile, paternal n-3 N and n-3 H diets altered the expression of the imprinted genes H19, Igf2, Igf2r, Plagl1, Cdkn1c, Kcnq1ot1, Peg3, and Grb10 in the adipose tissue of juvenile and adult F1 males, with almost no effects on F1 females, while more effects were observed in the adult F2 females than F2 males. Principal component analysis verified that Plagl1, Cdkn1c, and Kcnq1ot1 contributed the most to variation in adipose tissue expression in all offspring. Some of these genes (Plagl1, Cdkn1c, Kcnq1ot1, Peg3, and Grb10) were altered by the paternal n-3 N and n-3 H diets in the F1 and F2 generation testes as well. Furthermore, adipose Lep expression was positively correlated with expressions of H19, Igf2r, Plagl1, and Kcnq1ot1 in juvenile F1 males and females, negatively correlated with the Kcnq1ot1 expression in adult F1 males, and positively correlated with the Plagl1 expression in adult F2 females. These data imply that paternal Plagl1, Cdkn1c, and Kcnq1ot1 might be part of the pathways involved in offspring leptin programming. Therefore, a lower ratio of n-6: n-3 PUFAs, with higher intake of n-3 PUFAs in paternal pre-conception, may help maintain the offspring's optimal leptin pattern in a sex-specific manner through multiple generations, and thereby, be beneficial for the offspring's long-term health.

Polybrominated diphenyl ethers (PBDEs) are endocrine‐disrupting chemicals (EDCs) that are widely used flame retardants added to common household products. In humans, PBDEs are associated with neurodevelopmental deficits including ones associated with social competence and learning. We have previously shown that PBDE‐exposed F1 female progeny display autism‐relevant characteristics such as deficient social novelty preference and social recognition memory (SRM), exaggerated repetitive behavior and deficient social odor discrimination (Kozlova et al 2021). In this study, we show that developmental exposure (via dam GD0‐PND21) to the PBDE mixture, DE‐71 (L‐DE‐71; 0.1 mg/kg/d), reduces oxytocin immunofluorescence density in the paraventricular nucleus of the hypothalamus (PVN) that is critical for social recognition memory in male and female F1 offspring relative to VEH/CON (0 mg/kg/d). One of the well characterized actions of PBDEs is disruption of the hypothalamo‐pituitary‐thyroid axis (HPT). Thyroid hormones (TH) are critical for nervous system development and regulate the transcription of oxytocin. We show for the first time, that L‐DE‐71 alters central thyroid hormone species (T2, rT3, T3, and T4) relative to VEH/CON in F1 offspring but with a different temporal pattern in males (PND30) and females (PND15) as measured using liquid chromatography‐tandem mass spectrometry. This coincides with reduced plasma T4 in PND15 male but not female offspring. Supplementation of pregnant dams with levothyroxine, a synthetic analogue of thyroxine (T4), during gestation and lactation (GD12‐PND21), increased plasma oxytocin in exposed dams. More importantly, supplementation normalized PBDE‐induced deficiency in social recognition memory test, and PVN oxytocin levels in exposed female but not male offspring. These changes are specific to social behavior as were no changes in passive avoidance memory. Interestingly, L‐DE‐71 female but not male F1 also receiving supplementation showed a significant increase in plasma T4. Maternal behavior (retrieval of PND4 pups) was significantly reduced by supplementation with 6‐propyl‐2‐thiouracil, a drug that blocks the production of thyroid hormone by thyroid gland, but not by DE‐71 nor DE‐71 plus levothyroxine supplementation. These results provide novel insight into neuroendocrine disruption of the central oxytocin system by maternal transfer of environmental pollutants and its possible contribution to neurodevelopmental disorders such as autism.