Neonatal overfeeding alters the functioning of the mesolimbic dopaminergic circuitry involving changes in DNA methylation and effects on feeding behavior.

Mesolimbic dopaminergic circuit is essential for food reward and motivational behaviors and can contribute to weight gain and obesity. Litter reduction is a classical model for studying the effects of neonatal overfeeding and overweight. Litters of Wistar rats were reduced to 4 pups/dam for small litter (SL) and 10 pups/dam for normal litter at postnatal day (PND) 4. Immediately after performing the feeding behavior tests, the animals were sacrificed in PND21 and PND90. The ventral tegmental area (VTA), Nucleus Accumbens Core (NAcC) and Shell (NAcSh) were isolated from frozen brain sections using the Palkovits micropunch technique. RNA and DNA were extracted from these areas, gene expression was measured by RT-qPCR and DNA methylation levels were measured by MSRM-qPCR technique. SL-PND21 animals presented increased expression levels of Tyrosine Hydroxylase and Dopamine Receptor D2 in VTA, decreased expression levels of dopamine active transporter (DAT) in VTA, and higher expression levels of DAT in NAcC. On the other hand, SL-PND90 animals showed decreased expression levels of Dopamine Receptor D1 and higher expression of DAT in NAcSh. These animals also evidenced impaired sensory-specific satiety. In addition, altered promoter methylation was observed at weaning, and remained in adulthood. This work demonstrates that neonatal overfeeding induces disruptions in the mesolimbic dopaminergic circuitry and causes alterations in feeding behavior from weaning to adulthood, suggesting that the neonatal period is critical for the normal development of dopaminergic circuit that impact on feeding behavior.

Disruption of the developmental programming of the gonad of the broad snouted caiman (Caiman latirostris) after in ovo exposure to atrazine.

Environmental exposure to agrochemicals during early stages of development can induce subtle alterations that could permanently affect normal physiology. Previously, we reported that in ovo exposure to atrazine (ATZ) disrupts testicular histoarchitecture in postnatal caimans (Caiman latirostris). To assess whether such alterations are the result of disruption of gonadal developmental programming, this study aimed to evaluate the expression of histofunctional biomarkers (VASA, ER, PR, PCNA, and aromatase) and genes involved in gonadal development and differentiation (amh, sox-9, sf-1 and cyp19-a1) in the gonads of male and female caiman embryos and to assess the effect of ATZ exposure on these biomarkers and genes in the gonads of male embryos. Our results suggest that amh, aromatase and sox-9 play a role in sex determination and gonadal differentiation. In male caiman embryos, ATZ exposure increased aromatase expression and altered the temporal expression pattern of amh and sox-9 evidencing an ATZ-induced disruption of gonadal developmental programming. Since the effects of ATZ are consistent across all vertebrate classes, the ATZ-mediated disruptive effects here observed could be present in other vertebrate species.

Epigenetic Changes Associated With Exposure to Glyphosate-Based Herbicides in Mammals.

Glyphosate is a phosphonomethyl amino acid derivative present in a number of non-selective and systemic herbicides. During the last years the use of glyphosate-based herbicide (GBH) has been increasing exponentially around the world, including Argentina. This fact added to the detection of glyphosate, and its main metabolite, amino methylphosphonic acid (AMPA), in environmental matrices such as soil, sediments, and food, has generated great concern about its risks for humans, animals, and environment. During the last years, there were controversy and intense debate regarding the toxicological effects of these compounds associated with the endocrine system, cancer, reproduction, and development. The mechanisms of action of GBH and their metabolites are still under investigation, although recent findings have shown that they could comprise epigenetic modifications. These are reversible mechanisms linked to tissue-specific silencing of gene expression, genomic imprinting, and tumor growth. Particularly, glyphosate, GBH, and AMPA have been reported to produce changes in global DNA methylation, methylation of specific genes, histone modification, and differential expression of non-coding RNAs in human cells and rodents. Importantly, the epigenome could be heritable and could lead to disease long after the exposure has ended. This mini-review summarizes the epigenetic changes produced by glyphosate, GBHs, and AMPA in humans and rodents and proposes it as a potential mechanism of action through which these chemical compounds could alter body functions.

Temporary effects of neonatal overfeeding on homeostatic control of food intake involve alterations in POMC promoter methylation in male rats.

A small litter (SL) model was used to determine how neonatal overfeeding affects the homeostatic control of food intake in male rats at weaning and postnatal day (PND) 90. At PND4, litters were reduced to small (4 pups/dam) or normal (10 pups/dam) litters. At weaning, SL rats showed higher body weight and characteristic features of the metabolic syndrome. Gene expression of pro-opiomelanocortin (POMC), cocaine and amphetamine regulated transcript, neuropeptide Y (NPY) and leptin and ghrelin (GHSR) receptors were increased and POMC promoter was hypomethylated in arcuate nucleus, indicating that the early development of obesity may involve the GHSR/NPY system and changes in POMC methylation state. At PND90, body weight, metabolic parameters and gene expression were restored; however, POMC methylation state remained altered. This work provides insight into the effects of neonatal overfeeding, showing the importance of developmental plasticity in restoring early changes in central pathways involved in metabolic programming.

Cafeteria diet induces progressive changes in hypothalamic mechanisms involved in food intake control at different feeding periods in female rats.

We studied the effects of cafeteria diet (CAF) intake from weaning on mRNA levels and DNA methylation state of feeding-related neuropeptides and hormone receptors in individual hypothalamic nuclei at different feeding periods. Four weeks of CAF (short-term) increased energy intake and adiposity, without affecting neuropeptides' expression. Eleven weeks of CAF (medium-term) increased energy intake, adiposity, leptinemia, and body weight, with an orexigenic response of the lateral hypothalamus, paraventricular and ventromedial nuclei, given by upregulation of Orexins, AgRP, and NPY opposed by an anorectic signal of the arcuate nucleus, which displayed a higher POMC expression. The changes in neuropeptidic mRNA levels were related to epigenetic modifications in their promoter regions. Metabolic and molecular changes were intensified after 20 weeks of diet (long-term). The alterations in these hypothalamic brain nuclei could add information about their differential role in food intake control, and how their action is disrupted during the development of obesity.

Temperature- vs. estrogen-induced sex determination in Caiman latirostris embryos: Both females, but with different expression patterns of key molecules involved in ovarian development.

Caiman latirostris is a species with temperature dependent sex determination (TSD), which implies that the incubation temperature of the eggs is the main factor that determines the sex during a thermo-sensitive period (TSP). However, estrogens play a critical role in this process. The administration of 17ß-estradiol (E2) previous to TSP overrides the effects of male incubation temperature, producing phenotypic females. This effect has been defined as sex reversal or estrogen-induced sex determination (E2SD). The aim of the present study is to describe similarities and differences in the effects of TSD and E2SD treatment conditions on ovary development. Our results show that the two treatment conditions studied are able to produce different ovaries. Treatment with E2 modified the expression pattern of estrogen receptor alpha and progesterone receptor, and expression of the enzyme aromatase. Moreover, in E2SD females, the proliferation/apoptosis dynamic was also altered and high expression of TAp63 was observed suggesting the presence of greater DNA damage in germ cells. To the best of our knowledge, this is the first report that describes the morphology of the female gonad of C. latirostris in three stages of embryonic development and shows the expression of TAp63 during the gonad development of a reptile. It is important to emphasize that the changes demonstrated in E2SD female gonads of embryos show that environmental compounds with proven estrogenic activity alter the follicular dynamics of C. latirostris in neonatal as much as in juvenile animals, endangering their reproductive health and possibly bringing consequences to ecology and evolution.

Bisphenol A disrupts the temporal pattern of histofunctional changes in the female reproductive tract of Caiman latirostris.

Recently, we have described the ontogeny of histofunctional differentiation changes in the oviduct of Caiman latirostris. The expression of estrogen receptor alpha and progesterone receptor shows that the caiman oviduct could be a target of the action of xenoestrogens such as the widely environmentally present Bisphenol A (BPA), early in life. The aims of this study were: to complement oviduct characterization by establishing the ontogenetic changes in androgen receptor (AR) expression and assessing the effects of early postnatal exposure to 17-ß-estradiol (E2) or BPA on the histofunctional features of the oviduct. AR was expressed in all the stages studied. The spatial pattern of AR immunostaining changed from neonatal to juvenile caimans. In the luminal epithelium, changes were at the subcellular level, from cytoplasmic to nuclear. In the subepithelium, although both cytoplasmic and nuclear AR expression was observed, changes were mainly at tissue level, from the subepithelial compartment to the outer muscular layer. The oviduct was highly sensitive to E2 and BPA at the early postnatal developmental stage. E2- and BPA-exposed caimans showed increased luminal epithelial height and higher proliferative activity. Changes in histomorphological features (measured by a scoring system), steroid hormone receptors, collagen remodeling and muscle-associated proteins suggest a precocious oviduct histofunctional differentiation in E2- and BPA-exposed caimans. The modification of the temporal pattern of oviductal biomarkers suggests that organizational changes could impair C. latirostris reproductive health later in life. The alterations in the caiman female reproductive tract exposed to BPA highlight the importance of preserving aquatic environments from plastic pollution.

Dietary whey reduces energy intake and alters hypothalamic gene expression in obese phyto-oestrogen-deprived male rats.

Removing dietary phyto-oestrogens in adult male rats causes obesity and diabetes. As whey proteins have been reported to reduce food intake and improve glucose homoeostasis, we investigated whether they could attenuate susceptibility to obesity and diabetes due to phyto-oestrogen deprivation. To this end, thirty male Wistar rats were fed a high-phyto-oestrogen (HP) or a phyto-oestrogen-free (PF) diet for 10 weeks; six rats from each group were killed. The remaining HP animals (six animals) continued receiving the HP diet for 6 weeks. The remaining PF rats (twelve rats) were divided in two groups: one was given the PF diet and the other a variation of the PF diet plus whey protein (PF-W). Body weight, food intake and adipose tissue weights were recorded. Hypothalamic mRNA expressions of orexigenic (neuropeptide Y, agouti-related protein (AgRP)) and anorexigenic (pro-opiomelanocortin (POMC), cocaine-amphetamine-related transcript (CART)) neuropeptides were quantified by real-time PCR. Serum glucose, insulin and total thyroxine (T4), thyroid-stimulating hormone, testosterone and oestradiol were assessed. After 10 weeks of PF diet, increased body weight, adiposity and energy intake, with up-regulation of AgRP and down-regulation of POMC', were observed. Longer treatment exacerbated these results, increased total T4 levels, reduced oestradiol levels and impaired glucose homoeostasis. PF-W reduced energy intake and increased POMC expression; however, body weight and adiposity remained unchanged. PF-W could not prevent the hormonal changes or the high circulating glucose levels induced by phyto-oestrogen deprivation, but reduced fasting insulin. These data demonstrate that, although 6 weeks of whey administration could not prevent obesity in phyto-oestrogen-deprived rats, the reduction in energy intake and circulating insulin could be beneficial with longer treatments.

Histomorphological changes in testes of broad-snouted caimans (Caiman latirostris) associated with in ovo exposure to endocrine-disrupting chemicals.

Studies regarding the effects of endocrine-disrupting chemicals (EDCs) on the reproductive functions of wild animals have raised increasing concern. Thus, here we evaluated the consequences of in ovo exposure to endosulfan (END) and bisphenol A (BPA) in testes from neonatal to juvenile (Juv) caimans (Caiman latirostris). Caiman eggs were collected from areas with low to moderate anthropogenic intervention and incubated at male-producing temperature. At stage 20 of embryonic development (previous to gonad sex determination), eggs were exposed to either END (20 ppm) or BPA (1.4 ppm) and male gonad histomorphology examined in 10-day-old, 90-day-old, and Juv caimans. The relative seminiferous tubular area (RTA) was measured in testes and the proliferation index and the expression of estrogen receptor alpha (ERα) were quantified in intratubular cells. Regardless of the treatment, all eggs resulted in male hatchlings. The testes of EDC-exposed caimans presented tortuous seminiferous tubules with empty tubular lumens. The RTA of 10-day-old caimans exposed to BPA was decreased. The percentage of cells expressing ERα was not different after in ovo treatment with EDCs (compared to the Control group), although caimans exposed to END showed a different ERα distribution pattern. The proliferation index was lower in 90-day-old caimans exposed to END, and higher in Juv caimans exposed to BPA. In ovo exposure to END or BPA modified sensitive parameters of C. latirostris male gonads. The alterations described here might compromise not only the sexual maturation but also the reproductive performance of adult caimans. J. Exp. Zool. 325A:84-96, 2016. © 2015 Wiley Periodicals, Inc.