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 disruption of placental genes by chronic maternal cafeteria diet in rats.

Maternal diet has impact on reproduction, fetal development and offspring behavior, although molecular mechanisms remained unknown. Our aims were to assess (1) the effects of a cafeteria (CAF) diet (western diet habits) on female reproductive performance, fetal and placental parameters on gestational day 21 and litter size and pup weight at birth; and (2) placental messenger RNA (mRNA) expression and epigenetic regulation of Insulin-Like Growth Factor (Igf) and Vascular Endothelial Growth Factor (Vegf) and their receptors. Female Wistar rats were fed with control or CAF diet from weaning until parturition. At week 14 after diets started, females were mated and half of the animals were euthanized on gestational day 21 to evaluate reproductive parameters including the pregnancy rate, number of corpora lutea, implantation sites and resorption sites. Moreover, fetal weight and length, placental weight, and placental index were recorded. Placentas were collected for mRNA quantification and DNA methylation analysis. The remaining animals were allowed to give birth and the number and weight of the pups were evaluated. CAF diet did not affect reproductive performance or fetal weight and length. However, CAF-fed animals showed a decrease in placental weight and index and the pups exhibited a low birth weight. Additionally, we found an upregulation of Igf2 and a down regulation of Vegf placental mRNA expression in CAF dams, associated with methylation status changes of their promoters. We conclude that female chronic CAF diet consumption impairs feto-placental development and could be explained by an epigenetic disruption of Igf and Vegf systems.

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.

Epigenetic regulation of steroidogenic enzymes expressed in peripheral blood mononuclear cells from healthy individuals and from patients with chronic lymphocytic leukemia.

Sex hormone synthesis occurs in various organs and tissues besides the gonads, such as adrenal glands, brain, intestines, skin, fat, bone, and cells of the immune system. Regarding the latter, it is still not clear which pathways are active, and if they are modified in case of illness of the immune system. Our goal in this study was to determine mRNA expression of different steroidogenic enzymes in peripheral blood mononuclear cells (PBMCs) from healthy individuals of both sexes and of different ages, and then to compare their expression between healthy individuals and patients with Chronic Lymphocytic Leukemia (CLL). Furthermore, to elucidate possible mechanisms that regulate enzyme expression, we analyzed epigenetic events like promoter methylation. We determined that normal cells of the immune system, regardless of sex and age, expressed P450 side chain cleavage (P450scc), cytochrome P450 17α-hydroxylase/c17,20-lyase (P45017α), 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3ß-HSD), steroid 5 α reductase (5α-R) types 1, 2 and 3, 3α-hydroxysteroid dehydrogenase (3α-HSD) type 3, and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) types 1, 3 and 5. We also established that 5α-R 1, 5α-R 3, 3α-HSD 3, 17ß-HSD 1 and 17ß-HSD 5 expression was altered in CLL patients, and that promoter regions of 5α-R 1, 17ß-HSD 1 and 17ß-HSD 5 were diferentially methylated. These results suggest that steroidogenic pathways may be affected in CLL cells, and this could be related to disease pathogenesis.

Agrochemicals and neurogenesis.

Agrochemicals or pesticides are compounds widely used to prevent, destroy or mitigate pests such as insects, rodents, herbs and weeds. However, most of them also act as environmental estrogens, anti-estrogens and/or antiandrogenic chemicals. In addition, both herbicides (such as glyphosate and paraquat) and insecticides (such as pyrethroids, organophosphates, neonicotinoids and rotenone) have been shown to exert significant adverse effects on hippocampal neurogenesis. These effects are particularly important because neurogenesis dysregulation could be associated with cognitive decline and neuropathologies such as Alzheimer's disease. This review focuses on the most commonly used agrochemicals in Argentina and their effects on the hippocampal neurogenesis of mammals. It also discusses the disruption of hormone synthesis and action as a possible mechanism through which these chemical compounds could alter the brain functions. Finally, we propose some lines of research to study the potential endocrine mechanisms involved in the effects of agrochemicals on human health and biodiversity.

Perinatal exposure to bisphenol A (BPA) impairs neuroendocrine mechanisms regulating food intake and kisspetin system in adult male rats. Evidences of metabolic disruptor hypothesis.

Bisphenol A (BPA) is a compound used in the polymerization of plastic polycarbonates. It is an endocrine disruptor and it has been postulated to be an obesogen. Our objective was to determine the influence of perinatal exposure to BPA on body weight, hormone levels, metabolic parameters and hypothalamic signals that regulate food intake and kisspeptin system in adult male rats. Male rats were exposed to 50 µg/kg/day of BPA or vehicle from day 9 of gestation to weaning in the drinking water. Since weaning, they were fed with control or high fat diet for 20 weeks. Perinatal exposure to BPA impaired glucose homeostasis, induced obesity and increased food intake in adult male rats altering hypothalamic signals, partially mimicking and/or producing an exacerbation of the effects of feeding fat diet. We also observed an increase in kisspeptin expression by BPA exposure. Evidences shown in this work support the metabolic disruptor hypothesis for BPA.

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.

Sex- and age-associated differences in episodic-like memory and transcriptional regulation of hippocampal steroidogenic enzymes in rats.

The aim of this study was to evaluate the episodic-like memory (ELM) and the transcriptional regulation of the enzymes involved in hippocampal allopregnanolone synthesis in young adult and middle-aged male and female rats. Young adult males, but not middle-aged ones, showed a good performance in the ELM task. In contrast, neither young nor middle-aged females were able to discriminate the spatial order in which the objects were presented. In females, aging decreased the transcription of steroidogenic-related genes. In addition, the mRNA levels of 5α-reductase-1 were higher and the methylation of its promoter was lower in young adult females than in males, suggesting an epigenetic control. Further studies are needed to establish correlations between ELM and the transcriptional regulation of hippocampal steroidogenic enzymes. Our results contribute to the knowledge of sex differences in gene expression, methylation and memory during aging.

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.

Cafeteria diet differentially alters the expression of feeding-related genes through DNA methylation mechanisms in individual hypothalamic nuclei.

We evaluated the effect of cafeteria diet (CAF) on the mRNA levels and DNA methylation state of feeding-related neuropeptides, and neurosteroidogenic enzymes in discrete hypothalamic nuclei. Besides, the expression of steroid hormone receptors was analyzed. Female rats fed with CAF from weaning increased their energy intake, body weight, and fat depots, but did not develop metabolic syndrome. The increase in energy intake was related to an orexigenic signal of paraventricular (PVN) and ventromedial (VMN) nuclei, given principally by upregulation of AgRP and NPY. This was mildly counteracted by the arcuate nucleus, with decreased AgRP expression and increased POMC and kisspeptin expression. CAF altered the transcription of neurosteroidogenic enzymes in PVN and VMN, and epigenetic mechanisms associated with differential promoter methylation were involved. The changes observed in the hypothalamic nuclei studied could add information about their differential role in food intake control and how their action is disrupted in obesity.

Dietary withdrawal of phytoestrogens resulted in higher gene expression of 3-beta-HSD and ARO but lower 5-alpha-R-1 in male rats.

Removing dietary phytoestrogens causes obesity and diabetes in adult male rats. Based on the facts that hypothalamic food intake control is disrupted in phytoestrogen-deprived animals and that several steroids affect food intake, we hypothesized that phytoestrogen withdrawal alters the expression of hypothalamic steroidogenic enzymes. Male Wistar rats fed with a high-phytoestrogen diet from conception to adulthood were subjected to phytoestrogen withdrawal by feeding them a low-phytoestrogen diet or a high-phytoestrogen, high-fat diet. Withdrawal of dietary phytoestrogens increased 3ß-hydroxysteroid dehydrogenase and P450 aromatase gene expression and decreased those of 5α-reductase-1. This is a direct effect of the lack of dietary phytoestrogens and not a consequence of obesity, as it was not observed in high-fat-fed rats. Phytoestrogen withdrawal and high-fat diet intake reduced hypothalamic expression of estrogen receptor (ER)α correlated with low levels of ERα-O, ERα-OS, and ERα-OT transcripts. Variations in gene expression of steroidogenic enzymes may affect the content of neurosteroids. As neurosteroids are related to food intake control, the changes observed may be a novel mechanism in the regulation of energy balance in obese phytoestrogen-deprived animals. In rats, steroidogenesis and ER signaling appear to be altered by phytoestrogen withdrawal in the rat. The ubiquity of phytoestrogens in the diet and changing intakes or withdrawal suggest that aspects of human health could be affected based on the rat and warrant further research.

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.

Withdrawal of dietary phytoestrogens in adult male rats affects hypothalamic regulation of food intake, induces obesity and alters glucose metabolism.

The absence of phytoestrogens in the diet during pregnancy has been reported to result in obesity later in adulthood. We investigated whether phytoestrogen withdrawal in adult life could alter the hypothalamic signals that regulate food intake and affect body weight and glucose homeostasis. Male Wistar rats fed from conception to adulthood with a high phytoestrogen diet were submitted to phytoestrogen withdrawal by feeding a low phytoestrogen diet, or a high phytoestrogen-high fat diet. Withdrawal of dietary phytoestrogens increased body weight, adiposity and energy intake through an orexigenic hypothalamic response characterized by upregulation of AGRP and downregulation of POMC. This was associated with elevated leptin and T4, reduced TSH, testosterone and estradiol, and diminished hypothalamic ERα expression, concomitant with alterations in glucose tolerance. Removing dietary phytoestrogens caused manifestations of obesity and diabetes that were more pronounced than those induced by the high phytoestrogen-high fat diet intake.

Prenatal exposure to pesticides disrupts testicular histoarchitecture and alters testosterone levels in male Caiman latirostris.

The increased use of agrochemical pesticides, such as atrazine (ATZ) and endosulfan (END), may have a significant impact on ecosystem health and biodiversity. The aim of this study was to investigate the consequences of in ovum exposure to ATZ and END on Caiman latirostris gonadal histo-functional features. Caiman eggs were collected from environmentally pristine areas and incubated in controlled conditions at male producing temperature (33 degrees C). At stage 20 of embryonic development, the sensitive stage for gonadal sex determination, eggs were exposed to one dose of either END or ATZ. Gonadal histo-morphology was examined in caiman hatchlings and serum levels of testosterone were measured. Regardless of treatment condition, all eggs incubated at 33 degrees C resulted in male hatchlings. Tortuous seminiferous tubules with increased perimeter, disrupted distribution of peritubular myoid cells (desmin positive), and emptied tubular lumens characterized the testes of pesticide-exposed caiman. An imbalance between proliferative activity and cell death was observed in the testes of caiman exposed to the higher doses of END, mainly due to a high frequency of apoptosis in intratubular cells. This altered cell turnover was associated with decreased testosterone levels. Prenatal exposure to only one dose of END and ATZ disrupted neonatal male gonadal histo-functional features. Alterations described here could have detrimental effects on the sexual maturation of the caiman and, ultimately, on the success of male caiman reproduction.

Developmental exposure to endocrine disruptor chemicals alters follicular dynamics and steroid levels in Caiman latirostris.

Human and wildlife are exposed at critical periods of development to endocrine disruptor chemicals (EDC) that may be responsible for reproductive disorders. To test the hypothesis that in ovum exposure to EDC at a critical period for gonadal organogenesis alters post-hatching folliculogenesis and steroidogenesis in Caiman latirostris, we studied the impact of in ovum exposure to 17 beta-estradiol (E2), bisphenol A (BPA), endosulfan (END) and atrazine (ATZ) on gonadal differentiation, follicular dynamics and circulating levels of steroid hormones in neonatal and juvenile caiman. Since C. latirostris is a species with temperature dependent sex determination, eggs were incubated at male (33 degrees C) or female (30 degrees C) producing temperatures and the effect of EDC was evaluated. Neonatal ovaries exhibited germ cells mainly located in clusters evidencing proliferative activity and type I to III follicles. Juvenile ovaries exhibited germ cells and advanced stages of pre-vitellogenic follicles. Prenatal exposure to the highest doses of E2 (1.4 ppm) or BPA (140 ppm) overrode male temperature effect on sex determination. Neonatal females produced by sex reversion lacked type III follicles, while females prenatally exposed to the lowest doses of E2 (0.014 ppm) and BPA (1.4 ppm) or ATZ (0.2 ppm) showed an increase in type III follicles. Juvenile caiman prenatally exposed to E2 or BPA showed an augmented incidence of multioocyte follicles. Neonatal female caiman exposed in ovum to E2 or BPA had higher estrogen serum levels whereas exposure to E2, BPA, ATZ and END decreased T levels. Present data demonstrates that exposure to EDC during gonadal organogenesis alters follicular dynamics and steroid levels later in life. These effects might have an impact on caiman fertility.

Organochlorine levels in adipose tissue of women from a littoral region of Argentina.

Organochlorine compounds (OCCs), such as pesticides and polychlorinated biphenyls (PCBs), are persistent lipophilic chemicals identified as endocrine disruptors, mainly with estrogen-like effects. Northeastern Argentina, near the Paraná River, is characterized by intensive farming and agricultural activities and industrial development, and is therefore prone to high incidences of environmental and dietary exposure to OCCs. Hence, we conducted a study to (1) estimate the organochlorine residues present in mammary fat tissue in a population of women from this littoral region and (2) identify potential sources of exposure to OCCs. Our subjects were 76 women (residing in and around Santa Fe city and not occupationally exposed to organochlorines) who underwent excision biopsy of a breast lesion or had plastic surgery. Both frequency of occurrence and levels of organochlorine residues were high in breast adipose tissue of all the participants. The organochlorine residues most frequently found were p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) in all the subjects analyzed, hexachlorobenzene (HCB) in 86.8%, and beta-hexachlorocyclohexane (beta-HCH) in 75.0%. The incidence of PCB congeners was very low. p,p'-DDE and beta-HCH residues reached the highest levels, 4794 and 1780 ppb, respectively. The diet was a relevant source of exposure, consumption of animal fat and freshwater fish playing a significant role. Bioaccumulation was evidenced by the significant positive association between organochlorine levels and body mass index (p = 0.0003) and the age of the patient (p = 0.0002). The frequency and levels of OCCs found in our study population raise concerns regarding Argentinean exposure to these endocrine disruptors.

Vitellogenin detection in Caiman latirostris (Crocodylia: Alligatoridae): a tool to assess environmental estrogen exposure in wildlife.

Environmental pollution with endocrine disrupting compounds (EDCs) has adverse effects on the ecosystem's health. Caiman latirostris are widely distributed in South American aquatic ecosystems. Caimans have physiological and ecological characteristics that make them particularly vulnerable to EDCs exposure and suitable candidate as a sentinel species. Vitellogenin (Vtg) is a yolk pre-cursor protein synthesized by the liver of non-mammalian vertebrates and induced in response to estrogen. Purified plasma Vtg from caimans injected with estradiol-17beta (E2) was used to generate a polyclonal anti-body. Anti-body specificity was assessed using Western blot. The antiserum was also effective in detecting turtle Vtg, exhibiting high cross-reactivity with Vtg from Phrynops hilarii and Trachemys scripta dorbigni. We developed a specific and highly sensitive ELISA for caiman Vtg. This method has a detection limit of 0.1 ng/ml of plasma. The ELISA did not detect Vtg in plasma of non-induced male caimans. Induction of Vtg in male caimans was evaluated in response to one or two (7 days apart) doses of E2. Due to its high sensitivity, ELISA allows to measure the small increases in plasma Vtg after exposure to exogenous estrogen. A priming effect was observed following the second E2 dose, with a tenfold increase in circulating Vtg. Hepatic synthesis was confirmed by immunohistochemistry. The results presented herein suggest that detection of plasma Vtg in male caimans might become a valuable tool in biomonitoring xenoestrogen exposure in a polluted environment.