Perinatal protein malnutrition alters maternal behavior and leads to maladaptive stress response, neurodevelopmental delay and disruption on DNA methylation machinery in female mice offspring.

Deficiencies in maternal nutrition have long-term consequences affecting brain development of the progeny and its behavior. In the present work, female mice were exposed to a normal-protein or a low-protein diet during gestation and lactation. We analyzed behavioral and molecular consequences of malnutrition in dams and how it affects female offspring at weaning. We have observed that a low-protein diet during pregnancy and lactation leads to anxiety-like behavior and anhedonia in dams. Protein malnutrition during the perinatal period delays physical and neurological development of female pups. Glucocorticoid levels increased in the plasma of malnourished female offspring but not in dams when compared to the control group. Interestingly, the expression of glucocorticoid receptor (GR) was reduced in hippocampus and amygdala on both malnourished dams and female pups. In addition, malnourished pups exhibited a significant increase in the expression of Dnmt3b, Gadd45b, and Fkbp5 and a reduction in Bdnf VI variant mRNA in hippocampus. In contrast, a reduction on Dnmt3b has been observed on the amygdala of weaned mice. No changes have been observed on global methylation levels (5-methylcytosine) in hippocampal genomic DNA neither in dams nor female offspring. In conclusion, deregulated behaviors observed in malnourished dams might be mediated by a low expression of GR in brain regions associated with emotive behaviors. Additionally, low-protein diet differentially deregulates the expression of genes involved in DNA methylation/demethylation machinery in female offspring but not in dams, providing an insight into regional- and age-specific mechanisms due to protein malnutrition.

Role of the hypothalamus in ghrelin effects on reproduction: sperm function and sexual behavior in male mice.

In brief: Ghrelin signals to the hypothalamus inhibit reproduction during times of food scarcity. In this study, we demonstrate that ghrelin impairs sperm quality in male mice. Abstract: Ghrelin (GHRL) is an orexigenic peptide that has been investigated as one of the signals responsible for the reproductive performance of mammals under fluctuating metabolic conditions. Central GHRL administration impairs spermatogenesis in mice by regulating the hypothalamic-pituitary-gonadal axis function. In the present study, the hypothalamus role as a mediator of GHRL effects on sperm fertilizing capacity and male sexual behavior was evaluated. After 42 days of hypothalamic GHRL infusion or artificial cerebrospinal fluid, in vitro and in vivo sperm fertilizing capacity, testicular α-tubulin, speriolin gene expression and spermatic α-tubulin protein were evaluated. Hypothalamic expression of genes Kiss1, Gpr54 and Gnrh was also studied. The second group of animals was infused with one time only GHRL or artificial cerebrospinal fluid into the hypothalamus to evaluate the effects on sexual behavior. Results demonstrated that chronic GHRL administration to male mice significantly increased the percentages of pre-implantation embryo loss and the number of post-implantation embryo loss. In relation to the gene expression, our results show a relative decrease of Kiss1, Gpr54 and Spatc1. Although no significant differences were observed in the quantitative expression of α-tubulin protein, qualitative changes in its expression pattern were observed. In addition, a dual effect on sexual behavior was observed: 40% of the treated animals showed a significant reduction in the number of mounts and intromissions, while a 60% showed a significant decrease in ejaculation latency vs control animals. In conclusion, our results provide evidence that central GHRL administration possibly induces failure in embryo development and/or implantation in the females mated with treated males, possibly because of a negative effect in the α-tubulin pattern.

Comparative Stress Response of Black and Gold Howler Monkey (Alouatta caraya) in Urban and Rural Environments of Northern Argentina.

The expansion of human populations associated with urbanization results in extensive modification of natural habitats. While many species cannot persist in these highly modified environments, some species adopt new strategies, which contribute to their survival. Several primate species have persisted in altered habitats, including members of the genus Alouatta. To improve our understanding of this interplay, we compared glucocorticoid (GCC) levels in male and female black and gold howler monkeys (Alouatta caraya) in urban and rural areas in northeastern Argentina. Fecal samples (n = 60) were collected from adults and hormone extracts were analyzed by enzyme immunoassays (EIA). Monkeys living in urban areas (females: 109.15 ± 18.83; males: 106.15 ± 10.48) had lower GCClevels than monkeys living in rural areas (females: 152.01 ± 19.50; males:139,82 ± 10.85). Interestingly, males living in urban areas had lower GCC levels compared to those living in rural areas, whereas no differences were observed in GCC levels between females living in urban and rural areas. While these results suggest that urban areas may provide a release from intergroup competition for male howler monkeys, future work is needed to better understand the dynamics of this association to best inform management and conservation of this vulnerable species.

Validation of an enzyme immunoassay and comparison of fecal cortisol metabolite levels in black and gold howler monkeys (Alouatta caraya) inhabiting fragmented and continuous areas of the humid Chaco region, Argentina.

In the last years, the study of how environmental stimuli influence the physiology and specifically the endocrinology of an organism became increasingly important, relying mainly on the quantification of glucocorticoids to monitor animal welfare. Most studies investigating cortisol levels in primates were focused on the impact of social stressors; however, a major concern for the conservation of howler monkeys is the increased habitat fragmentation led by the advancement of the agricultural frontier. We compared fecal cortisol metabolite levels (FGCM) in howler monkeys (Alouatta caraya) living in fragmented and continuous forests of the Argentine humid Chaco region, throughout the warm season (spring-summer). Fecal samples (n = 114) were collected from adult individuals, and steroid extracts analyzed with an enzyme immunoassay also validated in this work. Parallel displacement curves were obtained between dilutions of pooled fecal extracts and the cortisol standard curve (r2 = 0.99; P = 0.23). Efficiency of the fecal extraction procedure was 79.4% ± 38%; recovery of exogenous hormone added to fecal extracts indicated a low interference of components in the feces with antibody binding. The exogenous administration of ACTH in captive-bred animals demonstrated a "cause-and-effect" relationship between the adrenal gland activation and increased FGCM levels. Contrary to our initial prediction, we were not able to demonstrate a significant difference in FGCM levels of caraya monkeys inhabiting the continuous versus fragmented habitats in our study site (83.2 ± 4.9 ng/g [n = 10 individuals] vs. 71.5 ± 4.9 ng/g [n = 7 individuals]; P = 0.29); this could be the result of low levels of disturbance imposed by a moderate and selective logging, which has proved to be beneficial for this species with high resilience by adjusting their diet to cope with feeding in degraded habitats but with new leaves and buds. Regardless of the habitat, cortisol metabolites were significantly higher in females than in males (86.4 ± 4.2 ng/g [n = 12 individuals] vs. 60.7 ± 5.0 ng/g [n = 5 individuals] respectively; P = 0.007). RESEARCH HIGHLIGHTS: Validation of an enzyme immunoassay and comparison of fecal cortisol metabolite levels in black and gold howler monkeys (Alouatta caraya) inhabiting fragmented and continuous areas of the humid Chaco region, Argentina. Contrary to our initial prediction, no significant differences in Alouatta caraya fecal cortisol metabolite levels were detected; cortisol metabolites were significantly higher in females. Probably, animals adjusted their diet to cope with feeding in degraded habitats, but with new leaves and buds.

Non-invasive endocrine monitoring of ovarian and adrenal activity in chinchilla (Chinchilla lanigera) females during pregnancy, parturition and early post-partum period.

The chinchilla is a rodent that bears one of the finest and most valuable pelts in the world. The wild counterpart is, however, almost extinct because of a drastic past and ongoing population decline. The present work was developed to increase our knowledge of the reproductive physiology of pregnancy and post-partum estrus in the chinchilla, characterizing the endocrine patterns of urinary progesterone, estradiol, LH and cortisol metabolites throughout gestation and post-partum estrus and estimating the ovulation timing at post-partum estrus. Longitudinal urine samples were collected once per week throughout pregnancy and analyzed for creatinine, cortisol, LH, estrogen and progesterone metabolite concentrations. To indirectly determine the ovulation timing at post-partum estrus, a second experiment was performed using pregnant females subjected to a post-partum in vivo fertilization scheme. Urinary progestagen metabolites increased above baseline levels in early pregnancy between weeks-8 and -11 respectively to parturition, and slightly declined at parturition time. Urinary estrogens showed rising levels throughout mid- and late pregnancy (weeks-9 to -6 and a further increase at week-5 to parturition) and decreased in a stepwise manner after parturition, returning to baseline levels two weeks thereafter. Cortisol metabolite levels were relatively constant throughout pregnancy with a tendency for higher levels in the last third of gestation and after the pups' birth. Parturition was associated with dramatic reductions in urinary concentrations of sex steroids (especially progestagens). Observations in breeding farms indicated that the females that resulted in a second pregnancy after mating, did so on the second day after parturition. These data were in agreement with an LH peak detected 24h after parturition. Urinary steroid hormone patterns of estrogen and progestagen metabolites provided valuable information on endocrine events during pregnancy and after parturition in the chinchilla. Results presented in this study enhance our understanding of natural reproductive dynamics in the chinchilla and support empirical observations of breeders that post-partum ovulation occurs ∼ 48 h after parturition.

Environmentally relevant DEHP exposure during gestational and lactational period inhibits filamin a testicular expression.

Toxicological studies have revealed that DEHP exposure during pregnancy may induce developmental disorders, especially in male offspring, leading to morphological and functional alterations in the reproductive system by mechanisms that should be investigated. Thus, the aim of this work was to analyze the testicular toxicity induced by an environmentally relevant DEHP dose during development and its impact on FLNA, a protein that participates in the blood-testis barrier assembly. We used male Wistar rats exposed to DEHP during pregnancy and lactation. The results showed that DEHP exposure during development and lactation increased body weight, decreased gonadal weight and shortened anogenital distance. This phthalate induced morphological changes in the testis, suggestive of hypospermatogenesis. DEHP exposure decreased the number of FLNA positive cells and the expression of FLNA and claudin-1 in prepubertal testes. Furthermore, DEHP inhibited FLNA and claudin-1 protein expression in adult male rats. These results indicated that exposure to DEHP during gestation and lactation perturbed testis development and suggested that FLNA is a target protein of DEHP, possibly contributing to the phthalate-induced damage on BTB.

Ghrelin misbalance affects mice embryo implantation and pregnancy success by uterine immune dysregulation and nitrosative stress.

Introduction: In a previous study we found that ghrelin (Ghrl) misbalance during the peri-implantation period significantly impaired fetus development. In this study we aimed to evaluate the putative mechanisms underlying these effects, including embryo implantation success, uterine nitric oxide synthase (NOS) activity, nitric oxide synthesis and the inflammatory/immune uterine profile. Methods: Ghrelin misbalance was induced by injecting 4nmol/animal/day of Ghrl (hyperghrelinemia) or 6nmol/animal/day of a Ghrl antagonist (Ant: (D-Lys3)GHRP-6) from day 3 to 8 of pregnancy. Control animals (C) were injected with de vehicle. Females were euthanized at pregnancy day 8 and their uteri excised in order to evaluate: the percentage of reabsorbed embryos (microscopically), eNOS, iNOS and nytrotirosine expression (by immunohistochemistry), nitrite synthesis (by Griess technique), VEGF, IL-10, IL-17, IL-6, MMP9 and GM-CSF expression (by qPCR) and leukocyte infiltration by flow cytometry (evaluating T cells, NK cells, granulocytes, dendritic cells and macrophages). Results: Ant-treatment significantly increased the percentage of reabsorbed embryos and the uterine expression of eNOS, iNOS and nytrotirosine. (D-Lys3)GHRP-6-treatment increased also the expression of the inflammatory cytokines IL-6, IL-17 and MMP9, and decreased that of IL-10 (anti-inflammatory). Moreover, Ant-treatment increased also the NK cells population and that of CD11b+ dendritic cells; and decreased T cells percentages. Similarly, hyperghrelinemia showed a significant increase vs. C on eNOS, iNOS and nytrotirosineuterine expression and a decrease in T cells percentages. Conclusion: Ghrl misbalance during the peri-implantation period induces pro-inflammatory changes and nitrosative stress in the gravid uterus, impairing significantly embryo implantation and/or development.

Fetal Programming Effects of a Mild Food Restriction During Pregnancy in Mice: How Does It Compare to Intragestational Ghrelin Administration?

To explore in mice if a 15% food restriction protocol during pregnancy programs the offspring postnatal development, with emphasis on reproductive function, and to assess if ghrelin (Ghrl) administration to mouse dams exerts effects that mimic those obtained under mild caloric restriction. Mice were 15% food-restricted, injected with 4 nmol/animal/day of Ghrl, or injected with the vehicle (control) thorough pregnancy. After birth, the pups did not receive further treatment. Pups born from food-restricted dams (FR pups) were lighter than Ghrl pups at birth, but reached normal weight at adulthood. Ghrl pups were heavier at birth and gained more weight than control pups (C pups). This effect was not associated with plasma IGF-1. FR pups showed a delay in pinna detachment and eye opening, while an advance was observed in Ghrl pups. FR pups showed also impairment in the surface-righting reflex. In both female FR and Ghrl pups, there was an advance in vaginal opening and, in adulthood, FR pups showed a significant decrease in their own litter size and plasma progesterone, and an increase in embryo loss. A delay in testicular descent was evident in male Ghrl pups. Changes in puberty onset were not associated with differences in the expression of Kiss1 in hypothalamic nuclei. Finally, in adulthood, FR pups showed a significant decrease in sperm quality. In conclusion, a mild food restriction thorough gestation exerted programming effects on the offspring, affecting also their reproductive function in adulthood. These effects were not similar to those of intragestational Ghrl administration.

Modulatory effects of ghrelin on sperm quality alterations induced by a fructose-enriched diet.

The objectives of this study were: 1) to evaluate the effects of a fructose enriched diet (FED) on rat sperm quality, epididymal function (i.e. oxidative stress and alpha-glucosidase expression) and testosterone concentrations; 2) to determine if the administration of ghrelin (Ghrl), reverses the effects induced by FED. After validating the protocol as an inductor of metabolic syndrome like-symptoms, adult male rats were assigned to one of the following treatments for 8 weeks: FED = 10% fructose enriched in water (v/v); FED + Ghrl = fructose enriched diet plus Ghrl (6 nmol/animal/day, s.c.) from week 6-8; or C = water without fructose (n = 5-10 animals/group). FED significantly decreased sperm concentration and motile sperm count/ml vs C (FED: 19.0 ± 1.6 × 106sperm/ml and 834.6 ± 137.0, respectively vs C: 25.8 ± 2.8 × 106 and 1300.4 ± 202.4, respectively; p < 0.05); ghrelin injection reversed this negative effect (23.5 ± 1.6 × 106sperm/ml and 1381.7 ± 71.3 respectively). FED resulted in hypogonadism, but Ghrl could not normalize testosterone concentrations (C: 1.4 ± 0.1 ng/ml vs FED: 0.8 ± 0.2 ng/ml and FED + Ghrl: 0.6 ± 0.2 ng/ml; p < 0.05). Ghrelin did not reverse metabolic abnormalities secondary to FED. FED did not alter epididymal expression of antioxidants enzymes (superoxido-dismutase, catalase and glutathione peroxidases -Gpx-). Nevertheless, FED + Ghrl significantly increased the expression of Gpx3 (FED + Ghrl: 3.47 ± 0.48 vs FED: 0.69 ± 0.28 and C: 1.00 ± 0.14; p < 0.05). The expression of neutral alpha-glucosidase, which is a marker of epididymal function, did not differ between treatments. In conclusion, the administration of Ghrl modulated the negative effects of FED on sperm quality, possibly by an epididymal increase in Gpx3 expression. However, Ghrl could not neither normalize the metabolism of FED animals, nor reverse hypogonadism.

Reproductive performance and weaning success in fur-chewing chinchillas (Chinchilla lanigera).

In captive chinchillas, one of the most challenging behavioral problems is the development of a stress-related abnormal repetitive behavior (ARB) known as "fur-chewing". We investigated whether there is a relationship between the severity of fur-chewing behavior and reproductive function in male and female chinchillas. Regardless of the severity of abnormal behavior, fur-chewing males did not show significant differences in seminal quality (sperm concentration, motility and viability; integrity of sperm membrane and acrosome) and the response to the process of semen collection (the number of stimuli needed to achieve ejaculation) when compared to those with normal behavior. Also, females showing normal or fur-chewing behavior presented similar reproductive performance in terms of number of litters per female per year and litter size. However, pup survival rate was lower (p=0.05) in fur-chewing females than in normal females. These results seem to be consistent with data suggesting non-significant effects of ARBs on reproductive performance.