Impact of diet-induced maternal obesity on the reproductive capacity of F1 female offspring and the early development of the second generation.

The aim of this study was to examine the impact of maternal obesity on the reproductive capacity of the female offspring (F1) and on the early development of the second generation (F2). To this end, rats were fed either standard (SD) or cafeteria (CD) diet. CD rats and their offspring were divided into two groups: rats with 18% and ≥25% overweight (CD18 and CD25, respectively) and offspring from CD18 and CD25 rats (OCD18 and OCD25, respectively). Both OCD groups achieved greater weight gain than controls, without changes in the serum levels of glucose, cholesterol or triglycerides. However, they showed increased gonadal cholesterol concentration and fat content compared to controls. Female OCD groups showed a slight prolongation of the estrous cycle and different pattern of changes in the weight gain during pregnancy. The OCD25 group displayed an increased fertility index and pre-implantation losses, and changes in some fetal measurements. Some OCD25 dams gave birth to a larger litter of pups and displayed a lower viability index and lactation rate than controls. OCD25 dams also showed an increase in estradiol and a decrease in testosterone and anti-Müllerian hormone. OCD25 rats showed increased mRNA levels of steroidogenenic enzymes. The offspring from OCD25 females (F2OCD25 offspring) showed early vaginal opening and higher ovulation rate in females, and lower ano-genital distances in males, compared to controls. In conclusion, these results reflect that maternal obesity impacts on the reproductive health of successive generations, probably as a result of epigenetic changes in different systems, including germ cells.

Diet-induced maternal obesity and overnutrition cause a decrease in the sperm quality of the offspring.

The present work aimed to study the changes caused by maternal obesity and overnutrition in both the quality and function of spermatozoa of the offspring. To this end, female rats received either a standard or cafeteria diet from 22 days of age until the weaning of their offspring, and the male offspring from rats fed the standard and cafeteria diet (OSD and OCD respectively) were used. Different endpoints in the offspring, as body weight, weight gain, and glycemia were recorded and the testes were removed at 60 days of age. Different spermatozoa parameters, such as mitochondrial function, functional integrity of the sperm plasma membrane, capacitation, and acrosome status, were evaluated. The OCD group was heavier than the OSD group and exhibited lower testis and epididymal indices. The OCD group also showed a decrease in the ability of the sperm tail to react in the presence of a hypoosmotic solution, deficiency in sperm mitochondrial function, a lower percentage of spermatozoa without acrosome when exposed to a capacitation medium, and a higher number of abnormal metaphases. In addition, compared with OSD, OCD rats had a higher number of TUNEL-positive cells in the histological sections of the testis, and greater presence of reactive oxygen species in the spermatozoa, evaluated by a fluorescent probe. However, the OCD group displayed lower protein levels of cytochrome c and caspase-3 in testis tissue than the control group. These results suggest that maternal obesity and overnutrition program the offspring to develop poor sperm quality and function, which may imply a condition of subfertility.

3-Methylcholanthrene impacts on the female germ cells of rats without causing systemic toxicity.

We have previously shown that daily exposure to the environmental pollutant 3-methylcholanthrene (3MC) alters the ovarian function by affecting follicle growth and ovulation. To extend our findings, the aims of this work were to study the effects of daily and non-daily exposure to 3MC on oocyte morphology and integrity and the meiosis process. To this end, immature female rats were daily (0.1-1.0 mg/kg) and non-daily (0.1 mg/kg, three times a week) exposed to 3MC and/or α-naphthoflavone (αNF) (80 mg/kg) for 19 and 20 days, respectively. The latter was used to study its ability to prevent the 3MC action. Follicular growth was examined by histology, apoptosis by in situ cell death detection, oocyte integrity by morphological parameters and fluorescent dyes, and the meiotic spindle by immunostaining. Compared with controls (C), and in a dose-dependent manner, all 3MC-treated rats showed i) increased presence of apoptotic cells in antral follicles and decreased percentage of healthy oocytes, ii) increased oocyte area, perimeter and perivitelline space and decreased thickness of the zona pellucida, and ii) increased percentage of oocytes with abnormal meiotic spindle. In addition, the non-daily dose of 3MC caused DNA damage in oocytes, but not in blood or bone marrow cells. All 3MC-induced changes were prevented with the co-treatment with αNF. These results suggest that low doses of 3MC severely disrupt the ovarian function and that germ cells seem to be more sensitive to this environmental pollutant than other cells such as peripheral blood and bone marrow cells.

Impact of maternal overweight on the sexual maturity of male offspring in rats.

The aims of the present work were to study the effect of maternal overweight on both the count and quality of sperm of the offspring and to assess whether this maternal condition is able to alter testicular integrity and spermatogenic process. To this end, male offspring from rats fed a standard (OSD) or cafeteria (OCD) diet were used. Body and testis weight, length, preputial separation and ano-genital distance (AGD) were recorded and testes were removed at 60 days of age. In addition, the number of germ, Leydig and Sertoli cells, spermatogenesis and sperm integrity were examined. The OCD rats were divided into two groups: offspring from rats with 25% and≥35% of overweight (OCD25 and OCD35, respectively). Both OCD groups showed higher body and testis weight, higher length, and greater AGD than OSD rats. OCD25 also showed early preputial separation and OCD35 exhibited a high level of testosterone with normal glycemia. Both OCD25 and OCD35 rats had a lower number of spermatozoa and Leydig cells than OSD rats, and OCD35 also exhibited a lower number of spermatogonia and Sertoli cells than OSD rats. In addition, both OCD groups exhibited lower number of sperm cells with normal morphology and sperm motility, and OCD35 showed changes in both the seminiferous epithelium and spermatogenic process. These results suggest that maternal overweight severely affects the reproductive capacity of male offspring, likely leading to a subfertility condition and a premature reduction of the reproductive life span.

Changes in the expression of genes involved in the ovarian function of rats caused by daily exposure to 3-methylcholanthrene and their prevention by α-naphthoflavone.

Daily exposure to low doses of 3-methylcholanthrene (3MC) during the pubertal period in rats disrupts both follicular growth and ovulation. Thus, to provide new insights into the toxicity mechanism of 3MC in the ovary, here we investigated the effect of daily exposure to 3MC on selected ovarian genes, the role of the aryl hydrocarbon receptor (AhR) and the level of epigenetic remodeling of histone post-transcriptional modifications. Immature rats were daily injected with 3MC (0.1 or 1 mg/kg) and mRNA expression of genes involved in different ovarian processes were evaluated. Of the 29 genes studied, 18 were up-regulated, five were down-regulated and six were not altered. To assess whether AhR was involved in these changes, we used the chromatin immunoprecipitation assay. 3MC increased AhR binding to promoter regions of genes involved in Notch signaling (Hes1, Jag1), activation of primordial follicles (Cdk2), cell adhesion (Icam1), stress and tumor progression (Dnajb6), apoptosis (Bax, Caspase-9) and expression of growth and transcription factors (Igf2, Sp1). Studying the trimethylation and acetylation of histone 3 (H3K4me3 and H3K9Ac, respectively) of these genes, we found that 3MC increased H3K4me3 in Cyp1a1, Jag1, Dnajb6, Igf2, Notch2, Adamts1, Bax and Caspase-9, and H3K9Ac in Cyp1a1, Jag1, Cdk2, Dnajb6, Igf2, Icam1, and Sp1. Co-treatment with α-naphthoflavone (αNF), a specific antagonist of AhR, prevented almost every 3MC-induced changes. Despite the low dose used in these experiments, daily exposure to 3MC induced changes in both gene expression and epigenomic remodeling, which may lead to premature ovarian failure.

The systemic and gonadal toxicity of 3-methylcholanthrene is prevented by daily administration of α-naphthoflavone.

In the present study, we investigated the effect of 3-methylcholanthrene (3MC) on sexual maturity and the ability of α-naphthoflavone (αNF) to prevent this action. To this end, immature rats were daily injected intraperitoneally with 3MC (0.1 or 1mg/kg) and/or αNF (80mg/kg). Body weight, vaginal opening and estrous cycle were recorded and ovaries were obtained on the day of estrus. Ovarian weight, ovulation rate (measured by the number of oocytes within oviducts), and follicular development (determined by histology) were studied. No differences were found in body weight, ovarian weight, day of vaginal opening, or the establishment of the estrous cycle among the different groups of rats. However, animals treated with 3MC, at both doses, exhibited a lower number of primordial, primary, preantral and antral follicles than controls. Also, 3MC inhibited the ovulation rate and induced an overexpression of both the Cyp1a1 and Cyp1b1 genes, measured by chromatin immunoprecipitation assay. The daily treatment with αNF alone increased the number of follicles in most of the stages analyzed when compared with controls. Moreover, the αNF treatment prevented completely not only the 3MC-induced decrease in all types of follicles but also the 3MC-induced overexpression of Cyp enzymes and the genetic damage in bone marrow cells and oocytes. These results suggest that (i) daily exposure to 3MC during the pubertal period destroys the follicle reserve and alters the ovulation rate; (ii) the 3MC action seems to be mediated by an aryl hydrocarbon receptor-dependent mechanism; (iii) daily administration of αNF has a clear stimulatory action on the ovarian function; and (iv) αNF may prevent both the systemic and gonadal 3MC-induced toxicity.

Regulation of the ovarian oxidative status by leptin during the ovulatory process in rats.

Leptin exerts both stimulatory and inhibitory effects on the ovulatory process. In this study, we investigated whether these opposite effects involve changes in the oxidative status in response to different levels of leptin. To this end, we performed both in vivo and in vitro assays using ovaries of immature rats primed with gonadotropins to induce ovulation. Superoxide dismutase (SOD) and catalase (CAT) activity, lipid peroxidation, glutathione (GSH) content, and reactive oxygen species (ROS) were studied as oxidative damage-related parameters. The expression of BCL2, BAX, and caspase 3 were measured by western blot as apoptosis-related biomarkers. The acute treatment with leptin, which inhibits ovulation, decreased SOD activity and increased active caspase 3 expression. No differences were found in CAT activity, lipid peroxidation, or total GSH. In contrast, the daily administration of leptin, which induces ovulation, decreased GSH content, ROS levels, and Bax and active caspase 3 expression, but caused no changes in other parameters. In addition, the daily administration of leptin induced follicular growth, measured by the number of antral follicles in ovarian sections. Using ovarian explant cultures, we found increased BCL2 expression and decreased SOD activity at low and high concentrations of leptin respectively. Thus, leptin can modulate the oxidative status of the ovarian tissue, during the ovulatory process, by acting on different targets according to its circulating levels. At low concentration, leptin seems to play a protective role against the oxidative stress, whereas at high concentrations, this protein seems to be involved in cell death.