Fernando Larrea

A low maternal protein diet during pregnancy and lactation has sex- and window of exposure-specific effects on offspring growth and food intake, glucose metabolism and serum leptin in the rat

Extensive epidemiological and experimental evidence indicates that a sub‐optimal environment during fetal and neonatal development in both humans and animals may programme offspring susceptibility to later development of chronic diseases including obesity and diabetes that are the result of altered carbohydrate metabolism. We determined the effects of protein restriction during pregnancy and/or lactation on growth, serum leptin, and glucose and insulin responses to a glucose tolerance test in male and female offspring at 110 days postnatal life. We fed Wistar rats a normal control 20% casein diet (C) or a restricted diet (R) of 10% casein during pregnancy. Female but not male R pups weighed less than C at birth. After delivery, mothers received the C or R diet during lactation to provide four offspring groups: CC (first letter maternal pregnancy diet and second maternal lactation diet), RR, CR and RC. All offspring were fed ad libitum with C diet after weaning. Relative food intake correlated inversely with weight. Offspring serum leptin correlated with body weight and relative, but not absolute, food intake in both male and female pups. Serum leptin was reduced in RR female pups compared with CC and increased in RC males compared with CC at 110 days of age. Offspring underwent a glucose tolerance test (GTT) at 110 days postnatal life. Female RR and CR offspring showed a lower insulin to glucose ratio than CC. At 110 days of age male RR and CR also showed some evidence of increased insulin sensitivity. Male but not female RC offspring showed evidence of insulin resistance compared with CC. Cholesterol was similar and triglycerides (TG) higher in male compared with female CC. Cholesterol and TG were higher in males than females in RR, CR and RC (P < 0.05). Cholesterol and TG did not differ between groups in females. Cholesterol and TG were elevated in RC compared with CC males. Nutrient restriction in lactation increased relative whole protein and decreased whole lipid in both males and females. RC females showed decreased relative levels of protein and increased fat. We conclude that maternal protein restriction during either pregnancy and/or lactation alters postnatal growth, appetitive behaviour, leptin physiology, TG and cholesterol concentrations and modifies glucose metabolism and insulin resistance in a sex‐ and time window of exposure‐specific manner.

Sex differences in transgenerational alterations of growth and metabolism in progeny (F2) of female offspring (F1) of rats fed a low protein diet during pregnancy and lactation

Compelling epidemiological and experimental evidence indicates that a suboptimal environment during fetal and neonatal development in both humans and animals may programme offspring susceptibility to later development of several chronic diseases including obesity and diabetes in which altered carbohydrate metabolism plays a central role. One of the most interesting and significant features of developmental programming is the evidence from several studies that the adverse consequences of altered intrauterine environments can be passed transgenerationally from mother (F0) to daughter (F1) to second generation offspring (F2). We determined whether when F0 female rats are exposed to protein restriction during pregnancy and/or lactation their F1 female pups deliver F2 offspring with in vivo evidence of altered glucose and insulin metabolism. We fed F0 virgin Wistar rats a normal control 20% casein diet (C) or a protein restricted isocaloric diet (R) containing 10% casein during pregnancy. F1 female R pups weighed less than C at birth. After delivery, mothers received C or R diet during lactation to provide four F1 offspring groups CC (first letter pregnancy diet and second lactation diet), RR, CR and RC. All F1 female offspring were fed ad libitum with C diet after weaning and during their first pregnancy and lactation. As they grew female offspring (F1) of RR and CR mothers exhibited low body weight and food intake with increased sensitivity to insulin during a glucose tolerance test at 110 days of postnatal life. Male F2 CR offspring showed evidence of insulin resistance. In contrast RC F2 females showed evidence of insulin resistance. Sex differences were also observed in F2 offspring in resting glucose and insulin and insulin: glucose ratios. These sex differences also showed differences specific to stage of development time window. We conclude that maternal protein restriction adversely affects glucose and insulin metabolism of male and female F2 offspring in a manner specific to sex and developmental time window during their mothers (the F1) fetal and neonatal development.

On the mechanisms of action of short-term levonorgestrel administration in emergency contraception

The effects of short-term administration of levonorgestrel (LNG) at different stages of the ovarian cycle on the pituitary-ovarian axis, corpus luteum function, and endometrium were investigated. Forty-five surgically sterilized women were studied during two menstrual cycles. In the second cycle, each women received two doses of 0.75 mg LNG taken 12 h apart on day 10 of the cycle (Group A), at the time of serum luteinizing hormone (LH) surge (Group B), 48 h after positive detection of urinary LH (Group C), or late follicular phase (Group D). In both cycles, transvaginal ultrasound and serum LH were performed from the detection of urinary LH until ovulation. Serum estradiol (E2) and progesterone (P(4)) were measured during the complete luteal phase. In addition, an endometrial biopsy was taken at day LH + 9. Eighty percent of participants in Group A were anovulatory, the remaining (three participants) presented significant shortness of the luteal phase with notably lower luteal P4 serum concentrations. In Groups B and C, no significant differences on either cycle length or luteal P4 and E2 serum concentrations were observed between the untreated and treated cycles. Participants in Group D had normal cycle length but significantly lower luteal P4 serum concentrations. Endometrial histology was normal in all ovulatory-treated cycles. It is suggested that interference of LNG with the mechanisms initiating the LH preovulatory surge depends on the stage of follicle development. Thus, anovulation results from disrupting the normal development and/or the hormonal activity of the growing follicle only when LNG is given preovulatory. In addition, peri- and post-ovulatory administration of LNG did not impair corpus luteum function or endometrial morphology.

Mechanism of action of hormonal preparations used for emergency contraception: a review of the literature

This paper focuses on the research efforts undertaken to understand how emergency contraception (EC) methods act to prevent pregnancy and to identify what is known and what are the important gaps that need to be addressed. Divided into five sections the first section presents a discussion on the background of the review and a brief description of the mode of use efficacy and most common side effects of the Yuzpe regimen levonorgestrel (LNG) and mifepristone. Section 2 includes studies on the effects of postcoital steroid administration upon fertility in nonprimate animal models. Section 3 focuses on the effects of estrogens progestins or the antiprogestin mifepristone administered in the preovulatory period to macaques and the New World monkey Cebus apella. Section 4 highlights clinical studies on the effects of the Yuzpe regimen administered before and after the luteinizing hormone surge and on progesterone-regulated endometrial proteins. Finally section 5 identifies some of the most difficult areas of the literature that need to be researched.

A maternal low protein diet during pregnancy and lactation in the rat impairs male reproductive development

Nutrient restriction during pregnancy and lactation impairs growth and development. Recent studies demonstrate long‐term programming of function of specific organ systems resulting from suboptimal environments during fetal life and development up to weaning. We determined effects of maternal protein restriction (50% control protein intake) during fetal development and/or lactation in rats on the reproductive system of male progeny. Rats were fed either a control 20% casein diet (C) or a restricted diet (R) of 10% casein during pregnancy. After delivery mothers received either C or R diet until weaning to provide four groups: CC, RR, CR and RC. We report findings in male offspring only. Maternal protein restriction increased maternal serum corticosterone, oestradiol and testosterone (T) concentrations at 19 days gestation. Pup birth weight was unchanged but ano‐genital distance was increased by maternal protein restriction (P < 0.05). Testicular descent was delayed 4.4 days in RR, 2.1 days in CR and 2.2 days in RC and was not related to body weight. Body weight and testis weight were reduced in RR and CR groups at all ages with the exception of CR testis weight at 270 days postnatal age (PN). At 70 days PN luteinizing hormone and T concentrations were reduced in RR, CR and RC. mRNA for P450 side chain cleavage (P450scc) was reduced in RR and CR at 21 days PN but was unchanged at 70 days PN. Fertility rate was reduced at 270 days PN in RC and sperm count in RR and RC. We conclude that maternal protein delays sexual maturation in male rats and that some effects only emerge in later life.

Protein restriction during fetal and neonatal development in the rat alters reproductive function and accelerates reproductive ageing in female progeny

Recent studies demonstrate long‐term programming of function of specific organ systems resulting from suboptimal environments during fetal life and development up to weaning. Nutrient restriction during pregnancy and lactation impairs overall fetal growth and development. We determined the effects of maternal protein restriction (MPR; 50% normal protein intake) during fetal development and/or lactation in rats on the function and ageing of the reproductive system of female progeny. Rats were fed either a control 20% casein diet (C) or a restricted diet (R) of 10% casein during pregnancy. After delivery mothers received either C or R diet until weaning to provide four groups, CC, RR, CR and RC. We report data on female offspring only. After weaning pups were fed the C diet. MPR increased maternal progesterone, corticosterone, oestradiol and testosterone concentrations at 19 days gestation. Reproductive and somatic phenotype was altered as pup birth weight was decreased, and ano‐genital distance was increased by MPR. Pup corticosterone was decreased at 2 days postnatal (PN) life. Vaginal opening and timing of the first oestrus were delayed in RR and CR and these differences were not related to body weight. At 21 days PN oestradiol in RR and CR and progesterone in RR were reduced; at 70 days PN luteinizing hormone (LH) in all restricted groups was reduced in dioestrus while follicle stimulating hormone (FSH) was unchanged. Cycle length increased between 140 days and 1 year in RR and CR but remained unchanged in CC, providing evidence of premature ageing of reproductive function. Fertility rates declined over the same period in the three experimental groups but not CC. MPR in one of the two experimental periods, either pregnancy or lactation, resulted in decreased pup survival compared with CC and RR. These data show that MPR results in delayed sexual maturation and premature ageing of reproductive function.

Estradiol and progesterone synthesis in human placenta is stimulated by calcitriol

Calcitriol exerts a diverse range of biological actions including the control of growth and cell differentiation, modulation of hormone secretion, and regulation of reproductive function. The placenta synthesizes calcitriol through the expression of CYP27B1, but little is known about local actions of this hormone in the fetoplacental unit. The objective of this study was to investigate the effects of calcitriol upon progesterone (P(4)) and estradiol (E(2)) secretion in trophoblasts cultured from term human placenta. Cells were incubated in the presence of calcitriol for 18 h and pregnenolone or androstenedione were subsequently added as substrates for the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) or P450-aromatase (CYP19), respectively. Calcitriol stimulated in a dose-dependent manner E(2) and P(4) secretion. The use of a selective inhibitor of PKA prevented the effects of calcitriol upon E(2) secretion, but not on P(4). These results show that calcitriol is a physiological regulator of placental E(2) and P(4) production and suggest a novel role for calcitriol upon placental steroidogenesis.

Calcitriol inhibits TNF-α-induced inflammatory cytokines in human trophoblasts

Elevated placental proinflammatory cytokine release is associated with miscarriage, preterm labor and preeclampsia. Specifically, tumor necrosis factor-alpha (TNF-alpha)-induced cytokines may threaten pregnancy outcome. Since trophoblasts produce calcitriol, a hormone with strong immunosuppressive properties, we assessed the effects of this secosteroid on inflammatory cytokines induced in trophoblasts by challenge with TNF-alpha. The effects of calcitriol on synthesis of mRNAs encoding interleukin-6 (IL-6), interferon-gamma (IFN-gamma), and TNF-alpha were measured by real time RT-PCR. Secreted cytokines were quantified by ELISA. The effects of TNF-alpha on CYP24A1, chorionic gonadotropin (hCG), 3beta-hydroxysteroid dehydrogenase (HSD3B1) and P(450)-aromatase (CYP19) mRNA expression were also studied. TNF-alpha stimulated IL-6, IFN-gamma and its own expression more than 3-fold over controls (P<0.05). Calcitriol inhibited the expression profile of inflammatory cytokine genes in a dose-response manner (P<0.05). This effect was prevented by addition of the vitamin D receptor antagonist TEI-9647. TNF-alpha also significantly inhibited expression of hCG, HSD3B1 and CYP19 genes, and stimulated CYP24A1 gene expression. These data show that calcitriol prevents TNF-alpha induction of inflammatory cytokines through a process likely to be mediated by the vitamin D receptor. We conclude that TNF-alpha inhibits placental hormone synthesis and stimulates calcitriol catabolism by regulating enzymes involved in these processes.

Calcitriol affects hCG gene transcription in cultured human syncytiotrophoblasts

BackgroundIn pregnancy, maternal serum concentrations of calcitriol significantly rise as a result of increased renal and placental contribution in order to assure calcium supply for the developing fetus. Considering that placenta is a site for vitamin D activation, and the versatility and potency of calcitriol, it is feasible that this hormone participates in fetal/placental development and physiology. In the present work we studied calcitriol actions upon human chorionic gonadotropin (hCG) secretion and expression in cultured trophoblasts, as well as vitamin D receptor (VDR) and CYP27B1 immunolocalization in placental villi.MethodsQuantification of hCG in culture media was performed by immunoassay. Expression studies were carried out by real time PCR. Analysis of CYP27B1 and VDR localization in placental slides were performed by immunohistochemistry. Statistical significance was established by one way ANOVA using Tukey test for comparisons.ResultsCalcitriol regulated hCG in a time-dependent manner: at 6 h the secosteroid stimulated hCG, whereas longer incubations (24 h) showed opposite effects. Interestingly, calcitriol stimulatory effects on hCG were accompanied by an increase in intracellular cAMP content and were abolished by pre-incubation of the cells with a selective protein kinase A inhibitor. Immunohistochemical techniques showed differential VDR localization in the syncytiotrophoblast layer or in the vascular smooth muscle cells depending on the epitope to which the antibodies were raised (specific for the carboxy- or amino-terminal regions, respectively). CYP27B1 was immunolocalized in the syncytiotrophoblast layer of placental villi.ConclusionThe presence and location of the vitamin D activating enzyme CYP27B1 as well as the specific receptor for vitamin D were shown in placental sections. The latter, together with findings demonstrating specific effects of calcitriol acting through the VDR and the cAMP/PKA signaling pathway upon hCG expression and secretion, indicate that there is a functional vitamin D endocrine system in the placenta, and recognize calcitriol as an autocrine regulator of hCG.

In vivo and in vitro estrogen bioactivities of alkyl parabens.

The alkyl esters of p-hydroxybenzoic acid known as parabens (Pbens) are used as preservatives in food, pharmaceutical and cosmetic formulations. They have been reported as estrogenic. Here, we present evidence for the in vivo and in vitro bioactivities and receptor binding affinities of methylparaben (MePben), ethylparaben (EtPben), propylparaben (PrPben), and butylparaben (BuPben) compared with those of estradiol (E2). Estrogenicity was studied using the uterotrophic assay in immature (Im) and adult ovariectomized (Ovx) CD1 mice, and in immature female Wistar rats (IW). Animals were subcutaneously (sc) treated for three consecutive days with different molar equivalent doses ranging from 3.62 to 1086 mmol/kg body weight of Pbens, E2 (0.036 mmol/kg), or vehicle. Pbens increased uterine weight in Im and Ovx animals and their relative uterotrophic effect to E2 (100) (RUEE2) were from 34 to 91. The relative uterotrophic potencies related to E2 (100) (RUPE2) of these compounds were from 0.003 to 0.007. The E2 ED50 for CD1 animals able to increase the uterine weight was 7 mg/kg (0.9 -55 confidence limits); and that of Pbens ranged from 18 to 74 mg/kg. In IW rats, the ED50 were from 33 to 338 mg/kg. All Pbens, except MePb, competed with [3H]E2 for the estrogen receptor binding sites. The uterotrophic effects of Pbens in Im mice have a positive correlation with the side-chain length of the ester group of these compounds. The E2 and Pbens relative binding affinities (RBA) and Ki values correlated to their estrogenic activity. The NOELs values for Pbens uterotrophic activity in Im were from 0.6 to 6.5 mg/kg per day; and Ovx from 6 to 55 mg/kg. The NOELs IW ranged from 16.5 to 70 mg/kg indicating that Im were more susceptible than Ovx and IW to these effects. The data shown here confirm the estrogenicity of Pbens.