The anti-estrogenic activity of indole-3-carbinol in neonatal rat osteoblasts is associated with the estrogen receptor antagonist 2-hydroxyestradiol.

PURPOSE: To gain new insight into the roles of cruciferous vegetable-derived bioactive phytochemicals in bone cells, we investigated the effects of indole-3-carbinol (I3C) on cell proliferation and differentiation in estradiol (E2)-exposed calvarial osteoblasts that were obtained from neonatal rats. METHODS: Osteoblast activity was assessed by analyzing cellular DNA, cell-associated osteocalcin (OC) levels and alkaline phosphatase (AP) activity. We also examined [(3)H]-estrone (E1) metabolism and estrogen-agonistic and estrogen-antagonistic activities of 2-hydroxy (OH) E1 and 2-OHE2 and their capacity to displace [(3)H]-E2 at ER binding sites using competition studies. RESULTS: I3C did not affect on cellular DNA, OC levels or AP activity. However, I3C completely inhibited E2-induced increases in cell proliferation and differentiation in neonatal rat osteoblasts. Metabolic studies demonstrated that I3C promoted the conversion of [(3)H]-E1 to 2-OHE1 and 2-OHE2 and those higher rates of conversion (twofold-threefold) were archived when a higher dose of I3C was applied. Proliferation and differentiation studies showed that 2-OHE2 but not 2-OHE1 inhibited E2-induced increases in cell proliferation and differentiation via an ER-mediated mechanism. Likewise, Esr1 was expressed at high level than Esr2. 2-OHE1 showed no activity or affinity for ER. CONCLUSIONS: This study is the first to show that a bioactive compound derived from cruciferous vegetables, I3C, abolishes the E2-mediated stimulation of cell activities including, proliferation and differentiation, in rat osteoblasts and increases the 2-hydroxylation of E1, resulting in the formation of inactive and anti-estrogenic metabolites. These results suggest that in neonatal rat osteoblasts, the anti-estrogenic effect of I3C is mediated by 2-OHE2 through ER-α.

A cis-acting element in the promoter of human ether à go-go 1 potassium channel gene mediates repression by calcitriol in human cervical cancer cells.

The human ether à go-go 1 potassium channel (hEAG1) is required for cell cycle progression and proliferation of cancer cells. Inhibitors of hEAG1 activity and expression represent potential therapeutic drugs in cancer. Previously, we have shown that hEAG1 expression is downregulated by calcitriol in a variety of cancer cells. Herein, we provided evidence on the regulatory mechanism involved in such repressive effect in cells derived from human cervical cancer. Our results indicate that repression by calcitriol occurs at the transcriptional level and involves a functional negative vitamin D response element (nVDRE) E-box type in the hEAG1 promoter. The described mechanism in this work implies that a protein complex formed by the vitamin D receptor-interacting repressor, the vitamin D receptor, the retinoid X receptor, and the Williams syndrome transcription factor interact with the nVDRE in the hEAG1 promoter in the absence of ligand. Interestingly, all of these transcription factors except the vitamin D receptor-interacting repressor are displaced from hEAG1 promoter in the presence of calcitriol. Our results provide novel mechanistic insights into calcitriol mode of action in repressing hEAG1 gene expression.

Exercise in obese female rats has beneficial effects on maternal and male and female offspring metabolism.

BACKGROUND: Maternal obesity (MO) impairs maternal and offspring health. Mechanisms and interventions to prevent adverse maternal and offspring outcomes need to be determined. Human studies are confounded by socio-economic status providing the rationale for controlled animal data on effects of maternal exercise (MEx) intervention on maternal (F0) and offspring (F1) outcomes in MO. HYPOTHESIS: MO produces metabolic and endocrine dysfunction, increases maternal and offspring glucocorticoid exposure, oxidative stress and adverse offspring outcomes by postnatal day (PND) 36. MEx in part prevents these outcomes. METHODS: F0 female rats ate either control or obesogenic diet from weaning through lactation. Half of each group wheel ran (from day 90 of life through pregnancy beginning day 120) providing four groups (n=8/group)--(i) controls, (ii) obese, (iii) exercised controls and (iv) exercised obese. After weaning, PND 21, F1 offspring ate a control diet. Metabolic parameters of F0 prepregnancy and end of lactation and F1 offspring at PND 36 were analyzed. RESULTS: Exercise did not change maternal weight. Before breeding, MO elevated F0 glucose, insulin, triglycerides, cholesterol, leptin, fat and oxidative stress. Exercise completely prevented the triglyceride rise and partially increases glucose, insulin, cholesterol and oxidative stress. MO decreased fertility, recovered by exercise. At the end of lactation, exercise returned all metabolic variables except leptin to control levels. Exercise partially prevented MO elevated corticosterone. F1 offspring weights were similar at birth. At PND 36, MO increased F1 male but not female offspring leptin, triglycerides and fat mass. In controls, exercise reduced male and female offspring glucose, prevented the offspring leptin increase and partially the triglyceride rise. CONCLUSIONS: MEx before and during pregnancy has beneficial effects on the maternal and offspring metabolism and endocrine function occurring with no weight change in mothers and offspring indicating the importance of body composition rather than weight in evaluations of metabolic status.

Human fertilization: epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3.

Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein-protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.

Maternal obesity in the rat programs male offspring exploratory, learning and motivation behavior: prevention by dietary intervention pre-gestation or in gestation.

We studied the effects of maternal high fat diet (HFD, 25% calories from fat administered before and during pregnancy and lactation) and dietary intervention (switching dams from HFD to control diet) at different periconceptional periods on male offspring anxiety related behavior, exploration, learning, and motivation. From weaning at postnatal day (PND) 21, female subjects produced to be the mothers in the study received either control diet (CTR - 5% calories from fat), HFD through pregnancy and lactation (MO), HFD during PNDs 21-90 followed by CTR diet (pre-gestation (PG) intervention) or HFD from PND 21 to 120 followed by CTR diet (gestation and lactation (G) intervention) and bred at PND 120. At 19 days of gestation maternal serum corticosterone was increased in MO and the PG and G dams showed partial recovery with intermediate levels. In offspring, no effects were found in the elevated plus maze test. In the open field test, MO and G offspring showed increase zone entries, displaying less thigmotaxis; PG offspring showed partial recuperation of this behavior. During initial operant conditioning MO, PG and G offspring displayed decreased approach behavior with subsequent learning impairment during the acquisition of FR-1 and FR-5 operant conditioning for sucrose reinforcement. Motivation during the progressive ratio test increased in MO offspring; PG and G intervention recuperated this behavior. We conclude that dietary intervention can reverse negative effects of maternal HFD and offspring outcomes are potentially due to elevated maternal corticosterone.

Maternal protein restriction in pregnancy and/or lactation affects seminiferous tubule organization in male rat offspring.

Maternal protein restriction (MPR) during pregnancy impaired the reproduction of male offspring. We investigated, during the first wave of spermatogenesis, whether MPR exerts deleterious effects on germ cell proliferation and differentiation, as well as androgen receptor (AR) protein expression, which was used as a marker for Sertoli cell (SC) maturation. At the beginning of pregnancy (day 0), dams were fed a control diet (C: 20% casein) or a restricted isocaloric diet (R: 10% casein). After birth, four groups were established: CC, RR, CR and RC (first letter diet during pregnancy and second during lactation). Male offspring were studied at postnatal days 14, 21 and 36. At birth, pup body weight was unchanged. Body weight and testis weight were reduced in RR and CR groups at all ages evaluated. MPR delayed the germinal epithelium development at all ages evaluated. On performing Western blot and immunohistochemistry, AR expression was found to be lower in the three restricted groups. The results suggest that MPR during pregnancy and/or lactation delays SC maturation and germ cell differentiation, and affects intratubular organization. These changes might be responsible for the lower fertility rate at older ages.

Prevalence and characterisation of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in healthy volunteers in Tunisia.

The objective of this investigation was to analyse the carriage rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faecal samples of healthy humans in Tunisia and to characterise the recovered isolates. One hundred and fifty samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 µg/ml) for ESBL-positive E. coli recovery. The characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, multilocus sequence typing (MLST) and phylogroup typing were performed by polymerase chain reaction (PCR) and sequencing. The presence and characterisation of integrons and virulence factors were studied by PCR and sequencing. ESBL-positive E. coli isolates were detected in 11 of 150 faecal samples (7.3%) and one isolate/sample was further characterised. These isolates contained the blaCTX-M-1 (ten isolates) and blaTEM-52c genes (one isolate). The ISEcp1 (truncated by IS10 in four strains) and orf477 sequences were found upstream and downstream, respectively, of all bla (CTX-M-1) genes. Seven different sequence types (STs) and three phylogroups were identified among CTX-M-1-producing isolates [ST/phylogroup (number of isolates)]: ST58/B1 (3), ST57/D (2), ST165/A (1), ST155/B1 (1), ST10/A (1), ST398/A (1) and ST48/B1 (1). The TEM-52-producing isolate was typed as ST219 and phylogroup B2. Six ESBL isolates contained class 1 integrons with the gene cassettes dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one). Healthy humans in the studied country could be a reservoir of CTX-M-1-producing E. coli.

Maternal protein restriction in the rat during pregnancy and/or lactation alters cognitive and anxiety behaviors of female offspring.

Maternal protein deficiencies can developmentally program offspring to lifelong dysfunction of many physiological systems. We hypothesized that maternal isocaloric low protein diet during fetal and early postnatal development would negatively affect female offspring anxiety, exploration, associative learning and motivation as measured by the elevated plus maze (EPM), open field test (OFT), operant conditioning and the progressive ratio task, respectively. Control mothers (C) received a 20% casein diet and restricted mothers (R) a 10% casein diet to provide four groups: CC, RR, CR, and RC (first letter pregnancy diet and second lactation diet) to enable evaluation of offspring effects influenced by maternal diet during pregnancy and lactation. Maternal protein restriction decreased open arm time and distance in RR and RC offspring, increased anxiety behavior, in the EPM. In the OFT, the RR and RC offspring displayed decreased exploration (increased stress) as indexed by decreased distance in the center zone. These behaviors in the EPM and OFT was associated with increased corticosterone levels during an immobilization test in the RR offspring with intermediary effects in the RC offspring. Learning impairment was observed in the RR, CR and RC offspring during fixed ratio 5 schedule of reinforcement. Motivational effects were measured in RR offspring responding less, decreased motivation, and CR offspring making more responses, increased motivation, than CC offspring. These findings reveal the negative effects of developmental protein restriction on female offspring behavior. The underlying basis for these negative outcomes remains to be elucidated.

Pre- and/or postnatal protein restriction in rats impairs learning and motivation in male offspring.

Suboptimal developmental environments program offspring to lifelong health complications including affective and cognitive disorders. Little is known about the effects of suboptimal intra-uterine environments on associative learning and motivational behavior. We hypothesized that maternal isocaloric low protein diet during pregnancy and lactation would impair offspring associative learning and motivation as measured by operant conditioning and the progressive ratio task, respectively. Control mothers were fed 20% casein (C) and restricted mothers (R) 10% casein to provide four groups: CC, RR, CR, and RC (first letter pregnancy diet and second letter lactation diet), to evaluate effects of maternal diet on male offspring behavior. Impaired learning was observed during fixed ratio-1 operant conditioning in RC offspring that required more sessions to learn vs. the CC offspring (9.4±0.8 and 3.8±0.3 sessions, respectively, p<0.05). Performance in fixed ratio-5 conditioning showed the RR (5.4±1.1), CR (4.0±0.8), and RC (5.0±0.8) offspring required more sessions to reach performance criterion than CC offspring (2.5±0.5, p<0.05). Furthermore, motivational effects during the progressive ratio test revealed less responding in the RR (48.1±17), CR (74.7±8.4), and RC (65.9±11.2) for positive reinforcement vs. the CC offspring (131.5±7.5, p<0.05). These findings demonstrate negative developmental programming effects due to perinatal isocaloric low protein diet on learning and motivation behavior with the nutritional challenge in the prenatal period showing more vulnerability in offspring behavior.

Polymorphism in pbp5 gene detected in clinical Enterococcus faecium strains with different ampicillin MICs from a Tunisian hospital.

The polymorphism in pbp5 gene was investigated in nine unrelated clinical gentamicin-resistant Enterococcus faecium strains with different minimal-inhibitory-concentration values for ampicillin (six ampicillin-resistant and three ampicillin-susceptible). Five alleles were detected when the pbp5 C-terminal region was analysed, two of them in the ampicillin-resistant strains showed a new allele characterised by the Thr416Ala and Val462Ala substitutions. Two different alleles were identified when the pbp5 N-terminal region was studied; one of them in the unique strain (E. faecium 83) that presented very low ampicillin MIC (<0.125 microg/ml) and a nucleotidic mutation implicating a stop codon at 451 position. RT-PCR experiments carried out on five E.faecium positive results indication the expression of this gene. Specific mutations in pbp5 gene could be responsible of the high MIC values of some of the E. faecium strains.

Use of the splenic artery for arterial reconstruction in living donor liver transplantation.

BACKGROUND: The present study sought to evaluate the possibility of using the splenic artery for arterialization of a living donor liver graft. PATIENTS AND METHODS: In the period between August 2004 and April 2006, we performed 31 adult-to-adult living donor liver transplantations. In 27 patients (group A), the right or left hepatic artery was used to arterialize the graft, whereas in the other four cases (group B), we used the recipient splenic artery. RESULTS: The Model for End-stage Liver Disease (MELD) score of the patients averaged 17 (17.2 and 15.2 for groups A and B, respectively) ranging between 7 and 28. We did not observe pancreatitis, splenic infarction, or other complications related to ligation of the splenic artery. Two cases (6.4%) of arterial complication were observed, both in group A patients. CONCLUSION: The use of the splenic artery is a safe, practical alternative for arterial reconstruction in living donor liver transplantation procedures, when the hepatic artery is not adequate or in cases of portal hypertension with splenomegaly.

Liver transplantation from deceased donors serologically positive for Chagas disease.

The high mortality rates among patients waiting for liver transplantation has motivated the use of "marginal livers", among which are included livers from deceased donors serologically positive for Chagas disease (CD). The present work describes the outcome of orthotopic liver transplantation in six patients with severe liver disease (Child Pugh C), with livers from donors serologically positive for CD. Transplantations were performed from November 2000 to January 2005, and the patients received prophylactic treatment with benznidazole for 60 days, as a recommended by the Brazilian Consensus in Chagas Disease. The transplantation procedures presented no technical problems, and all the patients were discharged from hospital. Five of them did not present side effects demanding interruption of the prophylactic treatment. Four of the patients were clinically well over 1 year after transplantation (mean follow-up of 42.1 months), with negative serological results for CD. Two patients died, one of them 6 months post surgery of sepsis due to biliary complication and other one due to pulmonary (tuberculosis) complications. They were both serologically negative for CD. These results suggest that liver transplantation from CD donors, followed by benznidazole prophylactic treatment, is an important therapeutic alternative for severe liver disease.

Diversity of structures carrying the aac(6')-aph(2") gene in clinical Enterococcus faecalis and Enterococcus faecium strains isolated in Tunisia.

The diversity of structures carrying the aac(6')-aph(2") gene was studied in 46 high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium clinical strains recovered in a Tunisian hospital during the period 2000-2003. The inclusion of the aac(6')-aph(2") gene within the Tn4001 composite element or in its truncated forms (lacking the IS256 at the right, the left or at both sides of the aac(6')-aph(2") gene) was investigated by PCR and sequencing. The aac(6')-aph(2") gene was included in the composite Tn4001 element in 19 of 34 high-level gentamicin-resistant E. faecalis strains (56%) and in 1 of 12 E. faecium strains (12%). A truncated form of Tn4001 lacking IS256 at the left-hand (in 10 E. faecalis and 8 E. faecium), at the right-hand (3 E. faecalis and 2 E. faecium) or at both sides of the aac(6')-aph(2") gene (in 2 E. faecalis and 1 E. faecium) was also detected in 26 of our enterococci. The transference by conjugation of the aac(6')-aph(2") gene, associated with other resistance genes, was demonstrated in seven of the high-level gentamicin-resistant E. faecalis strains.

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.

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.

Characterization of antibiotic resistance genes and virulence factors in faecal enterococci of wild animals in Portugal.

Antibiotic susceptibility was tested in 140 non-selected enterococci (73 Enterococcus faecalis, 45 E. faecium and 22 of other species) recovered from faecal samples of 77 wild animals in Portugal. Susceptibility testing for 11 antibiotics (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, kanamycin, chloramphenicol, tetracycline, erythromycin, quinupristin-dalfopristin and ciprofloxacin) was determined by disk diffusion and agar dilution methods. Forty-four isolates (31.4%) showed susceptibility to all the antibiotics tested (5.5% of E. faecalis; 62.2% of E. faecium; and 78.6% of E. hirae). Neither ampicillin-resistance nor acquired-vancomycin-resistance was detected and 1.4% of the isolates showed high-level-resistance for gentamicin or streptomycin. Tetracycline and erythromycin resistances were shown in 28.6% and 20.1% of the isolates, respectively. Antibiotic resistance genes were studied by polymerase chain reaction (PCR) and sequencing and tet(M) + tet(L), erm(B) or aac(6')-aph(2'') genes were detected in most of tetracycline-, erythromycin- or gentamicin-resistant enterococci respectively. Genes encoding virulence factors were studied by PCR and a wide variety of virulence genes were detected in most of E. faecalis isolates but were rarely found in E. faecium and not detected in the other species. The prevalence of genes encoding virulence factors in E. faecalis was as follows: cpd (98.6%), gelE (75.3%), agg (30.1%), fsr (17.8%), ace (9.6%) and esp (4.1%). Low percentages of antibiotic resistance was found in the faecal enterococci of wild animals but a wide variety of virulence genes were detected among E. faecalis isolates although were rare in the other species.

Wine volatile and amino acid composition after malolactic fermentation: effect of Oenococcus oeni and Lactobacillus plantarum starter cultures.

Red wine amino acids and volatile compounds were analyzed before and after malolactic fermentation carried out by four different starter cultures of the species Oenococcus oeni and Lactobacillus plantarum. The purpose of this study was to determine whether differences can be attributed to the lactic acid bacteria strain used in this important step of the wine-making process. The malolactic cultures selected for this study were indigenous wine lactic acid bacteria strains. The data were evaluated using different multivariate analysis techniques. Results showed different malolactic behaviors for O. oeni and L. plantarum and significant metabolic differences between both species. A degree of diversity was found within each lactic acid bacteria group, since wines presented specific characteristics depending on the lactic acid bacteria strain used. In all cases, malolactic fermentation seemed to modify the amino acid and volatile composition of the wine.

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 mother's (the F1) fetal and neonatal development.

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.

Neuroendocrine dopaminergic regulation of prolactin release in systemic lupus erythematosus: a possible role of lymphocyte-derived prolactin.

Prolactin (PRL) secretion by the pituitary is under the control of dopamine. Hyperprolactinemia has been found in patients with systemic lupus erythematosus (SLE) and seems to be associated with clinical activity. T-lymphocytes express PRL and those from SLE patients appear to secrete more PRL than controls. In this study, immuno-(RIA) and bio-(BIO) assayable PRL in both serum and culture media of peripheral blood mononuclear cells (PBMNC) from SLE and control subjects were evaluated in the basal state and in response to 10 mg oral administration of metoclopramide, a dopamine receptor antagonist. Prolactin size heterogeneity in serum and culture media and PRL gene transcription in PBMNC were also studied. Basal serum RIA-PRL, BIO-PRL and the BIO/RIA ratio were similar in both groups. The serum BIO-PRL response after metoclopramide was higher than RIA-PRL in SLE, and this increment was also greater than in control subjects. PBMNC from SLE subjects secreted and produced more BIO-PRL. After metoclopramide, secretion and production of PRL increased only in PBMNC from control women and not in those from SLE patients. Our results demonstrated an increased central dopaminergic tone in SLE and suggest that lymphocyte-derived PRL might contribute to alter the functional activity of the hypothalamic dopaminergic system in SLE attempting to maintain serum PRL within a physiological range.