Apoptosis in rat placenta is zone-dependent and stimulated by glucocorticoids

Apoptosis, or physiological cell death, is elevated in the placenta of human pregnancies complicated by fetal growth retardation, suggesting that placental apoptosis may be a key factor in the overall control of feto-placental growth. The present study used DNA internucleosomal fragmentation analysis to characterize apoptosis in the two morphologically and functionally distinct regions of the rat placenta, the basal and labyrinth zones, during the last week of pregnancy (Days 16, 22, and 23). In addition, because glucocorticoids are potent inhibitors of feto-placental growth and can stimulate apoptosis in other tissues, we examined whether dexamethasone treatment in vivo induces placental apoptosis. DNA fragmentation was clearly evident in both placental zones at each stage of pregnancy, with higher levels evident in the basal zone compared with the labyrinth zone on Days 22 and 23. TUNEL analysis, which identifies dying cells in situ, demonstrated positive staining of cells in the basal zone, particularly giant trophoblast cells. Dexamethasone treatment increased DNA fragmentation in the basal zone but not the labyrinth zone. Similarly, maternal treatment with carbenoxolone, which can enhance local concentrations of endogenous glucocorticoid by inhibition of 11 beta-hydroxysteroid dehydrogenase, also increased DNA fragmentation in the basal zone but not in the labyrinth zone. These effects of dexamethasone and carbenoxolone on placental apoptosis were associated with reduced placental and fetal weights. In conclusion, this study shows that apoptosis occurs in both zones of the rat placenta, particularly in the basal zone near term, and is elevated after increased glucocorticoid exposure in vivo. These data support the hypothesis that placental apoptosis is an important player in the regulation of feto-placental growth, and establish the rat as a useful model to study the endocrine control of placental apoptosis.     

Vasopressin receptor expression in the placenta

The arginine vasopressin (AVP) type 1a receptor (V1a) is well known to mediate vasoconstriction. In pregnancy, blood flow in the placenta is crucial for sustaining normal growth and development of the fetus. This is the first AVP receptor study in the placenta and fetal membranes. The aim was to compare, quantitatively, the level of V1a gene expression with that of a known marker for vascularization, aquaporin 1 (AQP1). V1a and AQP1 gene expression did not correlate; placental V1a mRNA levels were significantly upregulated at 45 and 66+/-1 compared with 27, 100+/-4, and 140 days (term approximately 150 days). V1a mRNA levels were much lower in fetal membranes in which no significant difference across gestation was observed. In situ hybridization histochemistry localized V1a gene expression in the maternal component of the placenta similar to the receptor-binding studies using 125I-labeled [d(CH2)5, sarcosine7] vasopressin. No AVP gene expression was observed in the placenta and fetal membranes, which eliminates local AVP production. This increase in V1a expression at 45 and 66+/-1 days of gestation correlates with the period of maximal placental growth in the sheep and suggests that AVP and V1a receptors may play a hitherto unrecognized role in placental growth, differentiation, and/or function, particularly in the deleterious effects of heat stress, early in pregnancy, on fetal growth.     

Physiological changes in rat hypothalamic CRF: Circadian,stress and steroid suppression

Hypothalamic CRF-like immunoreactivity was measured in the a.m. and p.m., after systemic dexamethasone administration or after either stress in adult male rats. Measurement of plasma corticosterone levels revealed the expected circadian rhythmicity, suppression after dexamethasone administration and increase after ether stress. The hypothalamic content of CRF-like immunoreactivity was significantly decreased in the p.m. and after dexamethasone administration. However, no change in hypothalamic CRF-like immunoreactivity was observed after ether stress. The results are consistent with an increased release in the p.m. and decreased synthesis of hypothalamic CRF after systemic dexamethasone administration. The observation that there is no change in content of hypothalamic CRF-like immunoreactivity after ether stress could be due to the fact that the animals were stressed by handling. The results show that this immunoreactivity present in the hypothalamus is altered by changes in the hypothalamic-pituitaryadrenal axis and thus suggest that this peptide is a physiologically significant CRF in the rat.     

Alterations in uterine and placental prostaglandin F and E with gestational age in the rat

Experiments were designed to determine the chronological alterations in placental and uterine prostaglandin F and E (PGF and PGE) during pregnancy in the rat. Pregnant rats (sperm in the vagina = day 0) were sacrified at days 15, 18,19, 20, 21 and delivery (day 21 ) and placental and uterine tissues assayed (RIA) for PGF and PGE immediately (“”) or after 1 hour incubation (“”). Uterine content of PGF and PGE (ng PG/mg DNA) was increased significantly by day 19 and further increases were seen through delivery. Incubation of uterine tissue resulted in enhanced net production of PGF and PGE (p <.05) per mg DNA (as judged by tissue content and release into the incubation medium) by day 18 of pregnancy vs. day 15. Net production peaked around the time of delivery thus paralleling the alterations in tissue content .By contrast, no differences with gestational age were found in placental content of PGF and PGE , the concentrations throughout late gestation remaining in the range of uterine PGs at day 15. However, production of PGs per mg placental DNA increased markedly during incubation with significant enhancement detected by day 19 vs. 15, achieving levels even greater than the uterus .The and findings for the uterus are consistent with the hypothesis that increases in uterine PGs levels at the end of pregnancy may play an important role in parturition. The experiences with placental tissue suggest that the potential for PG production per placental cell may also increase in late gestation and thereby contribute to the augmented intrauterine availability of PGs at that time.     

Killer Ig-like receptor expression in uterine NK cells is biased toward recognition of HLA-C and alters with gestational age

Immunogenetic studies suggest that interactions between maternal killer Ig-like receptor (KIR) expressed by uterine NK (uNK) cells, and fetal HLA-C molecules on trophoblast, influence the success of human placentation. However, the exact functional response of fresh uNK cells to trophoblast HLA-C molecules is unknown. In this study, we show by quantitative RT-PCR and FACS that both activating and inhibitory KIR specific for HLA-C are expressed at higher levels and on an increased proportion of NK cells in the human decidua compared with blood. In contrast, expression of KIR3DL1/S1, which is specific for HLA-B, is similar in both NK cell populations. Remarkably, there is also a temporal change in the expression pattern of HLA-C-specific KIR, with a decline in both intensity of expression and frequency on uNK cells throughout the first trimester of pregnancy. This selective up-regulation of KIR has functional consequences because uNK cells show increased binding of HLA-C tetramers compared with blood NK cells. Ab cross-linking shows that these KIR are functional and results in increased cytokine secretion. uNK cells, therefore, exhibit a unique KIR profile that enhances their ability to recognize trophoblast cells expressing HLA-C at the materno-fetal interface. This is the first report to demonstrate selective regulation of KIR expression over time in vivo in a normal physiological situation and suggests that KIR expression by uNK cells is regulated by the tissue microenvironment in the decidua.     

Leptin induces IL-1 receptor antagonist expression in the brain

Fructose has been shown to protect hepatocyte viability during hypoxia or exposure to mitochondrial electron transport inhibitors. We report here that the fructose metabolite D-glyceraldehyde (D-GA) is a good inhibitor of the mitochondrial permeability transition pore (PTP) in isolated rat liver mitochondria. We propose that a substantial portion of the protective effect of fructose on hepatocytes is due to D-GA inhibition of the permeability transition. Aldehydes which are substrates of the mitochondrial aldehyde dehydrogenase (mALDH) afford protection, while poor substrates do not. Protection is prevented by the ALDH inhibitor chloral hydrate. We propose that the NADH/NAD(+) ratio is the key to protection. The aldehydes phenylglyoxal (PGO) and 4-hydroxynonenal (4-HNE), which have previously been shown to inhibit the PTP, apparently function by a different mechanism independent of mALDH activity. Both PGO or 4-HNE are themselves potent inhibitors of ALDH, and their protective effect cannot be blocked by an ALDH inhibitor.     

Gene expression changes with age in skin,adipose tissue,blood and brain

Background

Previous studies have demonstrated that gene expression levels change with age. These changes are hypothesized to influence the aging rate of an individual. We analyzed gene expression changes with age in abdominal skin, subcutaneous adipose tissue and lymphoblastoid cell lines in 856 female twins in the age range of 39-85 years. Additionally, we investigated genotypic variants involved in genotype-by-age interactions to understand how the genomic regulation of gene expression alters with age.

Results

Using a linear mixed model, differential expression with age was identified in 1,672 genes in skin and 188 genes in adipose tissue. Only two genes expressed in lymphoblastoid cell lines showed significant changes with age. Genes significantly regulated by age were compared with expression profiles in 10 brain regions from 100 postmortem brains aged 16 to 83 years. We identified only one age-related gene common to the three tissues. There were 12 genes that showed differential expression with age in both skin and brain tissue and three common to adipose and brain tissues.

Conclusions

Skin showed the most age-related gene expression changes of all the tissues investigated, with many of the genes being previously implicated in fatty acid metabolism, mitochondrial activity, cancer and splicing. A significant proportion of age-related changes in gene expression appear to be tissue-specific with only a few genes sharing an age effect in expression across tissues. More research is needed to improve our understanding of the genetic influences on aging and the relationship with age-related diseases.     

Changes with gestational age in the nutritional requirements of postimplantation rat embryos in culture

Rat embryos explanted at 9.0, 9.5, and 10.5 days of gestation were cultured for periods of 61, 49, or 45 h, respectively, in extensively dialysed rat serum supplemented with various combinations of glucose, amino acids, and vitamins. Glucose was found to be a necessary and sufficient energy source for embryos of all three ages, and virtually no development took place in its absence. Only the youngest embryos required free amino acids for good development in dialysed serum, whereas at all three ages, vitamin supplementation was necessary. However, lack of vitamins had a much more marked deleterious effect on the younger embryos than on those explanted at 10.5 d. Experiments with media deficient in individual vitamins showed that for normal development, 9.0-d embryos required a number of vitamins--principally pantothenic acid, riboflavin, inositol, folic acid and niacinamide, whereas 10.5-d embryos needed only riboflavin. For embryos explanted at 9.5 d, the position was intermediate, with riboflavin and inositol the most significant vitamins. Inositol deficiency in embryos explanted at 9.5 d produced a characteristic neural tube defect--failure of closure at the level of the hindbrain. Thus it appears that both the range of micromolecular nutrients and the severity of developmental impairment in their absence decrease with advancing gestational age.     

Features of insulin receptor interaction in placenta from normal,overt and poorly controlled gestational diabetic patients

Features of insulin binding to trophoblast plasma membranes were studied in six normal pregnant women (NP), six overt diabetes (ODP) and six poorly controlled glycemic gestational patients (PCDP) i.e. women who did not strictly follow the management of diabetes mellitus during pregnancy. A decreased maximum specific insulin receptor binding per 0.1 mg membrane protein in placenta from PCDP (12%) was found comparing with that from ODP or NP (17.5% and 36.2%, respectively, P<0.01), The insulin binding in PCDP declined at a faster rate until it reached minimum when studied at a higher temperature (25–37°C). The binding equilibrium was likewise attained faster at this temperature than that at lower temperature of 4°C for all studied groups.The insulin receptor binding in all studied groups was pH dependent. The maximum binding in ODP and PCDP groups was attained at pH 7.8 while for NP maximum binding was at pH 7.4. The competitive dinding assay was carried out with 14 concentrations of unlabelled insulin and the half maximal displacement of¹²⁵I-insulin was at 8×10^–9 M, 6×10^–9 M and 4×10^–9 M for NP, ODP and PCDP, respectively (P<0.05) suggesting the differences in the effect of glycemic control on the insulin binding. Furthermore the binding yielded curvilinear Scatchard plots with the apparent affinity of the receptors being affected in the ODP and PCDP groups.The molecular characteristics of the receptors in the diabetic patients as revealed by the cross-linking technique used in this study did not reveal any changes in the subunit structures when compared with normals except that the¹²⁵I-insulin bound as shown by the band intensity was much less in PCDP. These findings indicate that control of hyperglycemia could optimize the outcome of insulin receptor function during diabetic pregnancy.     

Serum testosterone and dihydrotestosterone changes with age in rat.

Serum testosterone (T) and 5alpha-dihydrotestosterone (DHT) were measured in young, adult and old Albino Wistar male rats using a sensitive and reliable radioimmunoassay, after separating T from DHT by thin layer chromatography. The mean plus or minus S.E.M. for T in young, adult and old rats were 62 plus or minus 11, 250 plus or minus 27 and 125 plus or minus 25 (ng/100 ml) respectively. Serum T was increased in adults (P less than 0.001) and decreased in old rats (P less than 0.01). The mean plus or minus S.E.M. for serum DHT was 8 plus or minus 2, 19 plus or minus 2 and 17 plus or minus 1 (ng/100 ml) for young, adult and old rats respectively. DHT was increased in adults (P less than 0.001), but did not change in old rats.     

Modeling the pediatric paradox: Birth weight by gestational age

Abstract

The “pediatric paradox” of African versus European American infant mortality is often observed with respect to birth weight, but rarely to gestational age, even though the two measures are biologically related. This paper models the pediatric paradox by birth weight and gestational age simultaneously, using Covariate Density Defined mixture of logistic regressions (CDDmlr) fitted to 1985–1988 New York State births. The model controls for unobserved heterogeneity and isolates the pediatric paradox in the “compromised” subpopulation. The paradox is not limited to low birth weights and/or short gestational ages, but surrounds the normal birth range. Nevertheless, the pediatric paradox is only observed in the marginal distribution of birth weight and not the marginal distribution of gestational age. These results are consistent with the hypothesis that higher fetal losses in the “compromised” subpopulation may be responsible for the pediatric paradox and that African versus European American infant mortality differentials are underestimated.     

Enkephalin peptides in fetal sheep, changes with gestation, origin and production by the placenta.

Enkephalin-containing peptides have been followed in the circulation of fetal sheep between 118-143 days gestation. Using a combination of radioimmunoassay and hplc met5-enkephalin was found in the concentration range 60-500 pg/ml and proenkephalins containing met5-enkephalin had a concentration of 150-4000 pg/ml. The concentration of both increased towards term. The sources of the enkephalin peptides was investigated by measurement of differences across the umbilical circulation and by studying the effects of fetal adrenal demedullation and chemical sympathectomy. The placenta showed a continuous net output of enkephalin peptides which increased close to term. This placental output was increased sharply by reduction of uterine blood flow either using compression of the uterine artery or through infusion of adrenaline at 35 micrograms/min into the maternal circulation. Maternal hypoxia caused by breathing 9% O2 plus 3% CO2 also increased fetal plasma enkephalin levels, although not output from the placenta. Adrenal demedullation, particularly if accompanied by chemical sympathectomy depressed fetal plasma enkephalin concentrations and sharply suppressed the fetal peptide responses to maternal hypoxia. It is concluded that the placenta and the fetal adrenal are important sources of met5-enkephalin-containing peptides in the fetal circulation. The placental production appears to be closely tied to changes in uterine perfusion and adrenal output changes in response to fetal oxygenation.     

Segmental distribution and gestational changes of GABA-transaminase activity in the rat oviduct

The occurrence and the localization of 4-aminobutyrate:2-oxoglutarate transaminase (GABA-transaminase) in the non-pregnant and pregnant rat oviduct were examined using biochemical and enzyme histochemical techniques. Specific GABA-transaminase activity was detected in the ampullary and isthmic portions of the oviduct as well as in the utero-tubal junction. The enzymic activity was lower in the ampullary than in the isthmic or intramural segments of the oviduct. Pregnancy induced a significant increase of GABA-transaminase activity in each portion of the oviduct. Enzyme histochemistry showed the highest GABA-transaminase reactivity at the level of the epithelial cells of the oviduct irrespective of the portion of the tube examined. A faint specific activity was demonstrated in the smooth muscle of the oviduct while the serosa did not show specific staining. Our findings indicate that: the observed increase of GABA-transaminase activity in the oviduct of the pregnant rat may be responsible for the reduced GABA levels in the oviduct during gestation; and the extraneuronal localization of GABA-transaminase activity does not seem to support the suggestion of a possible GABAergic innervation of the oviduct.     

Leptin expression in the rat ovary depends on estrous cycle.

Leptin is a hormone originally identified in adipocytes. It is involved in the regulation of fat deposition and energy expenditure and in other functions, such as reproduction. The presence of leptin has been reported in several reproductive organs. However, few studies have addressed its expression in the ovary. Moreover, the existing information is not consistent with regard to the particular cell types responsible for leptin expression. In this work we studied the distribution of leptin in the rat ovary by immunohistochemistry (IHC) and in situ hybridization (ISH). Leptin staining was found in steroid-producing cells: thecal, luteal, and interstitial cells. The strongest signal with both techniques was found in the cytoplasm of oocytes. A weak reaction for leptin mRNA was detected in granulosa of all growing follicles, although leptin protein was found only in the mature follicle. Western blotting analysis detects a strongly reactive 16-kD band, giving further support to the presence of leptin in the rat ovary. Variations in this immunoreactive band were found throughout the estrous cycle. Localization of leptin in the ovary may contribute to a better understanding of female reproductive function.