Dynamic changes in maternal decidual leukocyte populations from first to second trimester gestation.

INTRODUCTION: Decidual leukocytes are critical to the development of the fetomaternal interface, regulating tolerance to the semi-allogeneic fetus and vascular transformation of the uterine spiral arteries. Despite the continuation of these processes beyond the first trimester of pregnancy, the second trimester has largely been unstudied, with investigation focusing on early gestation and term tissues. We sought to characterize changes in decidual leukocyte populations from first to second trimester. METHODS: Multicolor flow cytometry was performed on isolated decidual leukocytes from elective terminations of pregnancy between 6 and 20 weeks of gestation for study of first (6-12 weeks) and second trimesters (13-20 weeks). Specific subpopulations were identified by comparison to isotype and fluorescent-minus-one (FMO) controls. RESULTS: Decidual natural killer cells (CD56(+)CD16(-)CD3(-)) did not change in number, although a population of dNK with decreased CD56 brightness was observed in second trimester decidua. CD14(+)HLA-DR(+) macrophage numbers declined from first to second trimester (p = 0.031), yet a CD163(+)CD206(+) subset designating alternatively activated M2-like macrophages increased during the same period (p = 0.015). Intermediate CD205(+) dendritic cells demonstrated significant decline (p = 0.022), but immature CD209(+) and mature CD83(+) dendritic cells did not differ between trimesters. Total CD3(+) and CD3(+)CD4(+) T lymphocytes increased (p = 0.0079, p = 0.0028); CD3(+)CD8(+) T cells trended towards increase but did not differ significantly. CONCLUSION: Several changes in leukocyte subsets are observed in the second trimester that promote a tolerogenic and angiogenic decidual microenvironment through mid-gestation.

Umbilical cord diameter percentile curves and their correlation to birth weight and placental pathology.

OBJECTIVE: The aims of this study were to develop a nomogram of umbilical cord diameter (UCD) for pathologic examination of the placenta, to identify the umbilical cord components responsible for variations in UCD, and to examine the relationship between UCD and other placental pathologic features and perinatal outcome. STUDY DESIGN: We prospectively collected 497 umbilical cords between 18 and 41 weeks' gestation over a 1-year period. Fresh-tissue UCD were grouped according to gestational age and compared to sonographic and histological measurements. Associations between UCD percentile and placental pathologic findings or obstetrical outcomes were examined. RESULTS: Mean UCD increased with gestational age until a plateau at 1.0 cm in the third trimester, a value that was 0.56 cm less than sonographic measurements prior to delivery and 0.17 cm greater than UCD measured histologically. Umbilical cord components varied with UCD percentile, with umbilical vessel area increased in thick cords (p < 0.001) and Wharton's jelly area reduced in thin cords (p = 0.002). Thin umbilical cords were associated with at least one pathologic histological placental finding (p = 0.02), low placental weight (p < 0.001), single umbilical artery (p = 0.02), marginal cord insertion (p = 0.01), and low infant birth weight (p < 0.001). CONCLUSIONS: This study provides reference curves for post-delivery UCD from 18 to 41 weeks' gestation for use by perinatal pathologists. We show that increased UCD is a function of increased umbilical blood vessel volume and decreased UCD is a function of decreased Wharton's jelly volume. UCD shows a strong association with placental and infant birth weight.

Pathologic basis of echogenic cystic lesions in the human placenta: role of ultrasound-guided wire localization.

OBJECTIVE: The sonographic appearance of the placenta is normally homogenous throughout the second trimester. A variety of abnormalities in placental texture have been described, some of which may be pathologic and associated with adverse clinical outcomes. We characterized the pathologic basis of one lesion termed echogenic cystic lesions (ECLs) that may be a prognostic marker in intrauterine growth restriction (IUGR). STUDY DESIGN: We retrospectively correlated placental pathology in 50 pregnancies that had a total of 84 ECLs documented by ultrasound prior to delivery. Six additional women with placental ECLs prospectively underwent immediate post-delivery ultrasound-guided wire localization of 9 lesions followed by placental pathology. Obstetric outcome data were recorded. RESULTS: Severe pre-eclampsia (20%) and extreme IUGR (18%) were common outcomes. Of 93 ECLs identified, 46 (49%) gross lesions were found by placental pathology. Inter-villous thrombosis was the most significant lesion found (30/46, 65%) compared to all other lesions (35%; Z-Test, p = 0.007). Ultrasound guidance identified 8/9 (89%) lesions of which 6/8 (67%) were inter-villous thrombosis. Associated lesions (infarction, 36%; advanced villous maturation, 27%) and small placental weight (<10th centile, 38%) were present in 50%, but did not increase the risk of adverse perinatal outcome. CONCLUSIONS: ECLs are most commonly due to inter-villous thrombosis. The adverse clinical outcomes may be mediated by associated lesions not readily detectable by ultrasound. Ultrasound-guided wire localization is a promising research tool for future large-scale cohort studies needed to define the clinical utility of placental ultrasound findings.

The pattern of glucocorticoid and estrogen receptors may explain differences in steroid dependency of intrauterine prostaglandin production at parturition in sheep.

BACKGROUND: We have recently described two distinct pathways of intrauterine prostaglandin (PG) synthesis: a cortisol-dependent/estradiol-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an estradiol-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2(2alpha). We hypothesized that the differential effects of cortisol and estradiol on intrauterine PGH synthase-II (PGHS-II) expression and PG production may be because of the tissue specific expression of the glucocorticoid and estradiol receptors (GR and ER, respectively) within the intrauterine tissues. In addition, we suggest that these two pathways of PG production are linked through the expression of P450(C17hydroxylase) (P450(C17)) and subsequent increase in placental estradiol synthesis. METHODS: To test the hypotheses, we infused singleton, chronically catheterized fetal sheep beginning at day 125 of gestation (term 147 to 150 days) with (1) cortisol (0.45 mg/mL; n = 5); (2) cortisol and 4-hydroxyandrostenedione, a P450(aromatase) inhibitor (4-OHA: 1.44 mg/h; n = 5); (3) saline (n = 5); or (4) saline and 4-OHA (n = 5). PGHS-II, ER alpha, ER beta, and GR alpha were localized using immunohistochemistry. ER alpha, ER beta, P450(C17), and GR alpha protein expressions were determined by Western blot analysis. Data were analyzed by analysis of variance (ANOVA) (P < or =.05). RESULTS: Fetal cortisol infusion in the presence or absence of a rise in placental estrogen synthesis increased placental expression of GR alpha; both PGHS-II and GR alpha localized to the uninucleate trophoblast cells of the placentome and were excluded from the maternal stroma and binucleate cells. Both forms of ER were excluded from the trophoblast tissue of the placentome. ER alpha, ER beta, and PGHS-II showed a similar pattern of distribution within the luminal epithelium of the endometrium; there were no alterations in the level of the ER in the presence of cortisol +/- 4-OHA. Placental P450(C17) protein expression was increased in the presence of a rise in fetal cortisol independent of changes in placental estrogen synthesis. CONCLUSIONS: We concluded that the differential effects of cortisol and estradiol on intrauterine PGHS-II expression and PG production may be due to the tissue-specific expression of the GR and ER within the intrauterine tissues. Glucocorticoid effects on trophoblast PG production may be mediated in a positive feed-forward manner. We further suggest that either cortisol or a cortisol-stimulated intermediate, like PGE2, increased P450(C17) expression, leading to a rise in placental estradiol synthesis and triggering maternal intrauterine tissue PG production.

Ultrasound detection of placental insufficiency in women with 'unexplained' abnormal maternal serum screening results.

Serious placental insufficiency results in perinatal death or preterm birth from ischemic-thrombotic pathology, a process which has its origins in placental maldevelopment in the first trimester. A proportion of at-risk pregnancies may be identified from abnormalities in first or second trimester serum screening data, uterine artery Doppler waveforms or placental shape and texture at the time of the 18-20-week anatomical examination. In combination, these tests may be capable of recognizing a subset of at-risk pregnancies with 50% positive predictive values. Early recognition before fetal viability affords opportunities to direct women to regional perinatal care centres for enhanced maternal-fetal surveillance, corticosteroids to enhance fetal lung maturation, prophylactic measures to prevent pre-eclampsia and optimal decision making around the time of delivery. The creation of regional screening programs to use screening data with a placental focus is likely to be cost-effective, because existing patterns of care are utilized. More importantly, this strategy can direct women to participate in clinical research programs designed to reduce morbidity and mortality from this common group of conditions.

Effects of cortisol and oestradiol on hepatic 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus.

In the late-gestation sheep, increased fetal plasma cortisol concentration and placental oestradiol (E(2)) output contribute to fetal organ maturation, in addition to the onset of parturition. Both cortisol and E(2) are believed to regulate the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which interconverts bioactive 11-hydroxy glucocorticoids and their inactive 11-keto metabolites. 11beta-HSD1, abundantly expressed in fetal liver, operates primarily as a reductase enzyme to produce bioactive cortisol and thus regulates local hepatic glucocorticoid concentrations. Cortisol acts through the glucocorticoid receptor (GR) present in the liver. In this study, we examined the effects of cortisol and E(2) on hepatic 11beta-HSD1 and GR in the liver of chronically catheterized sheep fetuses treated with saline (n=5), cortisol (1.35 mg/h; n=5), saline+4-hydroxyandrostendione, a P450 aromatase inhibitor (4-OHA; 1.44 mg/h; n=5), or cortisol+4-OHA (n=5). Cortisol infusion resulted in increased plasma concentrations of fetal cortisol and E(2); concurrent administration of 4-OHA attenuated the increase in plasma E(2) concentrations. Using immunohistochemistry, we showed that fetal hepatocytes expressed both 11beta-HSD1 and GR proteins. Cortisol treatment increased GR in both cytosol and nuclei of hepatocytes; concurrent administration of 4-OHA was associated with distinct nuclear GR staining. Western blot revealed that cortisol, in the absence of increased E(2) concentrations, significantly increased concentrations of 11beta-HSD1 (34 kDa) and GR (95 kDa) proteins. 11beta-HSD1 enzyme activity was measured in the liver microsomal fraction in the presence of [(3)H]cortisone (10(-)(6) M) or [(3)H]cortisol (10(-)(6) M) and NADPH (reductase activity) or NADP(+) (dehydrogenase activity) respectively. 11beta-HSD1 reductase activity was significantly greater in the presence of cortisol. In summary, we found that, in sheep during late gestation, cortisol increased both 11beta-HSD1 and GR in the fetal liver, and these effects were accentuated in the absence of increased E(2).

Ontogeny and regulation of ovine placental prostaglandin E2 synthase.

Recent evidence suggests that ovine placental output of prostaglandin (PG) E2 rises through late gestation partly because of a direct effect of cortisol on PGH2 synthase 2 (PGHS-2) expression and activity within trophoblast tissue. Synthesis of PGE2 is also dependent, however, on PGE2 synthase (PGES), which converts PGH2 to PGE2. We hypothesized that PGES is expressed in the ovine placenta, and that, similar to PGHS-2, expression increases through gestation and is regulated positively by cortisol. Placental tissues from pregnant ewes in mid and late gestation, at term, and during early and active labor were analyzed to determine the gestational profile of PGES. The regulation of PGES expression was assessed in placental tissues from pregnant ewes in which intrafetal cortisol infusion was administered in late gestation, in the presence or absence of an aromatase inhibitor, to block the cortisol-stimulated rise in estradiol. Expression of PGES was analyzed by in situ hybridization, Western blot analysis, and immunohistochemistry. In the placentome, PGES localized to fetal trophoblast cells and endothelial cells in maternal blood vessels, consistent with its contribution to the rise in placental PGE2 output toward the onset of labor and with a role of PGE2 in the local regulation of uteroplacental blood flow, respectively. Expression of PGES mRNA and protein increased with gestation. However, there was no significant further change with labor or during cortisol infusion in the presence or absence of a rise in fetal plasma estradiol, in contrast to reported changes in PGHS-2. These results suggest that PGES is not coregulated with PGHS-2 in the sheep placenta at term. The progressive increase in PGES, however, likely contributes to the rise in circulating PGE2 in the fetus in late pregnancy.

The fetal placental hypothalamic-pituitary-adrenal (HPA) axis, parturition and post natal health.

A general characteristic of fetal endocrine maturation across different species is the enhanced activity of the fetal hypothalamic-pituitary-adrenal (HPA) axis during late gestation. Precocious activation of this axis may occur when the fetus is exposed to an adverse intra-uterine environment, such as hypoxemia. HPA development is associated with increased levels of ACTH(1-39) and adrenal corticosteroids (cortisol in sheep and human) in the fetal circulation, and increased expression of mRNA encoding corticotrophin releasing hormone (CRH) in the hypothalamus, proopiomelanocortin (POMC) in the pituitary, and key steroidogenic enzymes in the fetal adrenal. At term, increased levels of cortisol act on the placenta/trophoblast derived cells to increase expression of prostaglandin synthase Type II (PGHS-II). In human gestation, cortisol also decreases expression of 15-hydroxyprostaglandin dehydrogenase (PGDH) in chorionic trophoblast cells. Increased synthesis and decreased metabolism of prostaglandin (PG) results, during late gestation, in enhanced output of primary PG, which in turn increases the activity of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) in the human fetal membranes. Increased chorionic 11 beta HSD-1 results in increased local generation of cortisol from cortisone, with further paracrine/autocrine stimulation of PG output. Increased fetal cortisol contributes to the maturation of organ systems required for postnatal extra-uterine survival. However, excessive levels of feto-placental glucocorticoid, derived from maternal administration of synthetic corticosteroids or sustained endogenous fetal cortisol production, results in intrauterine growth restriction. Fetal sheep, exposed to maternal betamethasone in late gestation, develop insulin resistance and exaggerated adrenal responses to HPA stimulation by 6-12 months postnatal life. Thus, the level of fetal HPA activity is crucial not only for determining gestation length, but may also predict pathophysiologic adjustments in later life.

Understanding preterm labor.

Increased uterine contractility at term and preterm results from activation and then stimulation of the myometrium. Activation can be provoked by mechanical stretch of the uterus and by an endocrine pathway resulting from increased activity of the fetal hypothalamic-pituitary-adrenal (HPA) axis. In fetal sheep, increased cortisol output during pregnancy regulates prostaglandin H synthase type 2 (PGHS2) expression in the placenta in an estrogen-independent manner, resulting in increased levels of PGE2 in the fetal circulation. Later increases in maternal uterine expresssion of PGHS2 require elevations of estrogen and lead to increased concentrations of PGF2alpha in the maternal circulation. Thus, regulation of PGHS2 at term is differentially controlled in fetal (trophoblast) and maternal (uterine epithelium) tissue. This difference may reflect expression of the glucocorticoid receptor (GR), but not estrogen receptor (ER), in placental trophoblast cells. In women, cortisol also contributes to increased PG production in fetal tissues through upregulation of PGHS2 (amnion and chorion) and downregulation of 15-OH PG dehydrogenase (chorion trophoblasts). The effect of cortisol on chorion expression of PGDH reverses a tonic stimulatory effect of progesterone, potentially through a paracrine or autocrine action. We have interpreted this interaction as a reflection of "progesterone withdrawal" in the primate, in relation to birth. Other agents, such as proinflammatory cytokines, similarly upregulate PGHS2 and decrease expression of PGDH, indicating the presence of several mechanisms by which labor at term or preterm may be initiated. These different mechanisms need to be considered in the development of strategies for the detection and management of the patient in preterm labor.

Effects of cortisol and estradiol on pituitary expression of proopiomelanocortin, prohormone convertase-1, prohormone convertase-2, and glucocorticoid receptor mRNA in fetal sheep.

We hypothesized that in the late-gestation sheep fetus there is an interaction between the prepartum rise in cortisol and the increase in placental estradiol production that allows expression of key components of the fetal hypothalamic-pituitary-adrenal (HPA) axis. Therefore, the goal of this study was to investigate the effects of cortisol on the fetal HPA axis in the presence and absence of increased placental estradiol production. We obtained fetal plasma samples and pituitary tissue from animals that had received an infusion of either cortisol, cortisol and 4-hydroxyandrostenedione (40HA, an aromatase inhibitor), saline, or saline + 40HA controls. Cortisol significantly decreased plasma adrenocorticotropic hormone concentrations, and in the presence of 40HA reduced pituitary proopiomelanocortin (POMC) mRNA levels in the pars distalis. There was no effect of any treatment on the expression of the key POMC processing enzymes, prohormone convertase-1 or -2 in the fetal pituitary. Conversely, levels of glucocorticoid receptor (GR) mRNA in the pituitary were increased with cortisol treatment in the absence of increased estradiol. We suggest that in the late-gestation sheep fetus, cortisol and estradiol have opposite effects on pituitary POMC and GR mRNA expression, and interact to regulate these key components of the fetal HPA axis.

Glucocorticoid regulation of human and ovine parturition: the relationship between fetal hypothalamic-pituitary-adrenal axis activation and intrauterine prostaglandin production.

Birth in many animal species and in humans is associated with activation of hypothalamic-pituitary-adrenal function in the fetus and the increased influence of glucocorticoids on trophoblast cells of the placenta and fetal membranes. We suggest that in ovine pregnancy glucocorticoids directly increase fetal placental prostaglandin production, and indirectly increase prostaglandin production by maternal uterine tissues through the stimulation of placental estradiol synthesis. The events of ovine parturition are compared with those of human parturition. In the latter, we suggest similar direct effects of glucocorticoids on prostaglandin synthesis and metabolism in fetal membranes and similar indirect effects mediated by glucocorticoid-stimulated increases in intrauterine corticotropin-releasing hormone expression.

Prostaglandin production at the onset of ovine parturition is regulated by both estrogen-independent and estrogen-dependent pathways.

A current hypothesis of ovine parturition proposes that fetal adrenal cortisol induces placental E2 production, which, in turn, triggers intrauterine PG production. However, recent evidence suggests that cortisol may directly increase PG production in trophoblast-derived tissues. To separate cortisol-dependent and estrogen-dependent PG production in sheep intrauterine tissues, we infused singleton, chronically catheterized fetuses beginning on day 125 of gestation (term, 147-150 days) with 1) cortisol (1.35 mg/h; n = 5); 2) cortisol and 4-hydroxyandrostendione, a P450aromatase inhibitor (4-OHA: 1.44 mg/h; n = 5); 3) saline (n = 5); or 4) saline and 4-OHA (n = 5). Fetal and maternal arterial blood samples were collected at 12-h intervals starting 24 h before infusion and continuing during treatment for 80 h or until active labor. Uterine contractility was measured by electromyogram recording of myometrial activity. Plasma E2, progesterone (P4), PGE2, and 13,14-dihydro- 15-keto-PGF2alpha were quantified by RIA. PGHS-II messenger RNA (mRNA) and protein expression were determined by in situ hybridization and Western blot analysis, respectively. Data were analyzed by ANOVA (P < or = 0.05). Labor-type uterine contractions were present after 68 h of cortisol infusion and had increased significantly by 80 h. Labor-type uterine contractions were induced after 68 h of cortisol plus 4-OHA infusion, but the contraction frequency remained less than that in the cortisol-treated animals. Fetal cortisol infusion increased fetal and maternal plasma E2 concentrations and decreased the maternal plasma P4 concentration significantly; concurrent 4-OHA infusion attenuated the increase in fetal and maternal plasma E2, but not the decrease in maternal plasma P4. The fetal plasma PGE2 concentration increased after both cortisol and cortisol plus 4-OHA infusion. The maternal plasma 13,14-dihydro-15-keto-PGF2alpha concentration rose after fetal cortisol infusion, but not after cortisol plus 4-OHA infusion. Placental trophoblast PGHS-II mRNA and protein expression were increased significantly after both cortisol and cortisol plus 4-OHA infusion. Endometrial PGHS-II mRNA and protein expression increased after cortisol infusion, but not after cortisol plus 4-OHA infusion. Plasma steroid and PG concentrations, uterine activity pattern, and intrauterine PGHS-II expression were not altered in either control group. We conclude that these data suggest distinct pathways of intrauterine PG synthesis: a cortisol-dependent/E2-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an E2-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2alpha and appears necessary for uterine activity and parturition.

The characterization of human amnion epithelial and mesenchymal cells: the cellular expression, activity and glucocorticoid regulation of prostaglandin output.

The amnion, a single layer of epithelial cells (EC) overlying layers of mesenchymal cells (MC) has been identified as a source of intrauterine prostaglandins (PG). The objectives of the present study were: (1) to establish a technique for the isolation and culture of pure amnion EC and MC preparations, (2) to characterize the cellular expression of PGHS-II and PGHS activity within these separated amnion cells and (3) to characterize the pattern of glucocorticoid stimulation of these separated amnion cells. Term gestation human amnion was collected after elective caesarean section or vaginal delivery. A trypsin digestion was used to isolate EC and a mechanical digestion and collagenase dispersion was used to isolate MC. Following 48 or 96 h in culture, cells were incubated for 24 h in the presence or absence of 1 microm arachidonic acid and treated with cortisol (F: 10-1000 nm) or 1 microm dexamethasone (DEX). Cell types were identified by immunohistochemistry (IHC). Immunoreactive PGHS-II (ir-PGHS-II) and glucocorticoid receptor (ir-GR) were localized by IHC. PGHS activity was measured as PGE(2)output determined by radioimmunoassay. Mean PGE(2)production by MC at 72 h was 22-fold greater (P<0.05) and at 120 h was 32-fold greater (P<0.03) than PGE(2)output by EC. Administration of arachidonic acid stimulated a 5.0-fold increase in PGE(2)output (P<0.0002) by EC after 72 h and a 3.6-fold increase (P<0.05) after 120 h but did not alter MC PGE(2)output. Despite exogenous substrate, EC PGE(2)output remained significantly less than PGE(2)output by MC. There was no difference in PG production by EC and MC with the onset of labour. Ir-GR expression was found in both EC and MC. F and/or DEX with and without arachidonic acid (AA) stimulated PGE(2)output by EC. Only DEX and not F increased PGE(2)output by MC. These data suggest that relatively pure EC and MC preparations can be established from amnion. PG output and its regulation appears to differ within these two amnion cell types, dependent upon (1) substrate availability and (2) the regulation of PGHS activity.

Distribution of interleukin-1 receptors in term human fetal membranes and decidua.

Interleukin-1 (IL-1) is a dimorphic cytokine that acts on target cells through high-affinity receptors, type I and type II. It has been implicated in the onset of term and preterm labour with associated intrauterine infection. To define better the potential action of this cytokine in the human fetal membranes and decidua, the objective of this study was to define the type(s) of IL-1 receptors present in the tissues at term, examine the tissue and cellular distribution of the receptor(s) and determine if there were any changes in their expression or distribution with the onset of labour. Tissues were obtained following elective caesarean section (n=12) or normal labour delivery (n=11). Paraffin embedded and frozen sections were examined by immunohistochemistry and in situ hybridization for evidence of the type I and type II receptors and their corresponding mRNAs. In all tissues studied the type I receptor was localized mainly to the decidua and the type II receptor was localized to the decidua and scattered cells in the amnion-chorion mesenchymal layer. In situ hybridization localized type I receptor mRNAs and type II receptor mRNAs to the decidua. The type I and type II receptor protein in the decidua showed a similar pattern of staining as that found for CD-68, a macrophage marker. The pattern of receptor expression and distribution was unrelated to the mode of delivery. No evidence for the presence of the type I or type II receptor or their mRNAs in the amnion epithelial cells or chorion laeve trophoblast was found.

Nuclear matrix composition and in vitro cellular senescence.

The synthesis of nuclear matrix components from human diploid fibroblasts of different in vitro ages was analyzed. Radiolabeled nuclear matrices were prepared from human diploid fibroblasts at various stages of the cell cycle, and their components were separated by two dimensional electrophoresis. The same general electrophoretic pattern was observed at all cell cycle points analyzed, regardless of in vitro age. However, several of the more than 150 peptides that were observed exhibited some cell cycle or age-related variation in radiolabeling. Ten of these were chosen for further analysis. One peptide, with an approximate molecular weight of 47 kDa and pI of 6.8 exhibited the most significant cell cycle and age-related alterations. In matrices from younger cells, incorporation into this peptide was very low in GO but increased as these cells moved through the cell cycle, with maximum incorporation occurring in S phase. As cells neared the end of their in vitro lifespan, labeling of this peptide was elevated at all stages of the cell cycle. Since many of the functional alterations observed in senescent human diploid fibroblasts are nuclear-matrix-associated activities, these results suggest that the inappropriate expression of nuclear matrix components contribute to the functional changes which characterize in vitro senescence.

Influence of culture media on prostaglandin output by dispersed amnion cells.

The purpose of the study was to examine the influence of culture media on prostaglandin (PG) production by amnion cells and their response to phospholipase A2. Cells were dispersed from term tissue obtained by elective C-section; the PGE2 output was studied during the first 24 h of culture. The basal PGE2 production from cells cultured in Media 199 (M199) supplemented by 10% horse serum (HS) was significantly greater than that of cells cultured in F12:DME supplemented by 10% fetal calf serum (FCS). Furthermore, phospholipase A2 stimulated PGE2 production in cells cultured with M199 + HS but had no influence on PGE2 output by cells cultured with F12:DME + FCS. This effect was dependent on the presence of HS. The factor(s) in HS responsible was not removed by heating at 56 degrees C for 3 min, treatment with dextran coated charcoal or by ultrafiltration through 10,000 MW filters. Thus, culture media alters the in vitro production of PG and response to phospholipase A2 by amnion cells.

Histone gene stability during cellular senescence.

The extent to which human histone gene organization is conserved during the in vitro lifespan of human diploid fibroblast-like cells was determined by comparing the restriction patterns of a human H4 and an H3 histone gene from cells of various in vitro ages. No age related change in the organization of these two genes was detected.

Changes in the higher order organization of DNA during aging of human fibroblast-like cells.

Nucleosome spacing (DNA repeat length) was determined in human diploid fibroblast-like cells (HDF) of different in vitro ages following the electrophoretic separation of micrococcal nuclease digestion products. The results indicate that a heterogeneity of DNA repeat lengths is present in HDF of all in vitro ages. In older cells the organization of part of the DNA is conserved, but a greater proportion of shorter repeats is evident. The shorter repeat lengths are not due to nucleosome sliding, but result from the presence of shorter linker regions which are reduced by as much as 25% in part of the chromatin of high PDL cells.

Human histone gene organization. Identification of a histone gene polymorphism prevalent in a black population.

Analysis of the restriction enzyme digests of total genomic DNAs from a broad spectrum of human cell lines and from individuals with different genetic backgrounds, by hybridization with a series of cloned human histone sequences, indicated restriction site polymorphisms (RSPs) for two adjacent human histone genes which reside on chromosome 1. In most cell lines and individuals examined we observed a single 2.05 kb H4 histone HindIII fragment and a 7.0 kb H3 histone HindIII fragment. In contrast, the polymorphisms were manifested as a 2.15 kb H4 HindIII fragment and a 9.1 kb H3 HindIII fragment. From population studies, we were able to show that there is no linkage disequilibrium between these two polymorphic restriction sites. Nor was there any apparent correlation between the presence of the H3/H4 histone polymorphisms and maintenance of the transformed karyotype, passage in culture, transformation or tumor progression. These chromosome 1 H3 and H4 histone gene polymorphisms are common in the American Black population and, in our survey of individuals, were not found in the American Caucasian population. Among the American Blacks studied, the frequency of the H3 HindIII(-) allele is 43% and of the H4 HindIII(-) allele 30%. In limited family studies, we were unable to detect recombination between these two physically linked alleles.

The effects of histones, in vitro age, and culture state on the digestion of DNA by micrococcal nuclease and deoxyribonuclease I.

Nuclei from confluent and mitotically arrested populations of human diploid fibroblast-like cells were subjected to digestion by micrococcal nuclease and deoxyribonuclease (DNase I) following the removal of various histone components by salt extraction. There was no age or culture state variation in the susceptibility of DNA to micrococcal nuclease digestion. There was an age related inhibition of DNA digestion by DNase I in nuclei from older confluent cells before and after the removal of H1 histone but not after the removal of core particle histones. This inhibition was not detected in older arrested populations. These results indicate that an age-related masking by nucleosome core histones may limit the accessibility of DNA to enzymatic activities in older confluent cells. Since this inhibition was absent in older arrested populations, the importance of limited DNA accessibility as a primary cause of cellular senescence is questionable.