M.Linette Casey

Initiation of human parturition: XII. Biosynthesis and metabolism of prostaglandins in human fetal membranes and uterine decidua

Prostaglandin synthetase activity was assayed in homogenates prepared from human fetal membranes and uterine decidua tissues with [14C] arachidonic acid used as substrate. In amnion, chorion laeve, and decidua vera tissues obtained after the spontaneous onset of labor and vaginal delivery the mean specific activities of prostaglandin synthetase were respectively 1.47, 0.53, and 0.39 nmoles x hr-1 x gm-1 tissue; in these same tissues obtained at elective cesarean section before the onset of labor the mean specific activities of prostaglandin synthetase were respectively 0.61, 0.33, and 0.28. The specific activity of prostaglandin synthetase of amnion (but not chorion laeve or decidua vera) was significantly greater in samples obtained after labor than in those obtained before labor (p less than 0.02). Prostaglandin synthesis in amnion, chorion laeve, and decidua vera was inhibited by acetylsalicylic acid and by indomethacin. Prostaglandin E2 was formed in amnion and chorion laeve, whereas prostaglandin E2 and prostaglandin F2 alpha were formed in decidua vera. The activity of NAD+ -dependent 15-hydroxyprostaglandin dehydrogenase was also assayed and the specific activities of this enzyme in chorion laeve and decidua vera tissues obtained before and after labor were high, whereas 15-hydroxyprostaglandin dehydrogenase activity in amnion was low.

Regulation of interleukin-8 gene expression in human endometrial cells in culture

In this study, we investigated the regulation of interleukin-8 (IL-8) gene expression in separated endometrial stromal and epithelial cells of human endometrium. This research was conducted as part of an analysis of the role of these cells in regulating the recruitment of leukocytes to the endometrium. Well-characterized model systems were used to study the regulation of endometrial IL-8 gene expression, namely, stromal cells in monolayer culture after first passage and glandular epithelium in primary culture. The levels of IL-8 mRNA and the accumulation of immunoreactive IL-8 in the medium of endometrial stromal cells is culture increased in a time- and concentration-dependent manner upon treatment with IL-1 alpha, tumor necrosis factor-alpha, or serum. The effects of IL-1 alpha plus serum on IL-8 mRNA levels were at least additive. Serum treatment caused a modest stimulation of IL-8 gene transcription (evaluated after 6 h of treatment) in endometrial stromal cells, but serum also acted in these stromal cells to prolong the half-life of IL-8 mRNA by more than 2.5-fold. The regulation of the levels of IL-8 mRNA in endometrial epithelial cells is distinctly different from that in stroma. First, the levels of IL-8 mRNA in non-treated epithelial cells in serum-free medium were much greater than those in stromal cells under similar conditions. Second, whereas the levels of IL-8 mRNA in endometrial epithelial cells also increased in response to serum and to IL-1 in the absence of serum, in the presence of serum, IL-1 treatment caused no appreciable change in the levels of IL-8 mRNA as was the case in endometrial stromal cells.

A COMPARISON OF HUMAN AMNION TISSUE AND AMNION CELLS IN PRIMARY CULTURE BY MORPHOLOGICAL AND BIOCHEMICAL CRITERIA

SummaryProstaglandins, which are believed to mediate the initiation or maintenance of human labor or both, are synthesized from arachidonic acid. We have shown previously that the arachidonic acid content of two glycerophospholipids (diacyl phosphatidylethanolamine and phosphatidylinositol) in amnion tissue obtained during early labor is decreased compared with that in amnion tissue obtained before labor commenced. We and others have demonstrated that amnion tissue synthesizes prostaglandin E2 from exogenous and endogenous arachidonic acid. Thus, the amnion may be a source of prostaglandins involved in the initiation of parturition. We have investigated whether amnion cells in monolayer culture could be utilized as an in vitro model system for the study of glycerophospholipid and arachidonic acid metabolism in amnion. Several morphological biochemical, and enzymatic characteristics of amnion cells in culture were compared with those of amnion tissue. Morphologically, the amnion cells in culture and amnion tissue are similar. The lipid composition and arachidonic acid content of lipid fractions of amnion cells in culture and of amnion tissue also are similar. The specific activities of phospholipases A2 and C, the enzymes that initiate and thus probably regulate the release of arachidonic acid from phosphatidylethanolamine and phosphatidylinositol, are lower in amnion cells in culture than in amnion tissue obtained from term placentas. The specific activities of diacylglycerol lipase, monoacylglycerol lipase, and diacylglycerol kinase, enzymes that catalyze the release of arachidonic acid from diacylglycerol produced by the action of phospholipase C on phosphatidylinositol, are similar in amnion cells in culture and in amnion tissue. Therefore, we conclude that, based on morphology, lipid composition and enzymatic activities amnion cells in primary culture seem to be an appropriate in vitro model system for the investigation of the regulation of arachidonic acid metabolism in amnion.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity in human endometrium.

The specific activity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase was measured in human endometrial tissue obtained from ovulatory and anovulatory women. Employing PGE2 as substrate, the specific activity of this enzyme was found to be highest in endometrial tissue during the secretory phase of the cycle (ovarian cycle days 15-25) and lowest in menstrual (days 1-5) and premenstrual (days 26-28) endometrium. The specific activity of prostaglandin dehydrogenase in endometrium of anovulatory women was low, being similar to that found in proliferative endometrium (days 6-14) of ovulatory women. Prostaglandin dehydrogenase activity was found in the cytosolic fraction prepared from endometrial ti-sue, and was found principally in the glandular epithelium following separation of endometrial glands and stromal cells.

Endothelin increases cytoplasmic calcium and myosin phosphorylation in human myometrium

Endothelin, a recently discovered sarafotoxin-like peptide secreted by endothelial cells, is a potent stimulator of vascular smooth muscle contraction. We found that the action of endothelin is not restricted to the vasculature; we demonstrated that endothelin causes an increase in the concentration of intracellular Ca++ and phosphorylation of the 20 kd light chain of myosin in human uterine smooth muscle cells in culture. In the absence of Ca++ in the buffer medium of myometrial cells, the effects of endothelin on intracellular Ca++ and myosin light chain phosphorylation are attenuated but not abolished. Endothelin also increases the frequency of contraction of human uterine smooth muscle (longitudinal and circular). The contractile effects of endothelin on myometrial strips are diminished in the presence of nifedipine. We conclude that (1) human myometrium is responsive to endothelin, (2) endothelin promotes contraction in myometrium by effecting an increase in intracellular Ca++ and thus an increase in myosin light chain phosphorylation, and (3) endothelin acts in myometrium by stimulating Ca++ influx as well as Ca++ release from intracellular stores.

Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture

SummaryHuman myometrial cells were dispersed from uterine tissue by limited enzymatic digestion of myometrium that was obtained at the time of hysterectomy. The dispersed myometrial cells that are obtained in this manner can be maintained in monolayer culture in the presence of medium that contains fetal bovine serum. In primary culture, as well as after passage, the characteristics of these cells are morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue.

Regulation of monocyte chemotactic protein-1 gene expression in human endometrial cells in cultures

Bone marrow-derived leukocytes are present in human endometrium/decidua and are believed to serve a variety of functions in this tissue. The number and type of leukocytes in endometrium/decidua vary with the hormonal milieu of the ovarian cycle, with blastocyst implantation, and during pregnancy. The factors that regulate the recruitment of specific leukocytes to the endometrium and those that modulate the function or replication of leukocytes in this tissue are not well defined. In this study, we evaluated the potential for synthesis of monocyte chemotactic protein-1 (MCP-1), a polypeptide with monocyte/macrophage chemotactic and activating properties, in human endometrium and in separated endometrial stromal and epithelial cells. MCP-1 mRNA was readily detected by northern analysis of total RNA isolated from human endometrial tissue (n = 39 tissues from ovulatory women; n = 3 atrophic endometria from anovulatory women; n = 6 from women ingesting oral contraceptives or medroxyprogesterone acetate) and decidua parietalis at midtrimester (n = 6 pregnancies) and at term (n = 6 pregnancies). The levels of MCP-1 mRNA varied considerably among tissues; but in this relatively small number of samples, there was no apparent relationship between day of cycle, endocrine status, or duration of pregnancy and the level of MCP-1 mRNA. MCP-1 mRNA was detected in separated endometrial stromal cells and epithelial cells in culture. In confluent human endometrial stromal cells in the absence or presence of fetal bovine serum (10%, v/v), MCP-1 mRNA was detected by northern analysis of total RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Decidual activation in parturition: examination of amniotic fluid for mediators of the inflammatory response.

The accumulation of bioactive agents (characteristic of an inflammatory-type response) in amniotic fluid is common during term and preterm labor, viz., interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). In addition, prostaglandins, including PGE2, PGF2 alpha, and PGFM, also accumulate in amniotic fluid in some cases of term and preterm labor. From these observations, a number of critical questions arise. Namely, 1) what is the tissue source of origin of these agents?; 2) what are the stimuli that evoke this inflammatory response?; and, 3) are these bioactive agents of inflammation involved in the commencement of labor or else a natural accompaniment of the parturition process? It is reasonable to suspect that the decidua is activated during parturition as the membranes-decidua are exposed after cervical dilation to the vaginal/cervical secretions. Amnion and chorion laeve, in the human, are avascular tissues that produce PGE2 but not PGF2 alpha. Therefore, the accumulation of PGF2 alpha and PGFM in amniotic fluid during labor cannot be attributed to a fetal membrane origin. Moreover, the fetal membranes and decidua do not convert PGE2 to PGF2 alpha. In addition, the fetal membranes do not produce mature, i.e., secreted 17kD IL-1 beta. On the other hand, the decidua does produce PGF2 alpha and PGFM and is stimulated to do so by agents in the vaginal secretions, namely, bacterial endotoxin and IL-1 beta. After the fetal membranes and contiguous decidua are exposed during the time of cervical dilatation, these tissues are acted upon to cause 1) an influx of mononuclear phagocytes into the forebag compartment of the amniotic fluid; 2) to produce PGF2 alpha and PGFM; and 3) to produce cytokines, including IL-1 beta, IL-6, and TNF-alpha. Exposure of the fetal membranes-decidua to bioactive agents in vaginal/cervical secretions will effect an inflammatory response both in vivo and in vitro. We conclude that the accumulation of bioactive agents characteristic of the inflammatory response in amniotic fluid during term and preterm labor is usually an accompaniment of parturition and not its cause.

Expression and regulation of endothelin precursor mRNA m avascular human amnion

Posttranslational processing of preproendothelin in endothelial cells gives rise to endothelin, a 21 amino acid polypeptide that is a potent vasoconstrictor. Endothelin production is believed to be mediated principally by transcriptional mechanisms. Previously, preproendothelin mRNA expression has been detected only in vascular endothelial tissue and cells. In this study, we found that preproendothelin mRNA is expressed in an avascular human tissue, namely, amnion, an extraembryonic fetal membrane. Preproendothelin mRNA was not detected in avascular chorion laeve tissue (also an extraembryonic fetal membrane), in the highly vascularized fetal trophoblast, or in maternal uterine tissues. Furthermore, we found that preproendothelin gene expression is retained in human amnion cells maintained in primary monolayer culture. Using the amnion cells in primary monolayer culture to investigate the regulation of preproendothelin mRNA expression, we found that epidermal growth factor (EGF) and interleukin-1 (IL-1) act to stimulate preproendothelin mRNA levels; in addition, the induction of preproendothelin mRNA by either of these agents is enhanced upon simultaneous treatment with cycloheximide. These findings are indicative that preproendothelin gene expression in amnion is regulated positively by EGF and IL-1 and that inhibition of protein synthesis leads to superinduction of preproendothelin mRNA. In human umbilical cord endothelial cells, neither IL-1 nor EGF stimulate preproendothelin mRNA expression but inhibition of protein synthesis does lead to increased levels of preproendothelin mRNA. The amnion, therefore, provides a useful system for expansion of our understanding of the tissue specific expression and regulation of preproendothelin mRNA.

Responsiveness of human endometrial stromal cells to cytokines.

Cytokines are known to act in a variety of tissues, most commonly in a paracrine manner, to effect a number of biochemical processes. Previously, we found that human endometrial stromal cells respond to the action of interleukin-1 (IL-1) with an increase in the production of prostaglandins. In these investigations, we also found that IL-1 acts in endometrial stromal cells to stimulate the synthesis of IL-1 and IL-6 mRNA and protein. Specifically, in human endometrial stromal cells maintained in monolayer culture, treatment with IL-1 alpha leads to a striking increase in the synthesis of IL-1 beta mRNA and protein; this increase is IL-1 alpha-dose- and time-dependent. The pro-IL-1 beta produced, however, is not secreted into the culture medium but is retained within the stromal cell. The failure of secretion of IL-1 beta is characteristic of non-monocyte/macrophage cell types; this obtains because the enzyme that effects processing of pro-IL-1 beta (31 kDa) to the mature, secreted form of IL-1 beta (17 kDa) is believed to be present only in monocytes/macrophages. We also find that IL-1 and tumor necrosis factor-alpha (TNF-alpha) act in endometrial stromal cells to stimulate the synthesis of interleukin-6 (IL-6) mRNA and protein; the IL-6 produced by these cells is secreted into the culture medium. In addition, we find that IL-1 acts in endometrial stromal cells to inhibit the expression of mRNA for connexin43, a gap junction protein that is believed to be the principal component of gap junctions in cardiac and smooth muscle. Thus, it is likely that IL-1 action leads to a decrease in gap junction-dependent intercellular communication among endometrial stromal cells. Based on these findings, we conclude that endometrial stromal cells are responsive to the actions of IL-1 and TNF-alpha. These cells synthesize both IL-1 and IL-6; and, IL-6 is released into the extracellular medium. Thus, the possibility exists that the synthesis and action of cytokines may be involved in the mechanisms that serve to regulate the mesenchymal-epithelial interactions between endometrial stromal and glandular components; and, the formation and action of cytokines in decidua may serve to modulate immunological and infectious challenges encountered by this tissue in pregnancy.