Human Secretory Endometrium Research Articles

Immunohistochemical localization of transforming growth factor-alpha in human endometrium, decidua, and trophoblast.

Transforming growth factor-alpha (TGF-alpha) was localized immunohistochemically in human proliferative and secretory endometrium, decidua, and trophoblast from first, second, and third trimester pregnancies. In proliferative endometrium, TGF-alpha immunostaining was moderate to intense and localized predominantly to stromal cells, whereas glandular staining was absent to light. After ovulation, TGF-alpha staining was light within the stroma, but moderate to intense around spiral arterioles. Moderate to intense staining was also detected in glandular and surface epithelium in secretory endometrium, with no staining noted in subnuclear vacuoles. In hypersecretory endometrium, staining was predominantly epithelial. In decidua, TGF-alpha was detected in intermediate trophoblast and on the surface epithelium. In first trimester trophoblast, TGF-alpha was detected in both cytotrophoblast (CT) and syncytiotrophoblast. Cytoplasmic staining was light in CT and moderate to intense in ST, with particular staining of plasma membranes. Intense TGF-alpha staining of nuclear membranes in CT was noted. TGF-alpha staining was light to absent in second and absent in third trimester trophoblast. This study demonstrates immunoreactive TGF-alpha in tissues known to be responsive to epidermal growth factor, and also demonstrates the presence of immunoreactive TGF-alpha associated with nuclear membranes. Thus, TGF-alpha may play an autocrine/paracrine role in endometrial development and trophoblast function.

Platelet-activating factor mediates phosphatidylcholine hydrolysis by phospholipase D in human endometrium.

Human preimplantation embryos and endometrium secrete platelet-activating factor (PAF). The mechanism of phosphatidylcholine (PC) degradation stimulated by PAF was investigated in endometrial explants prelabeled with [methyl-3H]choline or preincubated with [3H]butan-1-ol. Analysis of the water-soluble metabolites of PAF-induced PC hydrolysis in secretory endometrium demonstrated that the stimulated generation of [3H]choline ([3H]Cho) precedes that of [3H]choline phosphate ([3H]ChoP) and [3H]glycerophosphocholine ([3H]GPC). Within 30 sec there was a rapid rise in PAF-induced [3H]Cho generation and by 2 min this had increased to 59.9% +/- 10.6% (p less than 0.02), with no effect upon [3H]ChoP and [3H]GPC during this period. Both [3H]GPC and [3H]ChoP, however, were increased at a later time point. The slower [3H]ChoP generation may suggest that PC-specific phospholipase C activation as well as delayed [3H]GPC rise may be due to PC-specific phospholipase A2 and lysophospholipase activation. Phospholipase D activity was confirmed by the incorporation of high-specific-activity [3H]butan-1-ol into [3H]phosphatidylbutanol ([3H]PBut). The rapid generation of [3H]PBut, which paralleled the rise in intracellular [3H]Cho, strongly suggests that PC breakdown is catalyzed by the phospholipase D pathway. It is proposed that PAF induces PC hydrolysis as a consequence of an early phospholipase D-catalyzed breakdown of PC in human secretory endometrium. This may be an alternative source for prostaglandin synthesis and an important pathway essential for long-term activation of local cellular events at the time of implantation.

The gene encoding human low-molecular weight insulin-like growth-factor binding protein (IGF-BP25): regional localization to 7p12–p13 and description of a DNA polymorphism

The low-molecular weight insulin-like growth-factor binding protein (IGF-BP25) is synthesized by human liver, secretory endometrium and decidua, and is also present in human serum. It binds insulin-like growth factors IGF-I and IGF-II with high affinity, and is proposed to act as a paracrine regulator of cell growth. In situ hybridization studies with a cDNA encompassing the entire protein coding region of IGF-BP25 localized the gene to bands p12-p13 on chromosome 7. Southern blot analysis with the enzyme BglII revealed a common restriction fragment length polymorphism: the presence of the polymorphic BglII site results in the formation of two fragments 4.6 kb and 1.6 kb in size whereas its absence produces a single 6.2 kb fragment. The frequencies of the two alleles were 0.73 and 0.27, respectively. IGF-BP25 constitutes a useful genetic marker for the proximal short arm of chromosome 7.

Interfering with implantation by postcoital estrogen administration II. Endometrium epithelial cell ultrastructure

Secretory human endometrium was studied by electron microscopy after high-dose ethinylestradiol administration, a method used for interception (morning-after pill). Medicated (experimental) cycles were compared to non-medicated (control) cycles in the same volunteers. The nucleolar channel system typically present in the nucleus of the epithelial cell in secretory phase of the human endometrium disappears completely as a result of this treatment, resulting in a nucleolar basket structure. Glycogen deposits were prominent at the basal membrane and in the apex of the cell during the experimental cycle. Giant mitochondria were observed to occur to similar extent during control and treatment cycles. This study confirms the morphological delay found in light microscopic studies, published earlier with diethylstilbestrol and with ethinylestradiol.

C19 adrenal steroids enhance prostaglandin F2α output by human endometrium in vitro

Dehydroepiandrosterone sulfate significantly increased prostaglandin F2 alpha output by fragments of human secretory endometrium during the first and second 24-hour periods of incubation in Ham's F-10 medium containing 10% charcoal-treated calf bovine serum. The effects of dehydroepiandrosterone sulfate were noted at 10(-6) mol/L concentrations, which are close to the normal plasma levels of this compound. For the purpose of comparison, the effects of estradiol at 10(-8) mol/L and those of unconjugated dehydroepiandrosterone, dehydroepiandrosterone acetate, 5-androstene-3 beta, 17 beta-diol, or 5 alpha-dihydrotestosterone were evaluated at 10(-8) to 10(-8) mol/L concentrations in parallel experiments. All of the delta 5-C19 steroids tested enhanced prostaglandin F2 alpha output at 10(-6) mol/L but not at 10(-8) mol/L; 5 alpha-dihydrotestosterone was inactive at 10(-6) mol/L but showed a stimulatory effect at 10(-5) mol/L in two experiments. The stimulation of prostaglandin F2 alpha production by the adrenal steroids was significantly reduced by the antiestrogen 4-hydroxytamoxifen at 10(-6) mol/L (a finding consistent with the reported utilization of the estrogen receptor for their actions on other systems) but was not affected by the antiandrogen 4-hydroxyflutamide at 10(-6) mol/L. Progesterone (10(-7) mol/L) also lowered the effects of dehydroepiandrosterone (10(-6) mol/L) and 5-androstene-3 beta,17 beta-diol (10(-6) mol/L), as well as those of estradiol (10(-8) mol/L). delta 5-C19 steroids at 10(-6) mol/L levels did not antagonize the effect of 10(-8) mol/L estradiol, whereas 5 alpha-dihydrotestosterone reduced it by 50% at these concentrations. The significant effect of 5 alpha-dihydrotestosterone points to potential antiestrogenic effects of the C19 compounds that may be manifested in vivo at particular C19/estradiol concentration ratios. The demonstration of direct estrogenic effects of delta 5-C19 steroids, which are not significantly converted to estrogens in vivo, justifies their use in estrogen replacement preparations and indicates that aromatase inhibitors may not eliminate completely the stimulation of estrogen-responsive breast and endometrial tumors by the patient's adrenal steroids.

Soluble 34K binding protein inhibits the binding of insulin-like growth factor I to its cell receptors in human secretory phase endometrium: evidence for autocrine/paracrine regulation of growth factor action.

The human secretory phase endometrium synthesizes and secrets a 34K insulin-like growth factor (IGF)-binding protein designated placental protein 12. We now report that membrane preparations of human endometrium possess IGF receptors that complete with the soluble binding protein for binding to IGF-I. Multiplication-stimulating activity and insulin were 1% and 0.1% as potent as recombinant (Thr59)IGF-I in inhibiting the binding of [125I](Thr59)IGF-I to endometrial membranes. Scatchard plots for the IGF-I binding data were curvilinear, and the apparent affinities [Ka = 1.4 +/- 0.2 ( +/- SEM) X 10(9) M-1] for (Thr59)IGF-I (high affinity site) did not change during the menstrual cycle. Affinity cross-linking of [125I](Thr59)IGF-I to endometrial membranes revealed two major bands with mol wt of 130K and 260K on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The 130K band is consistent with the alpha-subunit of the type I IGF receptor. The 260K band is either the type II IGF receptor or represents cross-linking (dimer) of two alpha-subunits of the type I receptor. A less intense band at about 40K was also seen in all membrane preparations. It comigrated with the cross-linked purified 34K binding protein. The band was more intensely labeled when the tracer was cross-linked to proteins in the cytosol fractions of late secretory phase endometria. By specific RIA, the 34K binding protein was detected in the cytosol of late secretory phase endometria only. Newly synthesized binding protein, which contaminated membrane preparations, caused an apparent increase in the binding of (Thr59)IGF-I to the membranes prepared from late secretory phase endometria when studied by competitive binding assay. In contrast, purified binding protein prevented the binding of [125I](Thr59)IGF-I to membrane receptors, as confirmed by affinity cross-linking. These results suggest that the 34K IGF-binding protein, secreted by the human endometrium in a cyclic fashion, has a significant role in inhibiting the receptor binding and, thus, the possible biological action of IGF-I in the endometrium in an autocrine/paracrine manner.

Progesterone and antiprogestins, a comparison of their effect on prostaglandin production by human secretory phase endometrium and decidua

The mechanism by which progesterone inhibits PG production is not clear. In systems using isolated human endometrial fragments, progesterone has been shown to inhibit PG production markedly. We have used such a system to test the action of two antiprogesterone steroids RU486 (Roussell-Uclaf) and ZK98734 (Schering) on isolated human endometrial and decidual tissue, with and without added progesterone. Progesterone (200nM) reduced PGF 2α production by the secretory phase endometrium from 10.9ng/mg tissue/24 hr to1.9 ng/mg/24hr on the third day of culture (p<01) and this effect was antagonised by the addition of either 200nM RU486 or 200nM ZK98734 (6.3 and 7.2 ng/mg /24 hr respectively). The antiprogestins on their own showed a slight inhibitory effect on day 3 and RU486 treatment resulted in a significant (p<.05) decrease in PG production from control. PGE and the main 13,14-dihydro-15-keto metabolites of E and F were also significantly decreased by progesterone and restored by the aMiprogestins. The PG production by decidua increased on days 2 and 3 in response to progesterone + antiprogestins but this was not significant. This data shows that the inhibition of PG production shown by progesterone, acting on secretory phase endometrium cultured as tissue fragments, is reversible by the receptor blocking antiprogestins.

The effect of estradiol-17β and actinomycin D on the release of PGF and PGE from separated cells of human endometrium

Estradiol-17β selestively stimulated the release of PGF from separated glandular but not stromal cells of human secretory endometrium (p<0.025) but had no effect on PGF release from either type of cells obtained from proliferative endometrium. PGE release was not affected by estradiol-17β. Actinomycin D did not antagonise the effect of estradiol-17β on PGF release from secretory, glandular cells. Basal release of PGF from these cells was stimulated by actinomycin D alone (100 ng/ml) (p<0.025) and PGE release stimulated in the presence of estradiol-17β. Actinomycin D had no effect on PGF or PGE release from proliferative endometrium. These findings suggest that estradiol-17β stimulates PGF release by a mechanism that does not affect PGE release and which is not dependent on the synthesis of new protein. The basal release of PGF and PGE by glandular cells of secretory endometrium in vitro is regulated by protein/proteins which reduce PG release.

Stress proteins in the human endometrium and decidua.

Protein synthesis in response to heat shock was induced in the proliferative and secretory human endometrium as well as in human decidua during a 2-h incubation period at 41 degrees C. A major 70 K stress protein and two minor stress proteins of 88 and 94 K were detected after [35S]methionine incorporation followed by SDS polyacrylamide gel electrophoretic separation and autoradiography of dried gels. Two dimensional isoelectric focusing followed by fluorography showed the major 70 K stress protein to consist of at least six polypeptides of pH 4.6 to 5.5, the 88 K to consist of at least four polypeptides, of pH 5.5 to 6.0, and the 94 K to consist of at least three polypeptides of pH 4.6 to 5.2. Stress proteins in the human endometrium and decidua may prove to be of physiological significance in reproductive events.

Progesterone-associated proteins PP12 and PP14 in the human endometrium

Two proteins, designated as PP12 and PP14 were originally isolated from soluble extracts of the human placenta and its adjacent membranes. We have shown that they are synthesized by decidualized/secretory endometrium and not by placenta. Both proteins occur at high concentrations in human amniotic fluid, which is therefore an excellent source for purification. PP12 is a 34-kDa glycoprotein, which has an N-terminal amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala. This is identical with that of somatomedin-binding protein purified from the amniotic fluid. PP12 too binds somatomedin-C, or IGF-I (insulin-like growth factor-I). Human secretory endometrium synthesizes and secretes PP12, and progesterone stimulates its secretion. PP14 is a 28-kDa glycoprotein. Its N-terminal sequence shows homology to that of beta-lactoglobulins from various species. We have found PP14 in the human endometrium, serum and milk. Immunologically, PP14 is related to progestagenassociated endometrial protein (PEP), alpha-2 pregnancy-associated endometrial protein (alpha-2, PEG), endometrial protein 15 (EP15), alpha-uterine protein (AUP) and chorionic alpha-2 microglobulin (CAG-2). In ovulatory menstrual cycles, the concentration of PP14 increases in endometrial tissue as the secretory changes advance. In serum, the PP14 concentration begins to rise later than the progesterone levels, and hih serum PP14 levels are maintained for the first days of the next cycle. By contrast, no elevation of serum PP14 level is seen in anovulatory cycles. Our results show that progesterone-associated proteins are synthesized by the human endometrium and appear in the peripheral circulation, where they can be quantitatively measured using immunochemical techniques.

Estradiol enhances prostaglandin synthase activity in epithelial but not in stromal cells of human endometrium

Exogenous estradiol (E 2) has been shown to elevate PGF 2α output by explants of human secretory endometrium and in monolayer cultures of glandular epithelial, but not of stromal cells isolated from endometrium. In this study, PGF 2α output was measured in each of these cultures in the presence of E 2 and the calcium ionophore A23187, added singly or in combination. The ionophore, known to liberate arachidonic acid (AA) by stimulating phospholipase activity, produced a calcium-dependent increase in PGF 2α output in the cultures of epithelial cells, whereas greater than additive effects were obtained with mixtures of E 2 and A23187. In contrast, PGF 2α levels were not elevated by A23187 in the stromal cell cultures even in medium supplemented with CaCl 2 or when E 2 was added. A calcium-dependent increase in PGF 2α output was also observed in fragments of secretory endometrium incubated with A23187. Effects on PGF 2α output by endometrial fragments incubated with E 2 and A23187 were essentially additive and intermediate between those of the two component cells types. Arachidonic acid produced similar increases in PGF 2α output in the epithelial and stromal cell cultures but only in the epithelial cell cultures was there greater utilization of AA in the presence of Ez. When mixtures of Ez and AA were added to the cultures of epithelial cells the increase in PGF 2α output was 2.5-fold greater than the sum of the increases elicited by E 2 or AA alone. In contrast, no enhancement of the AA effect by E 2 was observed in the stromal cell cultures. Extrapolation of these results from cell cultures to intact tissue suggests that the epithelium and not the stroma is the primary target for the effects of E 2 on PGF 2α output by secretory endometrium. The synergistic actions of E 2 and either AA, the obligatory precursor of PGF 2α, or A23187, an enhancer of AA release from phospholipid stores, point to a stimulatory effect of E 2 on prostaglandin synthase activity.

Uterine fluid and prolactin secretion in the ovulating cynomolgus monkey

Immunoreactive prolactin is produced by late secretory human endometrium in vitro. Human myometrium in explant culture also produces prolactin. A primate model with the use of the cynomolgus monkey is described that allowed repeated samplings of uterine secretions in vivo. The uterine secretory prolactin thus measured appears immunoreactively similar to human serum prolactin, and the pattern of secretions reflects the previously described pattern of endometrial prolactin production in vitro.

Prostaglandin F 2α output by human endometrium under superfusion and organ culture conditions

Specimens of proliferative and secretory human endometrium were incubated under organ culture or superfusion conditions and the levels of PGF 2α in the medium were measured by radioimmunoassay. Basal rates of PGF 2α output during short-term superfusions and long-term (1–2 day) batch incubations, performed on the same tissue specimens, were similar. Basal output of PGF 2α by proliferative endometrium (230–280 ng/mg protein × d) was significantly higher than that of secretory tissue under both experimental conditions. Estradiol (10 −8 M) increased PGF 2α output significantly (4-fold) only in secretory endometrium under organ culture conditions; Progesterone (10 −7 M) decreased it significantly (to 1 2 − 1 4 of the basal level) in both types of endometria during long-term incubations and in proliferative endometrium during superfusion. Glands isolated from proliferative and secretory endometrium produced PGF 2α during superfusion at a rate comparable to that of endometrial tissue under similar conditions. PGF 2α output by glands isolated from secretory endometrium increased significantly (3-fold) when estradiol was added to the superfusion medium.

Cholesterol esterification in human secretory endometrium and in endometrial cancer tissue. Demonstration of microsomal acyl-CoA-cholesterol acyl-transferase (ACAT) activity.

Human secretory endometrium and endometrial cancer tissue both contain acyl-CoA:cholesterol-acyltransferase (ACAT). The optimal assay conditions for measuring ACAT activity in microsomes prepared from these tissues were determined. The activity in normal secretory endometrium was 15.3 +/- 1.27 pmol cholesteryl ester formed per mg protein-1 per min-1, whereas 3 out of 4 endometrial cancers had 3-7-fold higher ACAT activity.

Rapid fluctuations in the levels of specific estradiol-binding sites in endometrial cells in culture.

Levels of specific estradiol (E2) binding sites were measured during 5–6 consecutive days in primary cultures of stromal and epithelial cells from 7 specimens of human proliferative and secretory endometrium. Large day to day variations were noted, as we previously reported. Levels of binding sites were also measured hourly (1–30 h after plating) in 11 cultures of an endometrial adenocarcinoma cell line (HEC). In these experiments, striking increases and decreases were found to occur within periods as short as 2 h. The rapidity of these changes suggests reversible unmasking or activation of binding sites for E2, rather than new synthesis of binders. Elevated levels were found immediately before periods of accelerated increase in the amounts of DNA per dish, both in cultures of endometrial fibroblasts and in partially synchronized cultures of HEC cells, indicating a possible relation between levels of specific binding sites and the cell cycle. However, since elevated levels have also been found in nongrowi...

STUDIES ON 17β-HYDROXYSTEROID DEHYDROGENASE IN HUMAN ENDOMETRIUM AND ENDOMETRIAL CARCINOMA

ABSTRACT Microsomal 17β-hydroxysteroid dehydrogenase obtained from the human secretory endometrium (17β-HSD) was solubilized with triton X-100. A 4-fold purification was achieved by ammonium sulphate precipitation and isoelectric focusing. In the presence of glycerol the partially purified enzyme was stable at 4°C for at least 48 h. Using crude microsomes, the conversion of oestradiol to oestrone was linear with time and with the concentration of protein. The optimum temperature was approximately 40°C and the optimum pH 9.4. For the reduction of oestrone the optimum pH was 6.5. With NAD, oestradiol was oxidized approximately three times more rapidly than with NADP. Km-values for oestradiol were nearly the same in endometrial carcinoma and in proliferative and secretory endometrium (i. e. approximately 3 × 10−6 m). The maximal velocity was highest in secretory endometrium. Testosterone and androstenedione could also serve as substrates but they were interconverted more slowly than oestradiol and oestrone. Sulphhydryl groups were shown to be essential for catalysis.

STUDIES ON 17β-HYDROXYSTEROID DEHYDROGENASE IN HUMAN ENDOMETRIUM AND ENDOMETRIAL CARCINOMA

Kinetic parameters, substrate specificity and stability of a cytoplasmic 17beta-hydroxysteroid dehydrogenase of human secretory endometrium were studied. Using oestradiol as substrate, oestrone formation was found to be linear with time and the concentration of protein. The optimum temperature was 40 degrees C and the optimum pH 9.5. For the reduction of oestrone the optimal pH was 6. With NADP the maximal velocity was about 1/3 of that with NAD (0.23 nmoles/mg protein/10 min). The Km for oestradiol was 3.3 times 10- minus 6 M. Testosterone and androstenedione also served as substrates but they were interconverted more slowly than oestradiol and oestrone. Sulphhydryl groups were shown to be essential for catalysis. The enzyme is cold sensitive but cold inactivation can be reduced by NAD, NADP, oestradiol or glycerol.

Effect of intrauterine copper on the nucleic acids, polysome pattern, and glycoprotein composition of the human endometrium

The modifications induced by the intrauterine release of copper in the macromolecular and glycoprotein composition of the proliferative and secretory human endometrium were studied. In addition, the endometrial changes produced in women users of copper-T intrauterine devices in the polysome pattern and in the content of ribonucleoprotein particles were determined during the secretory phase. A group of ten untreated normal women (control group) and 15 users of 200 mm. 2 copper-T intrauterine device were selected for this study from the outpatient clinic of the Hospital de Ginecologia y Obstetricia No. 2 del Instituto Mexicano del Seguro Social. The main changes observed in the copper-T users were: a significant decrease in the endometrial content of RNA in both phases of the menstrual cycle, a significant decrease in protein in the secretory phase, and drastic changes in the fucose-sialic acid ratios, which decreased during the proliferative and increased during the secretory phases. Normal human secretory endometrium contained 4.86 ± 0.28 (mean ± standard error [S.E.]) mg. ribonucleoprotein particle per gram wet weight, while the endometrium of the Cu-T users showed a significant decrease to 2.52 ± 0.17 (mean ± S.E.). In addition, the Cu-T induced a decrease in the heavy components of the polysome pattern with a concomitant increase in the lighter components.

Subcellular distribution of trace metals in the normal and in the copper treated human secretory endometrium

The subcellular distribution of some metals has been studied in the normal human secretory endometrium and in the secretory endometrium of women wearing a copper-T intrauterine device. The sustained release of copper inside the uterine cavity induces some significant changes in the metal composition of this tissue; Mg, Ca and Cu increase while Zn decreases. Neither Na nor K concentrations show any significant change. Under these conditions, endometrial nuclei concentrations seem to be able to passively come to equilibrium with the tissue concentrations of the elements studied, showing only a significant retention of Ca. On the contrary, mitochondria show differential concentrations of all metals. Ca is retained, but all the other elements remain at low levels. Copper is significantly increased in all fractions, but the relative increase is particularly noticeable in the microsomes (6 x). Zinc tends to decrease in all fractions but reaches statistical significance only in the microsomes and the supernatant. These results are analyzed in relation to the mechanism of action of the Cu-T.

Factors controlling intracellular levels of estrogens in human endometrium

The levels of estradiol 17β-dehydrogenase activity have been found to be more than 10 times higher in secretory human endometrium than in proliferative tissue. Since in vitro experiments indicated that the bulk of estradiol (E 2) entering endometrial cells is released back to the medium as estrone (E 1), the effect of changes in the E 217β-dehydrogenase activity on the intracellular concentrations of E 2 and E 1 could be predicted and verified experimentally. In the luteal phase of the menstrual cycle E 2 presented to endometrium contributes less to the intracellular E 2 pool and more to the E 1 pool than an equal amount of E 2 presented to endometrium in the proliferative phase. Estradiol and not E 1 showed tight binding to nuclear pellets after the incubation of slices of endometrium with labeled estrogens. The observed competitive effect of E 1 on the binding of E 2 could be accounted for by the intracellular conversion of E 1 to E 2. The total amount of estradiol receptor levels was found to be drastically reduced during the luteal phase. Preliminary in vivo experiments indicated that progesterone is responsible for this reduction and for the increase of the 17β-dehydrogenase activity. These observations point to means of regulating E 2 receptor levels and E 2 intracellular concentrations in normal tissue and possibly in endometrial cancer.