ABSTRACT

Pregnancy-associated endometrial α₁-globulin (α₁-PEG) is quantitatively the major secretory protein product, synthesized and secreted in vitro, of the human decidualized endometrium during pregnancy. This protein has been purified from cytosolic extracts of this tissue and has now been characterized as a 32 kDa somatomedin/insulin-like growth factor (IGF)-binding protein. Immunoreactive α₁-PEG isolated from amniotic fluid exhibited identical physiochemical properties and IGF-I-binding characteristics. In cytosolic extracts of pregnancy endometrium, in incubation medium of this tissue and in amniotic fluid, the 32 kDa protein represented the major α₁-PEG immunoreactive protein and major IGF-I-binding component. Purified α₁-PEG and incubation medium of pregnancy endometrium competed for IGF-I with placental membrane IGF receptors in vitro. The implications of the endometrial source of IGF-I-binding protein are dicussed with reference to the origin of the amniotic fluid and serum small M _r IGF-binding protein and to the suggested paracrine effect upon trophoblast proliferation.

J. Endocr. (1988) 118, 317–328

The time-course of cell hypertrophy and changes in in-vitro rates of secretion and synthesis of protein in intercaruncular and caruncular endometrium and maternal and fetal cotyledonary placenta have been examined during days 0–112 of pregnancy in the ewe. The concentrations of high-affinity receptors for oestradiol and progesterone in nuclear and cytosol fractions from these tissues were also determined.

Protein secretion by intercaruncular endometrium increased 25-fold between days 0 and 84. On day 84 10⁻⁵ m-colchicine blocked 75% of total secretion. Protein secretion did not increase in the other tissues. Protein synthesis and RNA: DNA ratio in intercaruncular endometrium increased steadily between days 0 and 112, whereas they did not change in caruncular endometrium between days 0 and 28 and declined in cotyledon between days 56 and 112. The levels of cytosol receptor for oestradiol and progesterone and of nuclear receptor for oestradiol in all tissues during days 56–112 were very low in relation to the corresponding levels in caruncular endometrium on day 0. The level of nuclear progesterone receptor in caruncular endometrium increased threefold between oestrus and day 28. The level of this receptor in cotyledon remained low on days 56–112, but in intercaruncular endometrium it increased to high values on days 84–112.

The results demonstrated a major surge in secretory activity by the intercaruncular endometrium at around mid-gestation, which was associated with a marked increase in nuclear progesterone receptor levels but only a low level of nuclear oestradiol receptor. The observations do not suggest any important role for oestradiol or progesterone in the growth of fetal and maternal cotyledon.

ABSTRACT

The present study was designed to determine the localization of the endometrial oxytocin receptor during the ovine oestrous cycle, particularly on day 14, the time of initiation of luteal regression in the ewe. Samples were obtained from 29 ewes at different stages of the oestrous cycle (several during the luteal phase and on every day between day 14 (− 2) and day + 3 of the oestrous period).

Oxytocin receptors were localized autoradiographically in sections of uterine tissue, using the ¹²⁵I-labelled oxytocin receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr²,Thr⁴,Orn⁸,Tyr⁹-NH₂]-vasotocin (¹²⁵I-labelled OTA). There was some variation in the pattern of ¹²⁵I-labelled OTA labelling between different uterine tissue samples from the same ewe and also between samples obtained from different ewes thought to be at the same stage of the oestrous cycle. A clear overall pattern did, however, emerge with ¹²⁵I-labelled OTA-binding sites distributed between luminal epithelial cells, glandular epithelial cells and caruncular stromal cells to varying extents on different days of the cycle.

During the luteal phase (days 5–12) clear specific labelling of endometrial tissue was generally absent. On day 14 labelling was evident on the luminal epithelium, but only in nine tissue samples out of a total of 18 studied, indicating that the entire luminal surface did not contain oxytocin receptors at this time. Between the day before oestrus and day 3 of the oestrous cycle the luminal epithelium was consistently labelled. The most extensive labelling of the remaining endometrial tissue was observed on the day of oestrus, with ¹²⁵I-labelled OTA-binding sites clearly present on the stromal cells within caruncles and on a large proportion of secretory epithelia. This contrasted with the day before and the day after oestrus when labelling of glandular tissue was confined to the superficial endometrium, and labelling of caruncular stromal cells, although sometimes evident, was never as intense as on day 0. On days 2 and 3 labelling varied between being similar to that found on day 1 and being confined to the luminal epithelium and very few superficial secretory glands.

The results of this study lead us to conclude that the oxytocin receptor shows a differential distribution between stromal cells, epithelial cells lining secretory glands and luminal epithelial cells during the oestrous cycle; that the steroidal regulation of the oxytocin receptor differs between endometrial cell types; and that control of the luminal epithelial oxytocin receptors is probably of particular importance to the regulation of prostaglandin F_2α release at luteal regression and during the maternal recognition of pregnancy.

Journal of Endocrinology (1991) 130, 199–206

SUMMARY

Human endometrium in the regenerative phase was maintained for 5 hr. in vitro with oestradiol-17β alone or together with actinomycin D. Qualitative and quantitative histochemistry of the tissue showed that the activities of glucose-6-phosphate, 6-phosphogluconate, and lactate dehydrogenases were increased by oestradiol, and that actinomycin suppressed the hormonal effect. The activities of succinate and iso-citrate dehydrogenases were unaffected by oestradiol. An attempt is made to correlate the metabolic roles of the enzymes affected by oestradiol. The suppression, by actinomycin, of the oestradiol effect suggests that the increased enzyme activity is due to the formation of new enzyme protein.

ABSTRACT

Phospholipase C and 1,2-diacylglycerol lipase activities were demonstrated in human endometrium using 1-stearoyl-2-[1-¹⁴C]arachidonyl phosphatidylinositol as substrate. Phosphatidylinositol is hydrolysed by phospholipase C to inositol phosphates and to 1,2-diacylglycerol which is then further metabolized by 1,2-diacylglycerol lipase to release free arachidonic acid. In the present study the radiolabelled products formed (1,2-diacylglycerol and arachidonic acid) were measured following chloroform/methanol extraction and thin-layer chromatography. Phospholipase C activity was calcium dependent and optimal at pH 5·0–5·5 and 7·5; 1,2-diacylglycerol lipase activity was also calcium dependent, with an optimum pH of 5·5. A significant increase in 1,2-diacylglycerol production was stimulated by steroid sulphates. Pregnenolone sulphate, oestrone sulphate, testosterone sulphate and dehydroepiandrosterone sulphate stimulated 4, 3·2-1·8- and 2·6-fold increases in release respectively. Oestradiol sulphate stimulated a 25% increase in diacylglycerol release which was not significantly different from the control value. Progesterone stimulated a fourfold increase but other free steroids had no effect. Arachidonic acid release was increased in the presence of oestradiol sulphate, oestrone and oestradiol but reduced by oestrone sulphate, dehydroepiandrosterone sulphate, progesterone, dehydroepiandrosterone and, to a lesser extent, by pregnenolone sulphate and testosterone sulphate. 5-Androstene-3β, 17β-diol had no effect on the liberation of either product.

This study demonstrates a potential route for the liberation of arachidonic acid from phosphatidylinositol in human endometrium. The opposing effects of steroids on phospholipase C and 1,2-diacylglycerol lipase activity could be important in regulating the release of arachidonic acid by this pathway.

J. Endocr. (1988) 117, 309–314

ABSTRACT

The purpose of this investigation was to assess the applicability of two well established procedures: (i) the product isolation assay and (ii) the radiometric ³H₂O assay for the determination of very low levels of aromatase activity. The methods were validated and used to assess the capacity of normal and neoplastic human endometrium to synthesize oestrogens from androgens. Using the product isolation assay, various specimens (n = 27) of normal and neoplastic endometrium were incubated with [1,2,6,7-³H]testosterone either by a standard incubation procedure or by a superfusion technique. Following the incubation, carrier oestrone and oestradiol or [¹⁴C]oestrone and [¹⁴C]oestradiol were added, and the oestrogens were isolated and purified by paper chromatography and high-performance liquid chromatography. The radiochemical purity of oestrone and oestradiol was checked by the isotope dilution technique. In all samples, the ³H associated with oestrone and oestradiol failed to recrystallize as oestrone and oestradiol. No radioactivity was detectable in the oestrone and oestradiol crystals after acetylation. Similarly, 16 endometrial samples were tested for aromatase activity by the ³H₂O release assay using [1β-³H]androstenedione as substrate. The results indicate that ³H₂O was indeed released during these incubations, but this activity could not be inhibited by the aromatase inhibitor 4-hydroxyandrostenedione, by excess substrate or by heat inactivation of the tissue. Furthermore, the release of ³H₂O from [1β-³H]androstenedione under the incubation conditions used (Dulbecco's modified Eagle's medium or RPMI-1640 containing fetal bovine serum and NADPH) also occurred in the absence of any tissue. This activity was not inhibited by 4-hydroxyandrostenedione nor by excess substrate. The results demonstrate that the human endometrium does not contain detectable levels of aromatase activity and that the radiometric assay can give rise to false-positive results if used for detection of very low levels of aromatase activity.

Journal of Endocrinology (1990) 127, 539–551

Increased prostaglandin production by tissues in the sheep uterus and placenta are thought to be important for the onset of parturition. In the sheep placenta, this is most likely due to increased expression of prostaglandin synthase type-2 (PGHS-2) rather than prostaglandin synthase type-1 (PGHS-1). However, there is no information concerning expression of PGHS isoenzymes in maternal uterine tissues during pregnancy. Therefore, the purpose of the present study was to examine the expression of PGHS-1 and PGHS-2 in the sheep myometrium and endometrium during late gestation using in situ hybridization and immunohistochemistry. Using (35)S-labelled oligonucleotide probes, which give specific hybridization signals in other tissues, we localized PGHS-2 mRNA to endometrial epithelium, and apparently to other cells in both endometrium and myometrium. This artefactual signal was still present with 100-fold excess unlabelled oligonucleotide probe and with sense probes, but was resolved with the use of (33)P-oligonucleotides. Using (33)P-labelled oligonucleotide probes we could not detect either PGHS-1 or PGHS-2 mRNA in myometrium, and found expression only of PGHS-2 mRNA in endometrium. PGHS-2 mRNA localized to the endometrial epithelium and was undetectable in glandular epithelium. The level of PGHS-2 expression rose significantly between days 80 and 85 of pregnancy and term, and this corresponded to the appearance of immunoreactive PGHS-2 protein, measured by immunohistochemistry, in the endometrial epithelium. Therefore we conclude that (33)P-labelled probes are preferred for detection of mRNAs encoding PGHS-2 in ovine uterine tissues. Expression of PGHS-2 mRNA is greater than that of PGHS-1, increases during gestation, and predominates in the endometrial epithelium, consistent with the site of PGHS-2 protein localization.

The effects of oestrogen are mediated by two specific intracellular receptors, oestrogen receptors (ER) alpha and beta, which function as ligand-activated transcriptional regulators. Ovariectomized macaques (Macaca fascicularis) were used to study the regulation of ERalpha and ERbeta in the endometrium by immunohistochemistry and in situ hybridization after long-term hormone treatment. Animals were treated continuously for 35 Months with either conjugated equine oestrogen (CEE), medroxyprogesterone acetate (MPA), combined CEE/MPA, or tamoxifen (TAM). Treatment with CEE/MPA down-regulated ERalpha in the superficial glands. In the superficial stroma the ERalpha level was lower in the CEE/MPA group than in the CEE and MPA groups. ERbeta immunostaining was faint with minor variation in response to treatment, but increased in the superficial stroma after MPA treatment. The ratio of ERbeta/ERalpha increased in superficial stroma and gland after CEE/MPA treatment, and also in stroma after MPA and TAM. Cystic endometrial hyperplasia was observed in TAM-treated animals, in combination with a high level of ERalpha protein expression. The present data show that long-term hormone treatment affects the ERalpha and ERbeta protein levels in the endometrium. The balance between ERalpha and ERbeta seems to be important for the proliferative response to oestrogen.

Up-regulation of endometrial oxytocin receptor (OTR) expression followed by an increase in pulsatile endometrial prostaglandin (PG) F(2alpha) secretion causes luteolysis in cattle. Inhibition of luteolysis is essential for the maternal recognition of pregnancy but also occurs in association with endometritis. The factors regulating OTR expression at this time are unclear. The OTR gene promoter region contains binding elements for acute phase proteins but their function has not been established. This study investigated the effects of various cytokines on OTR expression and on PGF(2alpha) and PGE(2) production in explant cultures of bovine endometrium. Endometrium was collected in the late luteal phase (mean day of cycle 15.4+/-0.50) or early luteolysis (mean day of cycle 16.4+/-0.24) as determined by the initial concentration of endometrial OTR. Explants were treated for 48 h with: (i) lipopolysaccharide (LPS) and/or dexamethasone (DEX), (ii) ovine interferon-tau (oIFN-tau), or (iii) human recombinant interleukin (IL)-1alpha, -2 or -6. OTR mRNA was then measured in the explants by in situ hybridisation and the medium was collected for measurement of PGF(2alpha) and PGE(2) by RIA. LPS treatment stimulated production of PGF(2alpha), whereas DEX either alone or in combination with LPS was inhibitory to both PGF(2alpha) and PGE(2). Neither of these treatments altered OTR mRNA expression. oIFN-tau reduced OTR mRNA expression but stimulated production of both PGF(2alpha) and PGE(2). In endometrial samples collected in the late luteal phase, IL-1alpha, -2 and -6 all inhibited OTR mRNA expression, but IL-1alpha and -2 both stimulated PGF(2alpha) production. In contrast, when endometrium was collected in early luteolysis, none of the interleukins altered OTR expression or caused a significant stimulation of PGF(2alpha) production but IL-2 increased PGE(2). Neither IL-1alpha nor -2 altered OTR promoter activity in Chinese hamster ovary cells transfected with a bovine OTR promoter/chloramphenicol acetyl transferase reporter gene construct. In conclusion, the action of interleukins on both OTR mRNA expression and endometrial prostaglandin production alters around luteolysis. Pro-inflammatory interleukins suppress OTR expression in the late luteal phase, while LPS stimulates PGF(2alpha) without altering OTR mRNA expression. IL-I and -2 and LPS are therefore unlikely to initiate luteolysis but may cause raised production of PGF(2alpha) during uterine infection.