Effects of injections of monoamine oxidase inhibitor or saline into the uterus in late pregnancy on uterine catecholamine levels related to abnormal parturition in rats.

Injection of a monoamine oxidase (MAO) inhibitor (nialamide) into the uterus of an anaesthetized and laparotomized rat on day 20 of pregnancy severely disturbed parturition. Injection of the solvent (0-9% isotonic NaCl solution) at the same stage of gestation produced the same but less frequent disturbances. When the rats were injected on days 19 or 21, impairment was less marked than on day 20. Therefore, day 20 seems to be a critical period for the onset of parturition. Injection of Ringer solution into the uterus on day 20 had effects analogous to those of saline injection at the same stage. Anaesthesia induced with ether, laparotomy of the pregnant rat on day 20, and handling of the uterine horns without injection of either Ringer or NaCl also disturbed parturition in 70% of the rats treated. Nevertheless, disorders were not as severe as those after injection. Laparotomy alone on day 20 did not disturb parturition. The effects on parturition of a saline injection into the uterus on day 20 were greatly decreased when the injection was performed on pregnant rats adrenalectomized on day 14, or on pregnant rats pretreated on days 18 and 19 with an agent blocking the adrenergic beta receptors (propranolol); 70-80% of the treated rats had normal deliveries. In control rats, uterine catecholamine levels were markedly modified between days 21 and 22 of gestation. These changes did not occur in rats injected with MAO inhibitor or saline.

Mitogenic activity of high molecular weight forms of insulin-like growth factor-II in amniotic fluid.

Amniotic fluid (AF) collected from ewes and goats at mid gestation displayed mitogenic activity in mouse fibroblasts. Upon fractionation of this material by size exclusion chromatography, the mitogenic activity was resolved into two peaks, whose activity was inhibited by an anti-IGF type 1 receptor blocking antibody. One of the peaks contained IGF-I and IGF-II (mature form), whereas the other contained high M(r) precursor forms of IGF-II. The presence in this latter fraction of IGF-binding proteins (IGFBP) suggests that the AF IGFBPs do not efficiently inhibit the mitogenic activity of the high M(r) forms of IGF-II. In agreement with this conclusion, exogenous IGFBP-1 failed to affect this activity. Analysis of IGF-II in sheep AF showed that the AF concentrations of both forms of IGF-II increased dramatically from mid pregnancy until 106-120 days of gestation, and fell thereafter. The amniotic IGFBPs followed a similar evolution. High M(r) forms of IGF-II were also found in human AF, with a pattern of electrophoretic migration different from that of sheep. We suggest that the precursor forms of IGF-II may play an important role in foetal development.

Human myometrial smooth muscle cells and cervical fibroblasts in culture : a comparative study.

Uterine contractility and cervical tonicity change throughout the menstrual cycle and pregnancy in response to modifications in hormonal environment and tissue receptivity to hormones. The uterine wall consists of a smooth muscle (myometrium) organized into three layers. the inner and outer layers, mainly composed of smooth muscle cells, and the richly vascularized intermediate layer The musculature is thick in the corpus uteri and vanishes at the level of the corpus/cervix Junction. The cervix itself is formed mainly by connective tissue.

Inhibition of prostaglandin E2 production in myometrial and amniotic cells in culture by human amniotic fluid. Loss of inhibition after intra-uterine fetal death.

OBJECTIVE: To study the effects of amniotic fluid obtained during early second trimester of normal human pregnancies on prostaglandin production in cultured myometrial and amniotic cells. STUDY DESIGN: Cultured human myometrial and amniotic cells were incubated with human amniotic fluid obtained by amniocentesis and fractionated by ultrafiltration. Prostaglandin E2 was measured in the incubation media. In one case, amniotic fluid was obtained during the days following intra-uterine fetal death (IUFD). RESULTS: PGE2 production in myometrial and amniotic cells was significantly decreased when incubated with the fraction of amniotic fluid containing molecules of molecular weight between 3 and 30 kD. This inhibition was still present after heating. After IUFD, the inhibitory activity of amniotic fluid was persistent for the first 3 days but had disappeared 6 days after IUFD. CONCLUSION: These data suggest that a factor contained in amniotic fluid, with a molecular weight within the 3- to 30-kD range, and possibly produced or controlled by the fetus, inhibits PG synthesis. Further work is necessary to characterize this factor and evaluate its physiological role in human parturition.

[Peptide-like substances in sheep amniotic fluid which regulate proliferation of BP-A31 cells]. / Peptidické látky amniónovej tekutiny oviec regulujú proliferáciu buniek BP-A31.

Cell proliferation and differentiation of developing fetus is influenced by hormones as well as insulin-like growth factors and their binding proteins contained in amniotic fluid. Our purpose was to study the actual mitogenic activity of proteins and peptides present in the sheep amniotic fluid. The cell cycle regulatory activity was estimated by using mouse fibroblasts BP-A31 as target cells. The whole amniotic fluid was inactive. However, after removal of small molecules on Sephadex G-10, the fraction eluted in the void volume (M(r) > or = 0.7 kDa) was able to induce the cell division cycle in a significant proportion of quiescent fibroblasts (Fig. 1, fraction A; Fig. 2). By further gel chromatography of this active fraction at acidic condition on Sephadex G-50, two components with mitogenic activity were separated. One component was eluted immediately after the void volume of the column, the other one was coeluted with 125I-IGF-I (Fig. 3). The functional characteristics of mitogenic signal of both components (sensitivity to mitogenic effectors) were similar to those of IGF-I and insulin (Fig. 4). We suppose that a component with higher molecular weight eluted in the vicinity of the void volume of Sephadex G-50 represents probably IGFBPs or other similar proteins.

Insulin-like growth factor binding proteins and mitogenic activity of partially fractionated sheep amniotic fluid.

Amniotic fluid collected from ewes on various days of gestation was examined for the presence of insulin-like growth factor (IGF) binding proteins. IGF-binding proteins with a molecular mass of 40-45 kDa appeared at day 41 of gestation. The level of these major IGF-binding proteins increased during pregnancy and reached a maximum at day 106. Smaller IGF-binding molecules with an approximate molecular mass of 35 kDa and 25 kDa appeared at day 90, also reaching a concentration peak at day 106. The mitogenic activity of sheep amniotic fluid after chromatography on Sephadex G-50 was separated into two peaks. The peak having lower molecular mass corresponded to an elution profile of 125I-IGF-I. The first peak, having higher molecular mass, was eluted immediately after the void volume of column. Electrophoresis and ligand blotting showed that proteins in the first peak had similar properties as IGF-binding proteins.

[Hypotheses concerning variations in sperm motility in peritoneal fluid]. / Hypothèses relatives aux variations de mobilité des spermatozoïdes dans le liquide péritonéal.

We have studied the effects on sperm motility in peritoneal fluid taken from fertile women at different times of the cycle as well as from sterile women in the pre-ovulatory phase. We have compared the effects on the movement of sperms in part in a solution of pure Tyrode and in part in a solution of Tyrode containing hydroxytoluene, which is an antioxydising agent, in order to try to find out the mechanism that immobilises sperms in these sterile women. In equal concentrations we have found that this substance can be protective against immobilisation, which itself was due to the liquids coming from abnormal pelvis, in the antioxydising agent that was used. Furthermore it was found, in liquid taken from the peritoneal cavities of fertile patients in the pre-ovulatory phase, that it behaves like a Tyrode medium to which one has added a complex antiperoxide comprised of bovine serum albumin and mercaptoethanol. This complex has been found to protect spermatozoa against loss of motility which leads to complete standstill when Tyrode solution alone is used after incubation for six hours under the conditions under which we conducted the experiment. From these observation it does seem to us that the female genital tract does undergo changes during the menstrual cycle which allow sperms to stay motile and that this is probably the same effect as the abumin mercaptoethanol complex. This equilibrium is broken when there are lesion in the pelvis such as endometriosis or inflammatory disease by secretion of peroxydising substances such as fatty acids and the oligopeptides that occur in inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

IGFBP-1 inhibits EGF mitogenic activity in cultured endometrial stromal cells.

The properties of the insulin-like growth factor-binding proteins (IGFBP-1 to 6) are not limited to modulation of IGF actions. IGFBP-1, which shares an Arg-Gly-Asp (RGD) motif in its C-terminal domain, modulates cell motility by binding to integrin alpha5beta1. The cross-talks between integrins and growth factor receptor signalling pathways are extensively documented, particularly in the case of the epidermal growth factor receptor (EGFR). However, whether IGFBP-1 can modulate growth factor signalling through its interaction with integrin alpha5beta1 has not yet been studied. As EGF is involved in the decidualisation of endometrial stromal cells (ESCs) and as decidualised ESCs are a source of IGFBP-1, we investigated if IGFBP-1 can modulate EGF effects on ESCs. RGD- and IGF-independent inhibition of EGF mitogenic activity and EGFR signalling by IGFBP-1 were demonstrated in ESC primary cultures, A431, cells and in mouse fibroblasts lacking IGF receptors.

The contractile proteins of the human myometrium.

The contractile proteins of the human myometrium were quantified and characterized in order to investigate their possible modifications during pregnancy. The myosin concentration was found to be 1 to 5 mg/g of tissue, while actin concentration ranged from 16 to 60 mg/g, leading to an actin/myosin ratio higher (Mean = 14) than in other smooth muscles. Purified myosin submitted to two-dimensional gel electrophoresis exhibited two isoelectric forms for the 17 KD Light Chain, the more acidic being predominant in the pregnant organ, the more basic in the non gravid one. The mobility of myosin of form filaments was studied using electron microscopy. Only the myosin purified from gravid uteri in its phosphorylated form did aggregate in long bipolar filaments. Actin was characterized in crude muscle extracts using two-dimensional gel electrophoresis. It appeared in three forms differing by their isoelectric points. The more basic form (gamma) predominates in the pregnant organ, as soon as 17 weeks of pregnancy, while in the non-pregnant uterus it is the intermediate (beta) form which is predominant.

A local signal from the gravid horn to the ovary for the onset of parturition in rats.

Rats were unilaterally ovariectomized on Day 17 of pregnancy and the times of onset of parturition were recorded. Unilateral ovariectomy did not delay the onset of parturition in bilaterally pregnant rats. In rats which were unilaterally pregnant (spontaneously or after ligation of one uterine horn), removal of the ovary on the side of the gravid horn led to delayed or partial parturitions. When fetuses and placentas were removed from one uterine horn on Day 13 and the ovary from the opposite side on Day 17, or when the uterine artery and/or vein were ligated on one side and the ovary removed from the other, about half of the parturitions were abnormal. It is suggested that a substance produced by the gravid uterine horn is transmitted to the ovary, primarily by a local circulatory system, and thus initiates the onset of parturition.

Characterization and comparison of the contractile proteins from human gravid and non-gravid myometrium.

Electrophoretic systems were used to quantify and characterize the main proteins involved in myometrium contraction, i.e., myosin, actin, and tropomyosin. Skeletin, a cytoskeletal component was also investigated. No difference in their respective concentrations could be found in 13 uteri representing various menstrual cycle stages, postmenopausal conditions, and different gestational ages. The great variability of their tissular level observed in each of the uteri does, however, reflect the heterogeneity of the muscular component of the uterine wall. Comparison of the different forms of actin in gravid and non-gravid uteri showed an increase of the gamma-form in gravid uteri. However, myosins purified from gravid and non-gravid uteri were shown to have identical ATPase activity and peptide patterns following limited chymotryptic proteolysis.

Isoforms of myosin and actin in human, monkey and rat myometrium. Comparison of pregnant and non-pregnant uterus proteins.

Using several electrophoretic procedures, we have compared the forms of myosin and actin in pregnant and non-pregnant uterus of woman, monkey (Macaca fascicularis) and rat. On non-dissociating gels, native myosin of the three species migrates as a single band, of identical mobility independently of the physiological state. Remigration of this band in dissociating conditions shows that it is constituted of two heavy chains of respectively 201 kDa and 205 kDa; the relative proportions of these two bands are different for the three animal species but do not vary during pregnancy. Using two-dimensional gel electrophoresis, we found that the 17-kDa light chain of purified uterus myosin exists under two isoelectric forms, the more acidic one becoming progressively predominant at the end of pregnancy in the human as in the monkey uterus, while we observed no changes in the rat. In two-dimensional gel electrophoresis, actin of human, monkey and rat uterus is present under three isoforms, the most basic one (the gamma form) increasing early in pregnancy in the two primate species but being always the most abundant form in the rat. The ATPase activity of human uterus myosin was found to be similar for the protein extracted from both pregnant and non-pregnant uterus. The changes observed in the 17-kDa light chain and in the actin isoforms might nevertheless participate in the modifications of contractility of the uterus during pregnancy of the primates.

Myosin heavy chain isoform expression in human myometrium: presence of an embryonic nonmuscle isoform in leiomyomas and in cultured cells.

We had previously found no myosin heavy chain (MHC) changes in expression during pregnancy in human myometrium. In the present work, we compared the MHC pattern of expression in normal human myometrium, pregnant and non-pregnant, to that in benign tumors of the uterine musculature and in cultured myometrial cells. We used a high-resolution gel electrophoretic system and monoclonal antibodies directed against smooth muscle and nonmuscle MHCs. Smooth muscle MHCs (SM1, 204 kDa, and SM2, 200 kDa, MHCs) and a nonmuscle MHC of 196 kDa (NM MHC) were detected in pregnant and nonpregnant human myometrium. Pregnant myometrium was found to differ from nonpregnant myometrium by its slightly lower content in NM MHC, whereas the ratio of SM1/SM2 was equivalent. In leiomyomas and in cultured cells grown from human myometrium explants, SM1, SM2, and NM MHCs were also expressed. In addition, a nonmuscle MHC of 198/200 kDa (SMemb MHC), which was present in a fetal human uterus but not in adult normal tissue, was observed in leiomyomas and in cultured cells. Expression of SM1 and SM2 MHCs was variable in the different leiomyomas studied. In cultured cells, SM1 and SM2 MHC content was low, but it was enhanced by suppression of serum after cell confluency. Present results confirm that pregnancy-associated smooth muscle cell hypertrophy is not accompanied by major changes in MHCs. In contrast, cell culturing and cell hyperplasia leading to leiomyoma formation induce substantial modifications in MHCs, including the occurrence of a second type of nonmuscle MHC.

Contractile proteins in human fetoplacental vessels.

OBJECTIVE: Our purpose was to compare the protein isoform composition of the contractile apparatus at different levels of the fetoplacental vessel musculature at term. STUDY DESIGN: Umbilical, chorionic, and stem villi vessel protein extracts were run on one- and two-dimensional gel electrophoresis; previously characterized human myometrium proteins were used as the smooth muscle proteins of reference. RESULTS: Fetoplacental vessel musculature exhibited a high actin/myosin ratio. The presence, in varying quantities, of myosin heavy chain and actin isoforms of smooth muscle type in the different vessels reflected their degree of differentiation. The presence of nonmuscle protein isoforms, particularly in stem villi vessels, indicated a certain degree of immaturity. CONCLUSIONS: The presence of smooth muscle contractile protein isoforms indicates that fetoplacental vessel musculature is highly differentiated. Regional modulation of fetoplacental blood flow could be, in part, the result of local differences in contractile apparatus protein composition.

Involvement of endothelin receptor subtypes in human myometrial activity.

We investigated the contribution of endothelin (ET) ETA and ETB receptors to ET-induced contractions of human myometrium and their distribution in cultured myometrial cells. ET-1 was more potent than ET-3 in evoking concentration-dependent increases in smooth muscle tension. A selective ETB agonist, BQ 3020, was inactive in eliciting contractions. In myometrial cell membranes, no specific [125I]ET-3 binding was observed. Inhibition of [125I]ET-1 binding by unlabeled compounds showed the following order of potency: ET-1 = FR 139317 > ET-3 >> S6c. Furthermore, ET-1 increased inositol phosphate (IP) production in a dose-dependent manner. ET-1-induced IP accumulation was totally abolished by FR 139317 (an ETA-selective antagonist) but was not altered by IRL 1038 (an ETB-selective antagonist). These results indicate that only ETA receptors, which mediate ET-1-induced uterine contraction, are present in cultured myometrial cells.

Characterization of type A endothelin receptors in cultured human myometrial cells.

The aim of the present study was to characterize endothelin (ET)-receptors in human myometrial cells in culture. 125I-labeled ET-1 binding to myometrial cells was specific and saturable, with a dissociation constant of 64.2 +/- 12.8 pM. Competition binding studies showed the following order of potency: ET-1 > ET-3, which is consistent with the presence of the ETA receptor subtype. FR-139317 and BQ-123, two ETA antagonists, both inhibited 125I-ET-1 binding. BQ-123 only elicited a partial inhibition. The fraction resistant to BQ-123 did not represent the ETB receptor subtype, since no specific 125I-ET-3 binding could be detected. ET-1 and ET-3 were found to stimulate [3H]inositol phosphate (IP) accumulation in cultured myometrial cells, with corresponding half-maximal effective concentration values of 0.26 +/- 0.04 and 87 +/- 17 nM, respectively. Both ETA antagonists inhibited ET-1-induced accumulation of [3H]IP. BQ-123 was only a partial inhibitor, whereas FR-139317 totally suppressed ET-1-stimulated production of [3H]IP. We conclude that human myometrial cells in culture exclusively possess ETA receptor subtypes coupled to phospholipase C.

Paracrine action of vascular endothelial growth factor in the human endometrium: production and target sites, and hormonal regulation.

Vascular endothelial growth factor (VEGF) is an endothelium-specific growth factor with potent angiogenic activity and a stimulator of microvascular permeability. Because endometrial cyclic development is associated with vascular growth, we examined the expression of VEGF protein throughout the menstrual cycle and studied the regulation of VEGF mRNA by ovarian steroids in isolated human endometrial stromal cells. VEGF was localized immunohistochemically in glandular epithelial cells and in the surrounding stroma, as well as in capillaries and spiral arterioles, a localization which has not been described before. The strongest immunoreactivity for VEGF on endothelial cells was detected in the late proliferative and secretory phases. The localization of VEGF bound to the endothelium correlates with the presence of flt-1 and flk/KDR receptors on vascular structures, including capillary strands which have not yet formed a lumen, present during the mid-secretory period, which corresponds to a high estroprogestin influence and to implantation. Heparinase treatment of the sections decreases the staining intensity of VEGF bound to endothelial cells, suggesting that VEGF also binds to heparin-like molecules on the cell surface. These new results demonstrate a major role of VEGF on capillary formation and on hyperpermeability and edema during the menstrual cycle. Consistent with these in vivo observations, the treatment of isolated endometrial stromal cells with estradiol (E2) or E2 plus progesterone, significantly increased VEGF mRNA over the control value in a dose-dependent manner; the VEGF mRNA response to E2 was rapid (3h) and persisted with continuous estradiol treatment up to 12 days. Three species, VEGF_121, VEGF165 and VEGF189, were observed upon hormonal stimulation. The estradiol up-regulation of VEGF response did not require de novo protein synthesis as it was not blocked by cycloheximide. Also, the ability of the pure anti-estrogen ICI 182,780 to significantly block induction of VEGF mRNA by E2 suggests estrogen receptor-mediated transcriptional regulation. These results demonstrate that VEGF is an estrogen-responsive angiogenic factor that acts on vascular endothelium in a paracrine fashion, as previously suggested. This growth factor controls angiogenesis and hyperpermeability required for adequate receptivity to implantation of the cycling human endometrium. These findings also raise the possibility that estrogen effects on uterine edema, proliferation and tumoral transformation may involve local increases in tissue VEGF production.

Characterization of a membrane glycoprotein having pharmacological and biochemical properties of an AT2 angiotensin II receptor from human myometrium.

The angiotensin II receptors of human myometrial tissue were characterized using ligand binding, cross-linking with radioactive label, detergent solubilization and partial purification by lectin-affinity chromatography. Human myometrial membrane preparations contained variable amount (5-650 fmol/mg protein) of high affinity (Kd = 44-65 pM) binding sites for 125I-CGP42112, a ligand specific for the AT2 subtype of angiotensin II receptors. Competition studies with AT1-specific and AT2-specific compounds indicated that angiotensin II receptors on these membranes were exclusively of the AT2 subtype. The binding sites for 125I-CGP42112 were efficiently solubilized by the detergent Chaps, albeit with a marked decrease in affinity (Kd = 1.2 nM). The proteins in the myometrial membrane preparation were cross-linked to 125I-[Sar1, Ile8]angiotensin II (Sarile) with disuccinimidyl suberate. When low concentrations of cross-linker were used, a single radiolabelled band of about 66-70 kDa was revealed on SDS/PAGE. At higher concentrations additional bands of about 105-120 kDa and 200 kDa were labelled. The 66-70-kDa and 105-120-kDa bands were separated by electroelution of SDS/PAGE gel slices and submitted to trypsin cleavage. The tryptic-peptide maps were identical for both products, suggesting that the additional bands are homodimers and trimers of the labelled polypeptide. The Chaps-solubilized receptor was retained on wheat-germ-agglutinin-Sepharose and specifically eluted by the competing sugar triacetylchitotriose, leading to a fivefold purification factor. Treatment of the 125I-Sarile-labelled protein with N-glycanase caused a shift in its apparent molecular mass on SDS/PAGE from 66-70 kDa to 33 kDa.