Heterogeneity in the bioactivity of LH secreted by peripubertal rats.

Treatment of immature rats with 5 iu equine chorionic gonadotrophin (eCG) on day 25 typically stimulates a preovulatory surge of LH on day 27 and ovulation on day 28. In rats weighing > 60 g at the time of treatment, an LH surge and ovulation occurred in 75% of the animals but, in rats weighing < 60 g, only 13% ovulated even though 69% showed an LH surge. Previous findings have shown that exogenous LH can stimulate ovulation in the rats < 60 g, indicating that the anovulation was not due to ovarian immaturity, but rather to an abnormal form of LH. Thus, it was important to determine whether the bioactivity of LH released at the time of the surge differs in rats < 60 g compared with rats > 60 g. Experiments showed that LH from both groups of eCG-treated animals were equipotent in stimulating testosterone production from incubated Leydig cells and progesterone production from cultured granulosa cells. Similarly the surge of progesterone in vivo, which occurs co-incident with the LH surge, was of similar magnitude in both groups of animals. Since prostaglandin synthesis increases at the time of ovulation and is also stimulated by LH, it was investigated whether the activity of ovarian phospholipase A2, the rate limiting enzyme in prostaglandin synthesis, and ovarian prostaglandin E2 concentrations differed in the animals > 60 g and < 60 g. Phospholipase A2 activities were similar in both groups of animals at the time of the LH surge, as were the prostaglandin E2 concentrations. However, in all animals that ovulated (15/20 in rats > 60 g and 2/15 in rats < 60 g), there was a threefold increase in ovarian prostaglandin E2 concentrations. The results show that, in underweight animals, the bioactivity of LH, in terms of its ability to stimulate steroidogenesis and phospholipase A2 activity, is similar to that released by animals > 60 g; however, the LH produced by the underweight animals fails to induce ovulation by failing to increase, either directly or indirectly, prostaglandin E2 production. Comparison of the profiles of plasma LH collected at the time of the LH surge on an anionic ion exchange column indicates that the LH from rats < 60 g possesses significantly less of the neutral or basic glycoform of LH than that from rats > 60 g. This finding provides a further index that the biopotency of LH produced by underweight animals is different from that of rats > 60 g.

Regulation of epidermal growth factor receptor by androgens in human endometrial cells in culture.

Women with polycystic ovaries (PCO) have a thicker endometrium than women with normal ovaries. This cannot be due to unopposed oestrogen, as it occurs in ovulatory cycles. Androgens may be involved, as these are raised in women with PCO. The effects of steroids are partly mediated by growth factors and their receptors. The aim of this study was to investigate the effect of androgens on epidermal growth factor (EGF) receptor in human endometrium. Endometrium was enzymatically dispersed and glands and stromal cells separated. Cells were incubated in Ham's F10 medium supplemented with 5% charcoal-stripped fetal calf serum and either androgens or vehicle. Specific binding of [125I]-labelled EGF was measured. Testosterone and dihydrotestosterone (DHT) (10 micromol/l) increased EGF receptor concentration (control 100 +/- 9%, testosterone 196 +/- 23% control; control 100 +/- 1%, DHT 244 +/- 6% control) but did not alter receptor affinity. The effect of testosterone was inhibited by the anti-androgen hydroxyflutamide, but not by the antioestrogen ICI182780 nor the aromatase inhibitor 4-hydroxyandrostenedione. EGF receptor levels were increased by androstenediol (control 100 +/- 2%, androstenediol 120 +/- 10% control) but not by androstanediol, dehydroepiandrosterone (DHA), DHA sulphate or androstenedione. Testosterone and DHT increased EGF receptor concentrations in glandular epithelium (control 100 +/- 24%, testosterone 147 +/- 5%, DHT 185 +/- 30% control). These data suggest that androgens may have an effect on the endometrium via an increase in EGF receptor concentrations.

Regulation of epidermal growth factor receptor synthesis by ovarian steroids in human endometrial cells in culture.

The aim of this study was to investigate the effect of oestradiol and progesterone on epidermal growth factor (EGF) binding in human endometrial glands and stromal cells in culture. Monolayers of isolated glands or stromal cells were cultured for 6 days in the presence or absence of steroids which were replenished daily. Binding of 125I-labelled EGF was measured in the presence and absence of unlabelled EGF. Over a 6 day period, oestradiol caused a dose-dependent increase in the number of EGF receptors in stromal cells, with a maximum effect of 150% control at a concentration of 10 nmol l-1. The effect of progesterone was also dose dependent and reached a maximum of 170% control at 100 nmol progesterone l-1. Oestradiol and progesterone in combination caused a greater increase in EGF receptor concentration than did either steroid alone (control, 100 +/- 11%; oestradiol, 144 +/- 11% control; progesterone, 200 +/- 20% control; oestradiol and progesterone, 288 +/- 6% control). Steroid treatment did not alter the affinity of the receptor for EGF. The stage of the cycle of the tissue did not affect the response to steroids. The effect of oestradiol was inhibited by the anti-oestrogen ICI182780, and that of progesterone by the anti-progestin RU486. In endometrial glands, neither oestradiol nor progesterone affected the number of EGF receptors, but the two steroids in combination induced an increase of 140 +/- 23% control where control was 100 +/- 15%. These data demonstrate that oestradiol and progesterone increase the number of EGF receptors in vitro, and suggest that EGF is involved in mediating the actions of these steroids on the processes of proliferation and differentiation in the human endometrium.

The epidermal growth factor receptor in the human endometrial adenocarcinoma cell line HEC-1-B.

The binding characteristics and steroidal regulation of the EGF receptor were investigated in the human endometrial adenocarcinoma cell line HEC-1-B. The cell line was shown to possess a single, high affinity binding site for epidermal growth factor receptor (EGF) with a Kd of 3.09 +/- 1.39 nM (mean +/- SD, n = 6) and binding of 845 +/- 311 fmol/mg protein (mean +/- SD, n = 6). The protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA) increased the Kd of the EGF receptor in a dose dependent manner (PMA: 0, 1, 10, 100 nM; Kd: 4.1, 5, 10, 50 nM, respectively). The effect of PMA (10 nM) was overcome by preincubating the cells with the protein kinase C inhibitor staurosporine (1 microM) prior to the addition of PMA. The effect of the ovarian steroids oestradiol and progresterone on EGF receptor accumulation was studied by pretreating the cells for 6 days with oestradiol or progesterone in phenol red free DMEM:F12, 1:1 supplemented with 5% charcoal stripped fetal calf serum. Both steroids were shown to increase EGF receptor number with a maximum 5- and 7-fold increase in the presence of 1 nM oestradiol or 1 microM progresterone, respectively. The study demonstrates the presence of a high affinity binding site for EGF in HEC-1-B cells which is regulated by oestradiol and progresterone.

Characterization of epidermal growth factor receptor in human endometrial cells in culture.

The aim of the study was to determine the binding characteristics of the epidermal growth factor (EGF) receptor in isolated human endometrial glands and stromal cells in culture. Stromal cells and glands were obtained from endometrial tissue by collagenase dispersion followed by sieve filtration. They were plated into 24-well multiwell plates in Ham's F10 medium supplemented with 5% fetal calf serum and used at 70-80% confluence. Scatchard analysis revealed a single class of high-affinity binding sites in both cell types with apparent dissociation constants of 1.17 +/- 0.6 (n = 15) and 1.20 +/- 0.3 (n = 8) nmol 1-1 for stromal cells and glands, respectively. The concentration of receptors was higher in stromal cells than in glands, 719 +/- 377 (n = 16) and 310 +/- 177 (n = 8) fmol mg-1 protein, respectively. Epidermal growth factor labelled with 125I was displaced from the receptor by EGF and transforming growth factor alpha, but not insulin, insulin-like growth factor, fibroblast growth factor, or platelet-derived growth factor. Binding was shown to be dependent on time and temperature. Downregulation of the receptor was demonstrated by preincubating cells with 5 nmol EGF I-1, which reduced receptor concentrations by 75%. 12-O-Tetradecanoylphorbol-13-acetate decreased the affinity of the receptor for EGF changing the dissociation constant from 1.8 to 3.9 nmol l-1. A suitable system for investigating the regulation of this receptor in human endometrium was established.

In vivo and in vitro effects of gamma-linolenic acid and eicosapentaenoic acid on prostaglandin production and arachidonic acid uptake by human endometrium.

This study examines the effect of a 6 month dietary supplement of either gamma-linolenic acid (GLA) or eicosapentaenoic acid (EPA) on the synthesis of prostaglandins E2 and F2 alpha by the endometrium of women with regular menstrual cycles. Samples of endometrium, obtained pre- and post-treatment, were incubated in vitro for 2 h and the prostaglandins E2 and F2 alpha released into the medium measured by radioimmunoassay. The ability of the tissue to take up 14C-arachidonic acid before and after treatment was also examined. Both GLA and EPA caused a marked decrease in the synthesis of prostaglandins E2 and F2 alpha (P < 0.001) but under the experimental conditions used, there was no consistent effect on arachidonic acid uptake. Body mass index, serum testosterone, fasting insulin and serum sex hormone-binding globulin (SHBG) concentrations did not change during the 6 month treatment period. An effect of GLA and EPA on arachidonic acid uptake into endometrial tissue explants was demonstrated in vitro. In the presence of both GLA and EPA, uptake into phospholipids (particularly phosphatidylcholine) decreased while uptake into triglycerides increased. Free 14C-arachidonic acid levels (that which could not be removed from the tissue by washing) also increased. Suppression of endometrial prostaglandin E2 and F2 alpha synthesis following GLA or EPA supplementation can be explained as direct competition between these fatty acids and arachidonic acid (the precursor of 2-series prostaglandins) for incorporation into membrane phospholipids. The amount of arachidonic acid available for 2-series prostaglandin synthesis will therefore be reduced. However, other mechanisms may exist which need to be investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal control of ovarian phospholipase A2 activity in rats.

The ovulatory effect of LH is mediated by prostaglandins, the synthesis of which involves prostaglandin synthetase and phospholipase A2 (PLA2). It is known that the activity of the former is regulated by LH in the ovary and the objective of this study was to establish the presence of PLA2 in the rat ovary and to determine whether LH has a role in regulating its activity. We demonstrated the presence of a calcium-dependent PLA2 in rat ovaries and shown that, in adult rats, the activity of this enzyme rises significantly on the evening of pro-oestrus (at the expected time of the preovulatory LH surge) and remains high over the evening hours, declining by the next morning. Furthermore, pregnant mares' serum gonadotrophin (PMSG), which when injected into 28-day-old rats induces an LH surge 54 h later, caused at the same time, a rise in ovarian PLA2 activity. Administration of 5 micrograms oestradiol benzoate S.C. to 25-day-old rats stimulated ovarian PLA2 activity 54 h later, at the time of the oestrogen-induced LH surge. Human chorionic gonadotrophin (hCG) had no effect on circulating concentrations of LH, FSH or oestradiol but stimulated ovarian PLA2 activity 2 h later. Gonadotrophin-releasing hormone (GnRH) given to 25-day-old rats (either untreated or primed with 0.5 mg oestradiol benzoate) had no effect on ovarian PLA2 activity but the resulting LH surge may have been too transient.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypersecretion of luteinizing hormone and ovarian steroids in women with recurrent early miscarriage.

Polycystic ovary syndrome is associated with hypersecretion of luteinizing hormone (LH) which has been implicated in the aetiology of early pregnancy loss. Although 82% of women with recurrent early loss have polycystic ovaries on ultrasound imaging, random serum LH concentrations are normal. In the present study, we have obtained further information from serial samples concerning the cyclical patterns of gonadotrophin and sex steroid secretion in these women. Twenty-one women with recurrent early pregnancy loss and 10 multiparous controls were investigated; 81% of them and one of ten control subjects had polycystic ovaries. Mean mid-follicular and mid-luteal serum LH and follicle stimulating hormone (FSH) levels were similar in both groups. Seventeen women with pregnancy loss had either raised urinary LH excretion or a premature LH surge; one control subject had a premature LH surge. Total LH excretion during the cycle and mean follicular phase serum testosterone was significantly greater with early pregnancy loss than in the control group, the difference in LH being greatest in the early luteal phase. Urinary oestrone-3-glucuronide excretion was raised in the early luteal phase of the cycle in the group with early pregnancy loss; there was no difference between the groups in pregnanediol-3 alpha-glucuronide excretion. These data demonstrate abnormalities in LH secretion in 81% of women with recurrent fetal loss. Inappropriately raised LH levels may have adverse effects on the developing oocyte or endometrium either directly, or indirectly by causing an elevation in testosterone and oestrogen levels.

Arachidonic acid release and inositol lipid metabolism in response to bradykinin and related peptides in human endometrial cells in vitro.

Prostaglandins of the 2 series are known to play a role in the regulation of menstruation and implantation but, more recently, other vasoactive peptides have been considered as potential regulators of these endometrial processes. The aim of the present study was to investigate the action of the potent vasoactive peptide bradykinin and the structurally related peptide, kallidin, on endometrial function by examining their effect on phosphoinositide hydrolysis and arachidonic acid release from endometrial cells in vitro. Primary cultures of endometrial glands and stromal cells were prelabelled with [14C]-arachidonic acid (AA) or [3H]-inositol to monitor arachidonic acid release and inositol phosphate accumulation respectively. Bradykinin and kallidin stimulated a dose and time-dependent release of arachidonic acid from stromal cells which, with 100 nmol/L bradykinin, was 30-150% above basal release and maximal at 5 min. Glands were less responsive; 100 nmol/L bradykinin (at 5 min) caused a release of AA of 30-69% above basal level. Bradykinin also stimulated a dose dependent increase in inositol monophosphate production. The maximum response with stromal cells was 8- to 10-fold and with glands, 2-fold (1 and 100 nmol/L bradykinin, respectively). Kallidin was equipotent to bradykinin with respect to both AA and inositol phosphate accumulation. The bradykinin analogue des Arg bradykinin (which acts through the B1 receptor) released AA from stromal cells but did not alter phosphoinositide hydrolysis, suggesting that these two cellular responses are mediated by different receptors (B1 and B2 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Endometrial phospholipases A2, polycystic ovaries and pelvic pain.

OBJECTIVE: To compare the activity of calcium independent phospholipase A2 in the endometrium of women with polycystic ovaries and in women with normal ovaries, and to investigate the influence of chronic pelvic pain on phospholipase A2 activity. DESIGN: A prospective descriptive study. SETTING: The Samaritan Hospital for Women and the Unit of Metabolic Medicine, St Mary's Hospital Medical School, London. SUBJECTS: 73 women attending the Samaritan Hospital for Women for clip sterilization, infertility, early recurrent miscarriage or pelvic venous congestion. 45 of these women had no pelvic pain, 22 had normal ovaries and 23 had polycystic ovaries diagnosed by ultrasound. The other 28 women had chronic pelvic pain, 14 of them had normal ovaries and 14 polycystic ovaries. INTERVENTIONS: Endometrial tissue was obtained at curettage or from the excised uterus in the proliferative or secretory phase of the menstrual cycle. The activities of calcium dependent (type 1) and calcium independent (type 2) phospholipase A2 isoenzymes were measured in all endometrial samples. RESULTS: In all the women phospholipase A2 type 1 activity, was significantly higher in the secretory phase than in the proliferative phase (P less than 0.001). There was no difference between women with normal ovaries and those with polycystic ovaries at either stage of the cycle irrespective of whether or not chronic pelvic venous congestion was present. In women with normal ovaries, both with and without chronic pelvic pain, phospholipase A2 type 2 activity was significantly higher in the secretory phase than in the proliferative phase (P less than 0.02 and P less than 0.05 respectively) but in women with polycystic ovaries, with and without chronic pelvic pain, there was no significant difference in activity between the two phases of the cycle. Women with polycystic ovaries had markedly higher proliferative phase type 2 activity than women with normal ovaries (P less than 0.001). Secretory phase type 2 activity was similar in all the women investigated. CONCLUSION: These data suggest that phospholipase A2 type 2 activity is increased in proliferative phase endometrium of women with polycystic ovaries but that the increase is not associated with chronic pelvic venous congestion.

Release of arachidonic acid from human endometrial cells in culture mediated by calcium ionophore (A23187) or fluoride.

Primary cultures of endometrial glands and stromal cells were labelled with [14C]-arachidonic acid for 4 h before exposure to either the calcium ionophore, A23187 (which activates phospholipase A2 (PLA2) by increasing intracellular calcium concentrations) or sodium fluoride (which activates a G-protein). Calcium ionophore (0.5-50 mumol/l) stimulated a dose- and time-dependent release of arachidonic acid from endometrial glands. Incubation with ionophore (10 mumol/l) for 1 h released 22% of the incorporated arachidonic acid. There was a corresponding decrease in phospholipids and no loss from triglycerides. Stromal cells were unresponsive to ionophore. Fluoride (10 mmol/l) stimulated a release of arachidonic acid from stromal cells and endometrial glands (6.5% of the total arachidonic acid incorporated). In stromal cells, arachidonic acid was released from triglycerides in Day-1 cultures and from phospholipids in Day-2 cultures. In both Day-1 and Day-2 cultures of endometrial glands, arachidonic acid was released from phospholipids, but not from triglycerides. Among the phospholipids, phosphatidylcholine was always the major source of arachidonic acid. Arachidonic acid release from endometrial glands and stromal cells may be mediated by activation of PLA2 (or phospholipase C) via a G-protein, but in glands calcium ionophore may have a direct effect on PLA2. The response to calcium ionophore may reflect the differences in calcium requirements of the two endometrial PLA2 isoenzymes.

Phospholipase activity in the endometrium of women with normal menstrual blood loss and women with proven ovulatory menorrhagia.

The activity of phospholipase A2 types 1 and 2 and phospholipase C was measured in the endometrium of women with ovulatory menorrhagia and in those with normal menstrual blood loss. In both groups of subjects phospholipase A2 type 1 activity was significantly higher in the secretory phase than in the proliferative phase (P less than 0.001). The median activity (pmol/mg protein/min) for the proliferative phase was 27.6 in normal subjects and 40.4 in women with ovulatory menorrhagia and for the secretory phase the median activity was 144.5 in normal women and 138.1 in women with ovulatory menorrhagia. There was no difference between the two groups of women at either stage of the cycle. Phospholipase A2 type 2 activity was also higher in the secretory phase than in the proliferative phase (P less than 0.05 for normal subjects and P less than 0.001 for women with menorrhagia). The median activity (pmol/mg protein/min) for the proliferative phase was 94.4 (normal subjects) and 56.6 (women with menorrhagia) and for the secretory phase 148.3 (normal subjects) and 142.5 (women with menorrhagia). The activity of phospholipase A2 type 2 was significantly lower in the proliferative phase of women with ovulatory menorrhagia compared with normal subjects (P less than 0.05). Phospholipase C activity (nmol/mg protein/min) was significantly higher in women with ovulatory menorrhagia (median 8.2) compared with women with normal blood loss (median 5.5) (P less than 0.01).

Starvation decreases insulin secretion, prostaglandin E2 production and phospholipase A2 activity in rat pancreatic islets.

Pancreatic islets, isolated from rats starved for 48 h, secreted significantly less insulin in the presence of 2 mmol glucose/l than islets of fed controls. In contrast, the insulin secretory response of islets from fed and starved rats to a challenge of 20 mmol glucose/l was similar. Concentrations of prostaglandin E2 (PGE2) in islets from starved rats incubated with 2 mmol glucose/l were significantly lower compared with those in control islets obtained from fed animals. Although glucose (20 mmol/l) stimulated PGE2 production in islets from starved and fed rats by 2.7- and 1.6-fold respectively, the concentrations achieved were the same as a consequence of the different prestimulated concentrations. Incubation with [14C]arachidonic acid of sonicated islet preparations from fed rats and separation of metabolites generated by high-pressure liquid chromatography, indicated the biosynthesis of a number of cyclo-oxygenase- and lipoxygenase-derived compounds, including 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and 12-hydroxyeicosatetraenoic acid. Metabolism of arachidonic acid to cyclo-oxygenase-derived compounds occurred with the same efficiency, but production of lipoxygenase-derived compounds was reduced by 50% in sonicated islets from starved compared with fed rats. Activity of phospholipase A2 of islets from starved rats was significantly less than that measured in islets from fed rats, although the degree of stimulation by 20 mmol glucose/l was the same in both types of islet. These alterations in the phospholipase A2/arachidonic acid cascade may contribute to the diminished insulin secretory response of islets from starved rats to relatively low concentrations of glucose.