Inhibition of maturation of isolated rat oocytes by porcine follicular fluid.
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Biomedical subjects
Publications and source records attributed to H R Lindner.
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A concomitant increase in the activity of LH-senstive adenylate cyclase and in the number of LH/hCG binding sites was induced in ovaries of immature rats upon administration of pregnant mare serum gonadotropin (PMSG), a hormone preparation known to have predominantly follicle stimulation (FSH-like) activity. When an optimal dose of PMSG (15 i.u./rat) was administered to 25-day-old rats, specific activity of LH-dependent adenylate cyclase and the number of binding sites for LH/hCG per mg protein remained unchanged during the first 24h, but 48h after injection a 2-to 4-fold increase in both parameters was observed. By contrast, there was no change in basal adenylate cyclase activity or in the response of the enzyme to the stimulatory action of guanosine-5'-(beta gamma-imino) triphosphate (Gpp (NH)p), GTP, or NaF. Specific activity of succinate cytochrome c reductase, glucose-6-phosphatase and 5'-nucleotidase were found to be unaffected by the hormonal pretreatment, although total protein determined in these homogenates increased 3-fold in the course of this treatment. It is inferred that during follicular maturation, FSH enhances the responsiveness of ovarian adenylate cyclase to LH by stimulating the insertion of LH/hCG-receptors into the cell membrane.
Culture of preovulatory rat follicles with luteinizing hormone, follicle-stimulating hormone or prostaglandin E2 for 24 h reduced the subsequent response of adenylate cyclase to the homologous by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E2, and those refractory to prostaglandin E2 could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8--2.0 mug/ml in the medium; a lower dose of luteinizing hormone (0.4 mug/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E2 caused partial refractoriness at dose levels of 0.1--0.25 mug/ml; higher dose levels were more effective. These findings suggest that continued exposure to the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.
1. High affinity macromolecular binding of the non-steroidal synthetic oestrogen [3H]diethylstilboestrol and of [3H] oestradiol-17beta in cytosol of Müllerian duct and uterus, and in blood plasma of perinatal rats, was investigated by sucrose density gradient sedimentation. 2. While [3H] oestradiol was bound to both the characteristic 8 S uterine cytoplasmic receptor and a 4 S component of uterine cytosol and plasma of 11-day-old rats, [3H] diethylstilboestrol was bound almost exclusively by the 8 S cytoplasmic receptor. 3. The greatly reduced binding of [3H] diethylstilboestrol to the 4 S plasma plasmic receptor in the Müllerian duct (precursor of the uterus) of 20-day-old foetuses.
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Reference preparations of ovine follicle-stimulating hormone (NIH-FSH-S8 and S9; 10-50 mug/ml) induced ovum maturation and stimulated cyclic AMP formation, as well as progesterone and 17beta-estradiol secretion, by rat Graafian follicles in vitro. These actions of NIH-FSH were retained after immunoabsorption of any contaminating luteinizing hormone (LH) present in the preparations, by treatment with an antiserum to the beta-subunit of purified ovine LH (anti-betaLH). In contrast, the corresponding biological actions of NIH-LH-S18 (0.5-10 mug/ml) were abolished by treatment with this anti-betaLH serum. A highly purified FSH preparation (64-96 CD, 0.25 mug/ml) also triggered oocytic meiosis and increased follicular progesterone secretion in vitro. Intraperitoneal (ip) administration of anti-betaLH-treated NIH-FSH-S9 (50 mug/rat at 1430 h) consistently induced ovulation in proestrous rats in which the endogenous gonadotropin surge had been blocked by ip injection of either Nembutal (1345 h) or antiserum to the LH-releasing hormone (1200 h). Injection (ip) of anti-betaLH serum on its own into proestrous rats at 1200 h prevented ovum maturation and follicular rupture. We conclude that currently available reference preparations of ovine FSH possess the capacity to stimulate follicular adenylate cyclase, steroidogenesis, and ovum maturation in vitro, as well as ovulation in vivo, in the rat, and that this capacity cannot be attributed to contamination with material immunochemically identical with LH. However, it is inferred that the physiological triggering of ovulation and related events in this species depends principally on LH.
Twenty-six patients with migraine attacks were treated for 3 to 16 months with flufenamic acid (125 mg four to six times per attack), an inhibitor of prostaglandin synthesis and action. In 25 patients the drug afforded symptomatic relief in 195 of 200 treated attacks. Side effects observed were mild dyspepsia (eight patients) and severe upper gastrointestinal symptoms (two patients). None of the eight patients treated with placebo reported any relief (20 attacks). The "common" antimigraine drugs afforded symptomatic relief in 12 of the patients, partial relief in seven, and no relief in seven. Treatment with flufenamic acid was based on the hypothesis that prostaglandins are involved in migraine attack and that the drug relieves migraine by inhibition of the vasoactivity of prostaglandins.
Gap junctions were identified in the membrana granulosa and cumulus oophorus, and between cells of the internal theca, of the preovulatory rat follicle. In replicas of freeze fractured follicles, the A face presented clusters of closely packed intramembrane particles, 7--9 nm in diameter, forming a mosaic pattern, while the B face showed a similar pattern of small pits. Optical diffraction analysis of these electron micrographs revealed that both the intramembrane particles and the corresponding pits were organized in hexagonal lattices with centre-to-centre spacing of 9-10 nm. In small junctions (up to 0.4 mum in diameter), both A and B faces generally consisted of a single lattice, while large junctions (0.5-2 mum) contained multiple lattices differing in orientation. Occasionally intramembrane particles and pits were more loosely arranged throughout the junctional area and failed to show a hexagonal pattern. Both granulosa and thecal cells often contained intracellular vesicles resembling annular junctions. These contained intramembrane particles whose assembly resembled that of the gap junctions with regard to periodicity and lattice organization. Examination of thin section of thin sections suggested that small gap junctions occur also between cytoplasmic processes of coronal cells and the oolemma. No tight junctions were detected between granulosa cells and between thecal cells.
Luteinizing hormone (NIH-LH-S18; 5 mug/ml) stimulated aerobic glycolysis in cultured Graafian follicles explanted from pro-oestrous rats before the preovulatory gonadotrophin surge: lactate accumulation in the medium was 70% above control levels during 6 h incubations. Iodoacetate (2.5 x 10(-5) M) prevented this effect, without impairing the ability of LH to induce resumption of oocytic meiosis. Enrichment of the medium with pyruvate (3.3 x 10(-4) M) or lactate (2.5 x 10(-2) M) did not in itself cause ovum maturation. The results do not support the hypothesis that termination of meiotic arrest by LH is due to stimulation of glycolytic activity in the follicle cells, resulting in increased availability of an energy source readily urilizable by the oocyte.
Steroid release by cultured Graafian follicles explanted from rat ovaries on the morning of pro-oestrus was measured by radioimmunoaso the medium reduced the basal level of steroid secretion (progesterone, androstenedione and oestradiol-17beta) and abolished the steroidogenic effect of luteinizing hormone (LH; 5mug/ml) and prostaglandin E2 (PGE2; 10 mug/ml). However, the induction of oocyte maturation by either LH or PGE2 was not impaired by total suppression of the steroidogenic response of the follicles to these hormones by cyanoketone or aminoglutethimide. It is concluded that the meiosis-inducing action of LH on the mammalian egg is not mediated by the effect of the hormone on the rate and pattern of follicular steroidogenesis.
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More than 40 plant species have been shown to contain substances that are active in biological assays for estrogenic activity. Such substances may be constitutive metabolic products of a plant, or be formed adaptively in response to environmental factors, such as fungal attack (e.g. coumestrol synthesis in alfalfa infected with Pseudopeziza medicagensis); in other instances estrogens may arise from microbial attack on plant material during storage (e.g. zearalenone formation from corn by Fusarium spp.) Phyto-estrogens may reach man through direct consumption of fresh fruit, vegetables and processed plant products (e.g. administration of olive or cornoil can induce vaginal keratinization in post-menopausal women); or---more relevant to this Symposium---by consumption of carcasses and products from animals fed estrogen-containing forage. Important pasture and forage plants shown to contain phyto-oestrogens include Trifolium subterraneum L, notably the cultivars Dwalganup, Mt. Barker, Yarloop and Marrar, T. pratense (red clover), T. fragiferum L. (strawberry clover), T. alexandrinum (berseem clover), Medicago sativa (alfalfa or lucerne) and Soya hispida (soya beans). A beneficial anabolic action of the estrogens contained in these plants has been implied, but not unequivically established. More attention has been paid to their noxious effects on livestock. On affected T. subterraneum pasture, castrated male sheep showed lactation, squamous metaplasia of the bulbo-urethral glands and urethral stenosis; infertility, variously attributed to suppression of gonadotrophin release and ovulation; faulty ovum transport; premature regression of corpora lutea; irreversible cystic hyperplasia of endometrial glands on prolonged exposure; dystocia and prolapse of the uterus. Sporadic incidence of phyto-estrogen induced infertility in cattle has been reported, attended by ovarian cyst formation. Estrogenic activity in forage plants has been reported from Australia, New Zealand, India, Sweden, Great Britain, Germany, Denmark, Holland, Finland, Egypt and Israel. The clover constituents chiefly incriminated for these effects are glycosides of the isoflavone derivatives genistein and its 4'-methyl ether biochanin-A, daidzein and its 4'-methyl ether formononetin, and pratensein; coumestrol and its 3'- and 4'-methyl ethers account for the estrogenic activity of alfalfa. The isoflavone content of subterranean clover may reach 3 percent of its dry weight, and the coumestrol content of lucerne may exceed 100 mug/g. Coumestrol and genistein compete with 17beta-estradiol for binding sites on the uterine cytoplasmic receptor and induce macromolecular synthesis in the uterus, but fail to induce ovum implantation in ovariectomized, gestagen-maintained rats. Uterotrophic activity of coumestrol and genistein given parenterally to sheep is approximately 10(-3) and 10(-5) times that of stilboestrol, respectively. Biological activity of ingested phytoestrogens is modified by ruminal micro-organisms and hepatic metabolism...
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The concentration of prostaglandins of the E-group (PGE) and F-group (PGF) and the activity of prostaglandin-synthetase in rat ovaries increased on the evening of the day of proestrus and reached a peak at 5.00 h on the following morning, i.e. about the time of ovulation. Enzyme activity and PG concentrations receded to basal levels by 10.00 h on the day of estrus. These changes were prevented when the proestrous gonadotropin surge was blocked by administration of nembutal, and could be restored by administration of either LH or of FSH freed of LH contamination. The spontaneous preovulatory rise in prostaglandin concentration was about 6-fold for PGF and 30-fold for PGE, compared with values observed during the remainder of the cycle, whichas the rise in prostaglandin synthetase activity was only about 1.7-fold. The LH effect on PG accumulation had a latency of 2-4 h, which argues for enzyme synthesis rather than activation of preformed enzyme as the mechanism responsible. The small magnitude of the change in enzymic activity suggests that LH may, in addition, augment the availability of PG precursors. The results are compatible with the concept that prostaglandins play a physiological role in the gonadotropin-induced process of follicular rupture.
Prostaglandin (PG) synthetase activity and tissue concentration were measured in unilateral deciduomata induced by traumatization of the pseudo-pregnant rat uterus and in the decidua of pregnancy. PG synthetase activity per unit weight of deciduoma tissue was 7-10 fold higher, throughout the life-span of the deciduoma, than that in the untraumatized control horn. The concentration of prostaglandins of the E-type in the deciduoma exceeded that found in the control uterine horn by a factor of 10-20 on days 3-4 after decidual induction, and about five-fold on days 9-10. The concentration of prostaglandins of the F-type in the deciduoma measured on days 4 and 8 did not differ significantly from that in the control horn.