U. Zor

Gonadotropin Action on Cultured Graafian Follicles: Induction of Maturation Division of the Mammalian Oocyte and Differentiation of the Luteal Cell

Publisher Summary This chapter discusses the nature of interactions between oocyte and granulose cells and the triggering effect of luteinizing hormone (LH). The mammalian oocyte embarks on its first reduction division in prenatal life or during the early postnatal period. This division has a protracted and complicated prophase. Just before or shortly after birth, depending on the species, the germ cell reaches the stage of diplotene. By this time, the oocyte has doubled its DNA complement, the chromosomes have condensed, and homologous chromosomes have become paired and are linked by chiasmata permitting the exchange of paternal and maternal genetic information. In murid rodents, the chromosomes decondense and resume their transcriptive activity. In the adult, during each estrous cycle, a number of oocytes characteristic of the species complete their first reduction division, resulting in the abstriction of the first polar body shortly before ovulation. This resumption of meiosis and its progress to the metaphase of the second meiotic division refer to as ovum maturation. Completion of the second meiotic division, with extrusion of a second polar body, occurs only upon penetration of the oocyte by a spermatozoon.

Physiological role of prostaglandins in the induction of ovulation

Abstract The administration of prostaglandin E2 at the dose levels of 0.7, 1.0 and 1.5 mg/rat on the afternoon of proestrus to adult rats in which the pre-ovulatory surge of LH was prevented by Nembutal induced ovum maturation in 58, 70 and 90% and ovulation in 42, 60 and 81% of the animals, respectively; the incidence of persistent uterine distension was reduced by the prostaglandin treatment, suggesting that ovarian progesterone secretion was stimulated. Injection of indomethacin, an inhibitor of prostaglandin synthesis, on its own at 14.30 on the day of proestrus (5 – 10 mg/rat) prevented follicular rupture in 78 – 89% of the animals, but maturation of the oocytes retained in the follicles was unimpaired. Concomitant treatment with indomethacin and Nembutal prevented both follicular rupture and ovum maturation. Administration of LH at a dose level adequate to induce ovulation in Nembutal-blocked rats (2.5 μg/rat), failed to overcome the indomethacin-induced block of ovulation, but prostaglandin E2 brought about follicular rupture in the majority of the indomethacin-treated animals. It is concluded that (i) indomethacin, under the conditions studied, does not block LH release, but exerts its anti-ovulatory action directly on the follicle: it prevents follicular rupture, but not ovum maturation; (ii) prostaglandins have an essential role in the mechanism by which LH brings about follicular rupture; (iii) though prostaglandin E2 is able to induce ovum maturation, prostaglandins are not indispensible for this action of LH.

The transduction of mechanical force into biochemical events in bone cells may involve activation of phospholipase A2

SummaryMechanical forces applied to cultured bone cells induce the production of cAMP via stimulation of the formation of prostaglandin E2 (PGE2) and its release into the medium, resulting in stimulation of adenylate cyclase. In this paper we show that either the antibiotic gentamycin (100 μg/ml) or antiphospholipid antibodies (0.1%) which bind to membrane phospholipids abolish cAMP formation induced by mechanical forces; exogenously added arachidonic acid or PGE2 stimulates cAMP formation, even in the presence of these agents. Addition of exogenous phospholipase A2 (but not phospholipase C) causes an increase in the formation of cAMP in bone cells, a response that is also inhibited by gentamycin or antiphospholipase antibodies. These observations suggest that mechanical forces exert their effect on bone cells via the following chain of events: (1) activation of phospholipase A2, (2) release of arachidonic acid, (3) increased PGE synthesis, (4) augmented cAMP production.

Radioimmunological assay of prostaglandin synthetase activity

Abstract A sensitive assay of prostaglandin synthetase activity is described, based on the radioimmunological determination of prostaglandin E 2 (PGE 2 ) formed during incubation of tissue preparations (homogenates or microsomal fraction) with exogenous unlabeled arachidonic acid. The incubation is carried out under conditions in which PGE 2 is the main product formed, and no chromatographic step is required. The antiserum used, produced by immunization of rabbits with a PGE 2 -albumin conjugate, preferentially bound PGE 2 and showed negligible cross-reaction with arachidonic acid ( 1 , A 1 , A 2 , B 1 and B 2 . Prostaglandin synthetase activity, as determined by PGE 2 formation (ng/mg tissue/5 min, mean ± S.E.M.) was 1.95 ± 0.15 (n = 9) in ovaries from 30-day-old rats and 1220±200 (n = 4) in ovine seminal vesicles; the activity in the microsomal fraction (μ g PGE 2 /mg protein/ 3 min) was about 0.03 for rat ovaries and 43 for ovine seminal vesicles. PGE 2 formation was abolished when indomethacin (10 μg/ml) was added to the incubation medium, when substrate was omitted or when the tissue was inactivated with citric acid. The assay is simple to perform, reasonably accurate (coefficient of variation about 20%), and should be useful in studies of the physiological role of prostaglandins and the regulation of their synthesis.

Production and characterization of an antiserum to synthetic gonadotropin-releasing hormone.

Abstract The synthetic decapeptide “luteinizing hormone-releasing hormone” (LH-RH) was rendered antigenic by reaction of its histidine or tyrosine residues (7 : 3 approx.) with p-diazonium phenylacetic acid and coupling of the azo-derivatives formed to bovine serum albumin (BSA). Immunization of rabbits yielded antisera that bound 125 I-labeled LH-RH (approx. 50 pg) at dilutions up to 1:200, 000 and showed no cross-reaction with unrelated hypothalamic and pituitary hormones, extracts from rat cerebral cortex, and with small fragments of LH-RH. Cross-reaction was minimal (0.2%) with the free acid analogue of LH-RH, and moderate with des -pGlu LH-RH (20%), des -pGlu-His-LH-RH (2.4%) and with LH-RH analogues in which a single residue (No. 4–6 or No. 8) was exchanged by an amino-acid of similar character (1.2–12%). Biologically active hypothalamic extract and LH-RH produced parallel 125 I-LH-RH-binding inhibition curves, providing immunochemical support for the identity of the native releasing hormone with synthetic LH-RH.

Mechanism of steroid action in inflammation: inhibition of prostaglandin synthesis and release.

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5-8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 mug/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 - 30 mug/ml) were ineffective. Aldosterone and progesterone (100 mug/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 mug/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 mug/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 mug/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.

Acute Effect of ▵1–Tetrahydrocannabinol on the Hypothalamo-Pituitary-Ovarian Axis in the Rat

Administration of delta1-tetrahydrocannabinol (delta1-THC), the principal psychoactive ingredient of cannabis, to proestrous rats (2 mg/rat, i.p., between 12.00 and 16.00 h) suppressed the proestrous rise in the plasma levels of LH, FSH and prolactin (Prl) and caused a 24 h delay in ovulation. Furthermore, the increased accumulation of prostaglandins of the E-type (PGE) in the ovaries, normally seen on the evening of proestrus, was prevented. Earlier (08.00-10.30 h) or later (18.00 h) administration of the drug on the day of proestrus was only partially effective in inhibiting ovulation. The suppressive effects of delta1-THC on ovulation and gonadotropin secretion were prevented by administration of gonadetropin releasing hormone (GnRH, 0.2 microgram/rat) 1 h after the drug, indicating that the central action of delta1-THC was exerted on the hypothalamus and not on the pituitary gland. Administration of ovine luteinizing hormore (oLH, 2.5 microgram/rat at 16.30 h on the day of proestrus restored ovulation and ovarian PGE accumulation in Nembutal-treated rats, but not in delta1-THC-treated rats; higher doses of oLH (5-10 microgram/rat) reversed the action of delta1-THC on these two parameters.

Suppression of gonadotropin secretion and prevention of ovulation in the rat by antiserum to synthetic gonadotropin-releasing hormone

Abstract Administration of an antiserum (0.10–0.25 ml/rat) to the synthetic decapeptide “luteinizing hormone releasing hormone” (LH-RH) suppressed the cyclic surge of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in proestrous rats and prevented ovulation; exogenous LH reversed the block of ovulation. Serum prolactin levels remained unaffected. In ovariectomized rats, the antiserum suppressed the elevated serum levels of both gonadotropins. These findings are compatible with the view that the synthetic decapeptide is identical with the natural hypothalamic hormone that regulates the secretion of both LH and FSH.

Pituitary cyclic AMP production and mechanism of luteinizing hormone release

Secretion of anterior pituitary luteinizing hormone (LH) is controlled by the hypothalamic luteinizing hormone-releasing hormone (LH-RH) which has recently been isolated [ 1,2] . Crude hypothalamic extract containing LH-releasing activity was reported to stimulate adenylate cyclase and to increase the cyclic AMP level in the pituitary gland [3] . Recently it was found that synthetic LH-RH increased adenylate cyclase activity [4] cyclic AMP level and concomitantly stimulated LH release [S-8] . Hence it was suggested that cyclic AMP is the mediator of LH-RH action on LH release [S-7]. This conclusion was further supported by the findings that analogues of LH-RH which increased cyclic AMP production also caused LH release [9] . The addition of dibutyryl cyclic AMP (DBC) to the incubation medium was reported to enhance the release of LH [7,10] , while other workers found no increase of LH upon the addition of this drug [ 11,121 . The present investigation was intended to clarify whether the increase in pituitary cyclic AMP production by LH-RH is an obligatory step in LH release.

Stimulatory effect of prostaglandin E2 on LH release in the rat: evidence for hypothalamic site of action.

The intravenous administration of prostaglandin E2 (PGE2; 100 mug/rat) to immature male and female rats increased the serum level of lutenizing hormone (LH). This effect was prevented by the prior administration of an antiserum to the hypothalamic gonadotropin-releasing hormone. LH release from rat pituitaries in vitro was not stimulated by PGE2 (10 mug/ml). It is inferred that the stimulatory effect of PGE2, on LH release is not a direct one on the pituitary gland, but is exerted at the level of the hypothalamus.