H.R. Lindner

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.

Specificity of antibodies to ovarian hormones in relation to the site of attachment of the steroid hapten to the peptide carrier

Estradiol-17β, estriol and progesterone were rendered antigenic by covalent attachment to bovine serum albumin through ring B or C of the steroid molecule. Coupling to lysine residues of the protein was effected via the 6-(O-carboxymethyl)-oximes of the estrogens and via the 6-(carboxymethylene) thioether or the 11α-hemisuccinate of progesterone, by use of the carbodiimide reagent. Antisera to the estradiol 6-conjugate raised in rabbits or goats distinguished estradiol-17β more efficiently from estrone, estradiol-17α and estriol than did antisera to estradiol-17β-hemisuccinate-BSA; neither serum reacted with non-phenolic steroids or non-steroidal estrogens. Antisera to estriol 6-conjugates showed minimal cross-reaction with estradiol-17β and estrone. Rabbit antisera against the progesterone 6-conjugate did not react with phenolic or C19-steroids, cr with corticosterone, and only feebly with 11-deoxycorticosterone and 20α-or 20β-dihydroprogesterone; significant crossreaction occurred with 17β-hydroxyprogesterone and 3β,17α-dihydroxypregn-5-en-20-one (3–4%), Δ5-pregnenolone (8–14%) and with 5β- and 5β-dihydro-progesterone (100%). Sera against the 11-conjugate were better able to recognize changes about the A-ring and failed to cross-react with 5α-dihydroprogesterone (< 3%). Both sera showed greater specificity than reported for antibodies to 20-conjugates of progesterone towards the D-ring and 17-sidechain and were similar in this respect to antibodies elicited by 3-conjugates described in the literature. It is concluded that the site of attachment of steroid haptens to the peptide carrier importantly affects the specificity of the antibodies produced.

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.

Kinetic of DNA synthesis in immature rat uterus: Age dependence and estradiol stimulation

Abstract 1. 1. The stimulation of DNA synthesis by estradiol-17β was studied in surving uteri of 1- to 30-day-old rats injected 24 h previously with estradiol-17β. 2. 2. Uteri from untreated immature rats showed an age-dependent decrease in incorporation of [Me−3H]thymidine into DNA during the first month after birth; incorporation decreased by a factor of 10 over this period. 3. 3. In rats less than 15 days old, a single estradiol-17β injection did not result in an increase in the rate of DNA synthesis; uterine wet weight, RNA content and protein content also showed no increase. At 15 days, estradiol caused an increase in uterine wet weight, but only slight increases in the other parameters measured. In uteri of 20-day-old rats, estradiol-17β caused a significant increase in the rate of DNA synthesis and in RNA and protein content. 4. 4. Amounts of estradiol-17β as low as 50 pg per 20-day-old rat (33 g in weight) significantly increased the rate of DNA synthesis. Dibutyryl cyclic AMP, administered in doses of 0.5 mg into the uterine lumen or 5 mg intraperitoneally, showed no effect on uterine nucleic acid or protein synthesis. 5. 5. In the 20-day-old rat uterus, the epithelial, stromal and myometrial tissues all showed a wave of cell division with a peak mitotic activity between 24 and 28 h after estradiol injection. In the epithelium, the increase was from 3 mitoses per 1000 cells to 164 per 1000 cells 26 h after hormone treatment.

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.

A synergistic effect of androgen on the stimulation of progesterone secretion by fsh in cultured rat granulosa cells

Granulosa cells from preovulatory follicles (PO) or from the enlarged preantral follicles of hypophysectomized immature diethylstilbestrol-treated (Hx-DES) rats were cultured with various combinations of FSH, androst-4-ene-3,17-dione (Ad), estradiol-17beta and dibutyryl cyclic AMP (dbcAMP). Progestin levels (progesterone and 20alpha-dihydroprogesterone) in the medium after 2 days of culture were assayed by radioimmunoassay. The control levels of the two progestins were lower for Hx-DES than for PO cells. Rat FSH (NIAMD-1-3;0.1 mug/ml) caused a 2-fold rise in progestin accumulation in both PO and Hx-DES cultures, dbcAMP (1 mM) increased progestin accumulation in PO cultures 4-5-fold, and to an even greater extent (10-20 fold) in Hx-DES cultures. Androstenedione (1.0 mug/ml) augmented progestin accumulation (1.5-3-fold), and synergized the steroidogenic action of FSH: in cells from Hx-DES rats, combined treatment with FSH and Ad caused a 5-10-fold increase over the values obtained with FSH alone. Testosterone and 5alpha-dihydrotestosterone, but not estradiol-17beta or estrone, mimicked these effects of Ad, Ad did not synergize the action of dbcAMP on progestin levels in Hx-DES cultures. It is proposed that androgen may play a role in the development of the FSH-responsive mechanism in preantral granulosa cells.

Ovulation rate and serum LH levels in rats treated with indomethacin or prostaglandin E2.

Abstract Serum LH levels were determined by radioimmunoassay at the normal time of the proestrous LH peak (17.30 – 18.00) and ovulatory performance was examined on the morning of estrus in rats treated with indomethacin, an inhibitor of prostaglandin synthesis. When the drug was administered at 14.30 on the day of proestrus, only 21% of the rats ovulated and the total number of ova shed was reduced to 4% of that found in the untreated control group, but there was no significant change in peak serum LH level (1122 ± 184 vs. 975 ± 240 ng/ml ± S.E., treated vs. control). Prostaglandin E2 (PGE2) given late on the day of proestrus (25 to 750 μ g/rat at 24.00) was effective in overcoming this antiovulatory action of indomethacin: 71–90% of the rats ovulated, though the number of eggs shed was low (24–55% of control value). Indomethacin was still effective in blocking ovulation when given at 20.00, that is after completion of the proestrous LH surge, but not at 24.00. Administration of PGE2 (2 × 750 μ g/rat) in the early afternoon of proestrus elicited a rise in serum LH levels in rats in which the cyclic LH surge had been blocked with Nembutal (470 ± 87 vs. 106 ± 17 ng/ml ± S.E.) and induced ovulation in two-thirds of these animals. The results confirm, by direct measurement, that indomethacin does not block LH release but interferes with a late phase of the ovulatory process. PGE2 reverses this action of indomethacin on the ovary. In addition, PGE2 has a central effect causing LH release.

Antigenic complexes of steroid hormones formed by coupling to protein through position 7: Preparation from Δ4 -3-oxosteroids and characterization of antibodies to testosterone and androstenedione

Abstract A general method for rendering Δ3-3-oxosteroids antigenic by coupling to a macromolecule through position 7 is described. It involves nucleophilic attack on the 6, 7-dehydroderivatives of the steroids by ambidentate reagents to form 7α-thioether alkanoic acids. These were covalently attached to bovine serum albumin (BSA) by use of the carbodiimide reagent. Addition products with mercapoacetic acid and β-mercaptopropionic acid and their BSA-conjugates, were thus obtained from testosterone androst-4-ene-3, 17-dione, progesterone and 17α-hydroxyprogesterone through the respective 4, 6-dienes. Immunization of rabbits with testosterone-7α-carboxymethyl-thioether-BSA and the homologous testosterone-7α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for testosterone (Ka=9. 4×109 1/mol) that showed little cross-reaction with androstenedione ( epit estosterone and dehydroepiandrosterone, or by 11-deoxycorticosterone, progesterone, 17α-hydroxyprogesterone, estrone and estradiol-17β. However, anti-testosterone sera cross-reacted with 5α-dihydrotestosterone (40–50%) and to a lesser extent with 5β-dihydrotestosterone (5%). Analogously, the anti-androstenedione sera cross-reacted with 5α-dihydroandrostenedione (71%) and to a minor extent with 5β-dihydroandrostenedione (8%). A radioimmunoassay procedure for the determination of testosterone in plasma is described, which makes use of the new anti-testosterone serum. Preliminary results suggest that it can be applied to ether extracts from human sera without Chromatographic purification.

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.

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.