Sara Bauminger

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.

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.

Periovulatory changes in ovarian prostaglandin formation and their hormonal control in the rat

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.

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland

The possibility that prostaglandin E2 (PGE2) may play a role in luteinizing hormone (LH) release was examined using an in vitro model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood. It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.

Steroid-independent effect of gonadotropins on prostaglandin synthesis in rat Graafian follicles in vitro

The content of prostaglandins of the E-group (PGE) or F-group (PGF) was determined by radioimmunoassay in rat ovaries and in homogenates of cultured Graafian follicles. Intraperitoneal administration of luteinizing hormone (NIH-LH-S18; 10 mug/rat) at 9.00 h on any day of the estrous cycle caused an increase in ovarian PGE content within 5 h. The response was greatest on the day of proestrus (940% rise), i.e. when the ovary contains large follicles, and least at metestrus (80%). Follicles explanted from proestrous rats before the preovulatory gonadotropin surge responded to addition of LH (1-5 mug/ml) to the culture medium with a 10 to 30-fold increase in PGE and a 5-fold increase in PGF accumulation over a 5-h-period. Follicle stimulating hormone (NIH-FSH-S9; 10 mug/ml) caused a similar rise in follicular PGE accumulation, even after treatment of the FSH preparation with excess of an antiserum to the beta-subunit of LH. Stimulation of follicular PG accumulation was unimpaired during suppression of progesterone and estrogen synthesis by aminoglutethimide. It is concluded that these steroids play no part in the mediation of the LH-effect on follicular prostaglandin formation.

Prostaglandin synthesis in decidual tissue of the rat uterus

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.

Stimulation of cyclic AMP production in the rat ovary by luteinizing hormone: Independence of prostaglandin mediation*

Abstract In cubation of intact juvenile rat ovaries with luteinizing hormone (LH; 10 μ g/ml) for 20 min caused a ten-fold rise in cyclic AMP concentration, without increasing the activity of “prostaglandin synthetase” in the tissue. Flufenamic acid [N-(α,α,α- trifluoro-m-tolyl) anthranilic acid], aspirin or indomethacin (100 μ g/ml of each added to the incubation medium) inhibited “prostaglandin synthetase” activity by 90%, 97% and 70%, respectively, but did not prevent the stimulatory effect of LH on cyclic AMP formation. Prostaglandin E 2 (10 μ g/ml) also stimulated cyclic AMP formation in vitro, but this action was abolished by flufenamic acid. These findings argue against the hypothesis proposed in the literature that prostaglandins of the E-type are essential for the LH effect on ovarian adenylate cyclase and thus serve as obligatory mediators of cyclic AMP-dependent actions of LH on the ovary.

Immune response to polypeptidyl proteins in rabbit tolerant to the protein carriers

Abstract : A study was made of the immune responses to polypeptidyl proteins in rabbits which were either naturally tolerant or made experimentally unresponsive to the protein carriers. Experimentally acquired tolerance to HSA could be terminated by immunization of the unresponsive rabbits with poly-Ltyrosyl derivatives of HSA. Two parameters determined the breakdown of tolerance: (1) the chemical nature of the peptides attached: polytyrosyl HSA was effective in the termination of tolerance to HSA, whereas polyalanyl HSA was not; (2) the degree of enrichment was tyrosine. There appears to be an optimal degree of enrichment, i.e. of molecular alteration of the HSA, which will confer on the altered antigen the maximal potency to terminate tolerance: a slight alteration (3 per cent enrichment of HSA with Tyr) and a severe alteration (13 per cent enrichment) were less effective in tolerance breakdown than an intermediate degree of alteration (7 per cent enrichment with Tyr). The level of tolerance breakdown obtained by polytyrosyl HSA, as measured by the ratio of anti-HSA/ anti-polytyrosyl HSA, was greater than the level obtained previously by other chemically altered antigens. The significance of this ratio was discussed in relation to the cellular basis of immunological tolerance. Antibodies were produced to the peptides per se, when attached to proteins towards which the animal is naturally tolerant (RSA), or to proteins to which the animal has acquired tolerance (HSA). In both systems, there was a similar pattern of antibody formation, which may reflect a similarity in mechanism between natural and actively acquired tolerance.

Differences in prostaglandin formation between thymocyte subpopulations

The concentration of prostaglandin E and the activity of prostaglandin synthetase were determined in mature and immature mouse thymocytes. Hydrocortisone resistant thymocytes, or thymocytes separated from the immature cell population after agglutination of the latter by peanut agglutinin served as a source of mature thymocytes. It was found that mature thymocytes contain a much higher concentration of prostaglandin E and have an increased activity of prostaglandin synthetase than immature cells.

Specificity of immunological tolerance to poly-dl-alanyl proteins

Abstract Animals rendered tolerant to poly- dl -alanyl RNase and poly- dl -alanyl HSA exhibited a marked degree of tolerance to the parent proteins. Animals rendered tolerant to polyalanyl RNase showed partial tolerance to the polypeptide determinant when immunized with polyalanyl HSA or polyalanyl BSA. Thus, neo-natal injections of polyalanyl protein conjugates induced tolerance both to the polyalanyl determinants and to the determinants of the native proteins. Animals injected at birth with the essentially non-immunogenic multi-chain poly- dl -alanine, became tolerant to the polyalanyl determinant of the immunogenic polyalanyl proteins. Thus, there is no correlation between the immunogenicity of a substance and its capacity to induce tolerance.