Analysis of bovine follicular fluid (FF) using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with a sensitive immunoblotting procedure resolved several components that were immunoreactive with an antiserum directed against the n-terminus of the \g=a\subunit of human inhibin (hI\g=a\(1\p=n-\32) In conclusion, the present study has revealed that the bovine ovary secretes considerable quantities of monomeric inhibin \g=a\subunit. The unexpected presence of this material in peripheral blood is likely to hinder attempts to obtain physiologically relevant data on circulating levels of inhibin in cattle using conventional radioimmunoassays.
The effect of heating on plasmin activity in various media, including phosphate buffer pH 7-0, skim milk, blood plasma, solutions of casein and solutions of whey proteins were investigated. Plots of log residual activity v. heating time were linear at all temperatures from 63 to 143 °C. In buffer solutions the presence of casein led to substantial substrate protection, the Arrhenius plots being linear both in the presence and absence of casein. The activation energy, E a , for the inactivation reaction, was 62-4 kj/mol in buffer alone and 58-4 kJ/mol with casein present at 25 mg/ml. In skim milk, despite the presence of casein at a similar concentration, plasmin was no more stable to heat than in buffer alone, and a curved Arrhenius plot was obtained indicating a more complex inactivation mechanism. Heating in the presence of proteins having free -SH groups accelerated the inactivation of plasmin. The role of -SH groups was confirmed by experiments with added a-lactalbumin, in which no free -SH groups occur, and reduced carboxymethylated /?-lactoglobulin, both of which were without effect. In blood plasma, plasmin was less stable to heat than in buffer (pH 7 -0) or in skim milk. Plasminogen behaved very similarly to plasmin either when activated to plasmin with urokinase before heating or when activated afterwards. A hypothesis is presented to describe the heat inactivation and denaturation of plasmin. Technologically important findings are that in skim milk plasmin was largely unaffected by pasteurization conditions and 30-40% of its activity remained even after ultra high temperature processing conditions.
Primary monolayer cultures of bovine theca cells isolated from pooled ovarian follicles (3-10 mm diameter) were used to examine the effects of various granulosa cell-derived substances on basal and luteinizing hormone (LH)-induced androgen and progesterone secretion. After an overnight pretreatment period, cells were incubated with a range of treatments including LH, oestradiol-17 beta, inhibin, activin and follistatin. Media were collected after 48 h and assessment of androstenedione and progesterone secretion made by radioimmunoassay. Addition of LH (5-50 ng/ml) to the cells resulted in a dose-dependent stimulation of both androstenedione (2.5- to 3-fold rise; P < 0.01) and progesterone (approximately 1.6-fold rise; P < 0.001) production. Secretion of androstenedione was also raised (up to 5-fold; P < 0.001) by addition of oestradiol-17 beta (0.3-300 ng/ml), whilst levels of the androgen in the presence of both LH (20 ng/ml) and oestradiol (300 ng/ml) were up to 12-fold higher (P < 0.001) than control values. In contrast, oestradiol treatment inhibited by up to 50% both basal (P < 0.001) and LH-stimulated (P < 0.001) secretion of progesterone. Exposure of cells to purified bovine inhibin (5-125 ng/ml) consistently raised androstenedione secretion by up to 42% over basal levels (P < 0.001). Inhibin also enhanced both LH-stimulated (approximately 35%; P < 0.001) and oestradiol-stimulated (approximately 20%; P < 0.05) secretion of androstenedione. In direct contrast, treatment of theca cells with human recombinant activin-A (1-50 ng/ml) inhibited both LH-stimulated (approximately 50%; P < 0.001) and oestradiol-stimulated (approximately 30%; P < 0.005) androstenedione secretion. Activin also reversed the positive effect of inhibin on basal (P < 0.01), LH-stimulated (P < 0.001) and oestradiol-stimulated (P < 0.001) androstenedione secretion, though activin alone did not affect basal steroid output. Simultaneous addition of human recombinant follistatin reversed the inhibitory effects of activin on LH- and oestradiol-induced androstenedione secretion but did not modify the effects of inhibin. Follistatin alone did not alter either basal or LH-stimulated androstenedione output. Neither basal nor LH-stimulated secretion of progesterone were consistently affected by inhibin, activin or follistatin. As well as confirming the stimulatory effects of both LH and oestradiol on bovine thecal cell androgen production, these observations are indicative of opposing intrafollicular paracrine roles for granulosa cell-derived inhibin and activin in modulating thecal cell responses to gonadotrophins and steroids in the bovine ovary.(ABSTRACT TRUNCATED AT 400 WORDS)
Two experiments were conducted to explore the effectiveness of synthetic peptide-based vaccines for active and passive autoimmunization of sheep against inhibin. In the first experiment, adult Romney ewes (n = 20) were actively immunized against a synthetically produced peptide that corresponded to the N-terminus of the alpha-subunit of bovine inhibin (bI alpha(1-29)-Tyr30). This peptide was conjugated to tuberculin purified protein derivative (PPD) to increase its antigenic properties. Control groups comprised non-immunized (n = 10) and PPD-immunized (n = 10) ewes. Primary immunization (400 micrograms conjugate/ewe) was followed by two booster immunizations (200 micrograms conjugate/ewe), given 5 and 8 weeks later. Following synchronization of oestrus using progestagen sponges, ovulation rates were assessed by laparoscopy. Weekly blood samples were taken throughout the experiment. All inhibin-immunized ewes produced antibodies which bound 125I-labelled bovine inhibin (Mr 32,000), and ovulation rate in inhibin-immunized ewes (2.15 +/- 0.22; mean +/- S.E.M.) was significantly (P less than 0.01) greater than in both non-immunized (0.90 +/- 0.23) and PPD-immunized (1.20 +/- 0.13) control groups. Immunization against the peptide, but not against PPD alone, resulted in a modest rise in plasma FSH, with mean levels after the second boost being significantly (P less than 0.025) higher (22%) than those before immunization. Moreover, when blood samples were taken (2-h intervals) from randomly selected groups of control (n = 7) and inhibin-immunized (n = 7) ewes for an 84-h period following withdrawal of progestagen sponges, the mean plasma concentration of FSH during the 48 h immediately before the preovulatory LH surge was 37% greater (P less than 0.025) in immunized than in control animals. However, more frequent blood sampling (every 15 min for 12 h) during follicular and mid-luteal phases of the oestrous cycle revealed no significant differences between treatment groups in mean plasma concentrations of FSH. In addition, neither mean concentrations of LH nor the frequency and amplitude of LH episodes differed between immunized and control ewes. However, the mean response of LH to a 2 micrograms bolus of gonadotrophin-releasing hormone, given during the luteal phase, was significantly (P less than 0.05) less in immunized than in control ewes. These findings indicate that active immunization of Romney ewes against a synthetic fragment of inhibin can promote a controlled increase in ovulation rate, but this response cannot be unequivocally related to an increase in plasma levels of FSH.(ABSTRACT TRUNCATED AT 400 WORDS)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.