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DM de Kretser, MP Hedger and DJ Phillips

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AJ Tilbrook, DM de Kretser and IJ Clarke

Three experiments were conducted with castrated Romney Marsh rams (wethers) to investigate the ability of testosterone and inhibin to suppress the secretion of LH and FSH during the breeding and the non-breeding seasons. In Experiment 1, wethers (n=5/group) were treated every 12 h for 7 days with oil or 16 mg testosterone propionate (i.m.) and were then given two i.v. injections either of vehicle or of 0.64 microg/kg human recombinant inhibin A (hr-inhibin) 6 h apart. Blood samples were collected for 4 h before inhibin or vehicle treatment and for 6 h afterwards for the assay of LH and FSH. In Experiments 2 and 3 wethers underwent hypothalamo-pituitary disconnection (HPD) and were given 125 ng GnRH i.v. every 2 h. In Experiment 2, HPD wethers (n=3/group) were injected (i.m.) every 12 h with oil or testosterone and blood samples were collected over 9 h before treatment and 7 days after treatment. In Experiment 3, HPD (n=5/group) wethers were treated with vehicle or hr-inhibin, as in Experiment 1, after treatment with oil, or 4, 8 or 16 mg testosterone twice daily for 7 days. Blood samples were collected over 4 h before treatment with vehicle or hr-inhibin and for 6 h afterwards. Treatment of wethers with testosterone (Experiment 1) resulted in a significant decrease in the plasma concentrations of LH and number of LH pulses per hour but the magnitude of these reductions did not differ between seasons. Testosterone treatment had no effect on LH secretion in GnRH-pulsed HPD wethers in either season and treatment with hr-inhibin did not affect LH secretion in wethers or HPD wethers in any instance. Plasma concentrations of FSH were significantly (P<0.05) reduced following treatment with testosterone alone during the breeding season but not during the non-breeding season. FSH levels were reduced to a greater extent by treatment with hr-inhibin but this effect was not influenced by season. During the non-breeding season, the effect of hr-inhibin to suppress FSH secretion was enhanced in the presence of testosterone. These experiments demonstrate that the negative feedback actions of testosterone on the secretion of LH in this breed of rams occurs at the hypothalamic level and is not influenced by season. In contrast, both testosterone and inhibin act on the pituitary gland to suppress the secretion of FSH and these responses are affected by season. Testosterone and inhibin synergize at the pituitary to regulate FSH secretion during the non-breeding season but not during the breeding season.

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AJ Tilbrook, DM de Kretser and IJ Clarke

We have investigated the effectiveness of human recombinant inhibin A (hr-inhibin A) to suppress the secretion of follicle-stimulating hormone (FSH) in ram lambs from 1 to 18 months of age. Seventeen rams (nine castrated, eight intact) were used. At 1, 3, 6, 9, 12 and 18 months of age the rams were given an i.v. injection of either vehicle or hr-inhibin A (0.64 microgram/kg). Blood samples were taken over 24 h. Plasma concentrations of FSH were suppressed in castrated and intact rams following injection of hr-inhibin A with maximal suppression occurring 6 h after injection. Vehicle injection had no effect. At 12 months of age the suppression in FSH was most rapid in castrated rams and was maximal in intact rams. The clearance rate of inhibin was greatest at older ages but during the period of seasonally induced testicular activity, there was a significant decrease in the inhibin clearance. The testicular weight was reduced in rams treated with hr-inhibin A, indicating the importance of FSH for testicular development during the pubertal period. There was no effect of hr-inhibin A on plasma concentrations of luteinizing hormone. We conclude that inhibin is capable of suppressing the secretion of FSH in rams from 1 month of age onwards and that the pituitary gland becomes maximally responsive to the actions of inhibin by the age of puberty.

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KL Jones, DM De Kretser and DJ Phillips

Activin A and follistatin are normally present in relatively low amounts in the circulation. Heparin administration elicits a rapid and robust release of these proteins, although this phenomenon is poorly defined. In the present studies, the response to heparin administration was evaluated in the plasma of adult ewes in terms of whether it was dose-dependent, could be neutralized, was responsive to multiple stimulation, and the nature of the activin A and follistatin released. Activin A and follistatin were rapidly released by heparin in a dose-dependent manner (25, 100 or 250 IU/kg), with differences in the response as adjudged by peak concentration, timing of the peak and area under the curve. The heparin response could be blocked by pretreatment with protamine; conversely protamine injection alone (2 mg/kg) elicited release of follistatin but not activin A. Repeat administration of heparin at three-hourly intervals resulted in activin and follistatin responses to each injection, but each subsequent stimulation increased and extended the responses, consistent with saturation of the heparin clearance mechanism. Size exclusion chromatography of plasma samples confirmed that the majority of activin and follistatin released by heparin was a complex, whereas follistatin released by protamine was unbound. These data are consistent with a large pool of activin A and follistatin resident on extracellular matrices, with the rapid response implicating the vascular endothelium as the prime site of release following administration of these commonly used anticoagulant therapies.

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DJ Phillips, McFarlane JR, MT Hearn and DM de Kretser

alpha 2-Macroglobulin (alpha 2-M), a major serum glycoprotein, has been implicated as a low-affinity binding protein for inhibin and activin. In serum, alpha 2-M exists as two major species, a native form that is abundant and stable, and a transformed ('fast') species that is rapidly cleared from the circulation via alpha 2-M receptors. In this study inhibin, activin and their major binding protein follistatin were investigated for their ability to bind to the native or transformed species of purified human alpha 2-M. Using native PAGE and size exclusion chromatography, radiolabelled inhibin, activin and follistatin bound to the transformed alpha 2-M. Inhibin and follistatin did not bind significantly to native alpha 2-M, whereas activin was able to bind to the native species but with a lower capacity compared with that to transformed alpha 2-M. Under reducing conditions, binding of these hormones to alpha 2-M was abolished. These findings implicate alpha 2-M as a mechanism whereby inhibin, activin and follistatin may be removed from the circulation through alpha 2-M receptors, but also whereby activin can be maintained in the circulation through its ability to bind to native alpha 2-M.

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DJ Phillips, DM de Kretser, A Pfeffer, WN Chie and LG Moore

The effects on plasma follistatin concentrations of an inflammatory episode, induced by the intrathoracic injection of yeast, were examined in growing lambs; this model results in acute loss of appetite, food intake and liveweight and the activation of the acute-phase pathway for several weeks as adjudged by the production of haptoglobin and other acute-phase proteins. In these animals (n = 8) there was a biphasic response in follistatin concentrations, with an initial 200% increase (P < 0.001) in follistatin within 24 h of injection of yeast. Thereafter, follistatin concentrations were depressed to 70% of pretreatment levels 48 h after injection (P < 0.01), followed by a gradual recovery of concentrations to pretreatment values. In another group of lambs (n = 16) that were feed-restricted to mimic the reduced food intakes and liveweight changes in the yeast-injected group, plasma follistatin was also reduced to around 70% of pretreatment levels (P < 0.01) within 1 day of the dietary regimen being implemented, followed by a gradual return to pretreatment values as food intakes were increased. Plasma follistatin correlated significantly (r = 0.57, P < 0.0001) with food intake, but not with liveweight changes. Plasma follistatin concentrations were unchanged in a third group fed ad libitum (n = 8), except during two periods when food intakes were significantly (P < 0.05) reduced, when follistatin concentrations also decreased (P < 0.01). Plasma follicle-stimulating hormone (FSH) concentrations in the three groups of lambs were not significantly affected by the treatment regimes or changes in follistatin concentrations. These findings indicate that peripheral follistatin concentrations are modulated by both inflammatory and nutritional mechanisms, and that significant fluctuations in follistatin levels can occur without detectable perturbations in FSH secretion.

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DJ Phillips, JN Brauman, AJ Mason, DM de Kretser and MP Hedger

A new in vitro bioassay for activin was developed using the mouse plasmacytoma cell line, MPC-11. Human recombinant (hr) activin A dose-dependently inhibited the proliferation of these cells, whereas a range of other factors, including inhibin, follistatin and transforming growth factor-beta1, -beta2 and -beta3 had no effect. Conditioned medium containing activin B induced an inhibition similar to hr-activin A. The inhibitory influence of activin A could be blocked by follistatin, but not by hr-inhibin A. This bioassay had a sensitivity for activin A of around 0.4 ng/ml, an ED50 response of 3.5 ng/ml, and an intra-assay coefficient of variation of <11%. It offers substantial advantages over existing in vitro activin bioassays in terms of ease of use, specificity and throughput. The utility of the MPC-11 bioassay was demonstrated in the purification of activin from amniotic fluid, where an almost identical profile of bioactive activin A was detected compared with the pituitary cell bioassay of activin. Bioactive activin could also be detected in unpurified ovine allantoic and amniotic fluids and bovine follicular fluid. Measuring activin in untreated and heat-treated human sera or seminal plasma was hampered by a non-specific inhibitory effect, so that several serum samples did not run parallel with the hr-activin A standard. This inhibitory effect by serum could not be overcome by addition of follistatin, suggesting it is not activin-like bioactivity. This new bioassay for activin demonstrates widespread applicability for monitoring of purified or partially purified samples during purification procedures, bioactivity measurements, receptor-binding studies and assays of cell culture medium.

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KL Jones, DM de Kretser, IJ Clarke, JP Scheerlinck and DJ Phillips

A series of experiments were conducted in adult ewes to delineate the release profile of activin A and its relationship to other cytokines following an i.v. injection of the bacterial cell wall component, lipopolysaccharide (LPS). Following this challenge, plasma activin A increased rapidly and appeared to be released in a biphasic manner, slightly preceding the release of tumour necrosis factor-alpha (TNFalpha) and before elevation of interleukin (IL)-6 and follistatin levels. The concentration of activin A was correlated with body temperature during the response to LPS. A second experiment compared cytokine concentrations in matched blood and cerebrospinal fluid (CSF) samples. This revealed that activin A was not released centrally in the CSF following a peripheral LPS injection, nor was TNFalpha or the activin binding protein, follistatin, but IL-6 showed a robust elevation. In a third experiment, the stimulus for activin A release was examined by blocking prostaglandin synthesis. Flurbiprofen, a prostaglandin synthesis inhibitor, effectively attenuated the fever response to LPS and partly inhibited cortisol release, but the cytokine profiles were unaffected. Finally, the bioactivity of TNFalpha and/or IL-1 was blocked using soluble receptor antagonists. These treatments did not affect the initial release of activin A, but blockade of TNFalpha depressed the second activin peak. These studies define more rigorously the release of activin A into the circulation following acute inflammatory challenge. The response is rapid and probably biphasic, independent of prostaglandin- mediated pathways and does not depend upon stimulation by TNFalpha or IL-1. The data suggest that activin A release is an early event in the inflammatory cascade following the interaction of LPS with its cellular receptor.

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D D'Antona, FM Reis, C Benedetto, LW Evans, NP Groome, DM de Kretser, EM Wallace and F Petraglia

Activin A levels are elevated in maternal serum of pregnant women with hypertensive disturbances. Because follistatin is a circulating binding protein for activin A, the present study was designed to evaluate whether serum follistatin and activin A levels also change in patients with hypertensive disorders in the last gestational trimester. The study design was a controlled survey performed in the setting of an academic prenatal care unit. Healthy pregnant women (controls, n=38) were compared with patients suffering from pregnancy-induced hypertension (PIH, n=18) or pre-eclampsia (n=16). In addition, the study included a subset of patients with pre-eclampsia associated with intrauterine growth restriction (IUGR, n=5). Maternal blood samples were withdrawn at the time of diagnosis (patients) or in a random prenatal visit (controls), and serum was assayed for follistatin and activin A levels using specific enzyme immunoassays. Hormone concentrations were corrected for gestational age by conversion to multiples of median (MoM) of the healthy controls of the same gestational age. Follistatin levels were not different between controls and patients, while activin A levels were significantly increased in patients with PIH (1.8 MoM), pre-eclampsia (4.6 MoM), and pre-eclampsia+IUGR (3.2 MoM, P<0.01, ANOVA). The ratio between activin A and follistatin was significantly increased in patients with PIH (1.5 MoM) and was further increased in patients with pre-eclampsia (4.5 MoM) and in the group with pre-eclampsia+IUGR (2.6 MoM). Follistatin levels were positively correlated with gestational age in control subjects (r=0. 36, P<0.05) and in patients with PIH (r=0.46, P<0.05) or pre-eclampsia (r=0.61, P<0.01), while activin A correlated with gestational age only in the healthy control group (r=0.69, P<0.0001). The finding of apparently normal follistatin and high activin A levels in patients with PIH and pre-eclampsia suggests that unbound, biologically active, activin A is increased in women with these gestational diseases.

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R.G. Forage, R.W. Brown, K.J. Oliver, B.T. Atrache, P.L. Devine, G.C. Hudson, N.H. Goss, K.C. Bertram, P. Tolstoshev, D.M. Robertson, D.M. de Kretser, B. Doughton, H.G. Burger and J.K. Findlay

ABSTRACT

Seven Merino–Border Leicester cross–bred ewes were immunized with a purified fusion protein, produced by recombinant DNA methods, of the a subunit of bovine inhibin. Four animals were immunized with the fusion protein alone and three with a conjugate made by coupling the fusion protein to keyhole limpet haemocyanin (KLH) using glutaraldehyde. Each animal received four injections of the fusion protein over 93 days. The animals were synchronized using progestagen sponges and subjected to laparoscopy for the determination of ovulation rates in two consecutive cycles (days 115 and 135). The immunized animals had overall mean ovulation rates for each cycle of 3.4 and 3.4 which was significantly (P < 0.001) above the rates of 1.1 and 1.4 determined for the controls, which had either received no treatment (n=5) or had been immunized with 300 μg KLH (n=4). Analysis of antisera taken on day 115 showed significant fusion protein antibodies and iodinated inhibin–binding capacity in the test but not control groups. Furthermore, antisera to the fusion protein in four out of seven ewes neutralized the inhibin bioactivity of ovine follicular fluid in an in–vitro bioassay. These data demonstrate that neutralization of inhibin can be effected by immunization with bovine inhibin a subunit and that such immunization results in increased ovulation rates as predicted from the biological role of inhibin as a suppressor of FSH.