ovariectomized ewes - Reproduction

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gland (de Jong & Robertson, 1985) and it has been isolated and sequenced using ..... Dr T. M. Farley for advice on statistical analysis; and Professor Henry Burger and Professor David de ... Bremner, W.J.,Findlay, J.K., Lee, V.W.K., de Kretser,.
Influence of dose and route of administration of bovine follicular fluid and the suppressive effect of purified bovine inhibin (Mr 31 000) on plasma FSH concentrations in ovariectomized ewes J. K.

Findlay, D.

M. Robertson and I. J. Clarke

Medical Research Centre, Prince Henry's Hospital, Melbourne, Victoria 3004 and * Department of Anatomy, Monash University, Clayton, Victoria 3168, Australia

Summary. Ovariectomized Merino ewes were used to develop an in-vivo bioassay for purified bovine inhibin of Mr 31 000. Various doses (0\m=.\25,0\m=.\5,1 or 2 ml) of bovine follicular fluid, given either by the intravenous (i.v.) or intracarotid route (i.c.) resulted in significant linear dose-related suppression of plasma FSH and interval to maximum suppression. Control ewes (1\m=.\0ml steer plasma) showed no significant change in FSH over the same period. Doses of 470 and 2590 U of pure inhibin given i.v. caused a significant suppression of FSH in plasma in all ewes. The in-vivo potency estimate of the high dose (2760 U, 1420\m=.\4690fiducial limits) agreed well with the in-vitro assay of potency. There were no significant changes observed in mean plasma LH after treatment with the higher dose of pure inhibin. There were no rebound effects of treatment with bovine follicular fluid or pure inhibin on FSH concentrations above that of controls. It is concluded that the form of bovine inhibin of Mr 31 000, which is believed to be the predominant circulating form, is biologically active when administered in vivo. Introduction

Inhibin is a gonadal peptide which selectively suppresses the secretion of FSH by the pituitary gland (de Jong & Robertson, 1985) and it has been isolated and sequenced using cloning techniques from pig (Miyamoto et al., 1985; Ling et al., 1985; Rivier et al., 1985; Mason et al., 1985) and cow follicular fluid (Robertson et al., 1985, 1986; Forage et al., 1986). At least two forms of inhibin have been identified, of Mr58 000 and 31-32 000 (Robertson et al., 1986; Fukuda et al., 1986), each consisting of two subunits of 43 000 and 13-15 000, 20-21 000 and 13-15 000, respectively. Incub?" of an iodinated form of the Mr 58 000 inhibin with serum but not follicular fluid led to generation of the Mr 31 000 form, whereas the latter form is not further processed (McLachlan et ., 1986). The inhibin of M, 31 000 is therefore most likely to be the predominant circulating form. The main objective of this study was to establish the biological activity in vivo of inhibin of Mr 31 000, purified from bovine follicular fluid. The suppression of plasma concentrations of FSH in ovariectomized ewes was used to test the biological activity of the pure inhibin. Materials and Methods Animals. Ewes selected from a Merino flock were ovariectomized 6 weeks before the study and had plasma concen¬ trations of FSH >20ng/ml. In 12 ewes one carotid artery was exteriorized in a skin loop (Bone et al., 1962) to facilitate intracarotid administration of the test materials. The experiment was conducted between February and July, and some ewes were treated on 2-3 occasions but with a 2-month intervening period. On each occasion, ewes were allocated to treatment groups such that the mean liveweight and range for each group was similar.

Ewes were housed in individual pens and fed chopped hay and water ad libitum. Blood samples were withdrawn from the jugular by venepuncture, collected into heparinized tubes, and the plasma harvested by centrifugation and then stored at 15°C. Blood samples were taken at —60, —30, —5, +30 min and +60 min and then at hourly intervals until 18-30 h after treatment. —

Injection material. Bovine follicular fluid and steer plasma were treated with dextran-charcoal (1 mg/ml final concentration) and stored at 15°C in tuberculin syringes at the appropriate doses. Follicular fluid stored in this way contained 9550U inhibin/ml, and