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H. M. RADFORD
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C. D. NANCARROW
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J. K. FINDLAY
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Prostaglandin analogues were used to induce luteal regression simultaneously in a number of ewes, thereby synchronizing the final stages of follicular maturation in these animals. Some of the ewes were anaesthetized for 24 h immediately after the injection of prostaglandin (experiment 1), and others for 15 h, starting 24 h after the injection of prostaglandin (experiment 2). In both experiments administration of anaesthetic significantly delayed the onset of oestrus and the time of ovulation relative to prostaglandin-treated control animals. The results from assays of blood samples collected at regular intervals in experiment 1 indicated that the preovulatory peak in the concentration of LH and the periovulatory changes in the concentration of FSH were similarly delayed and that during anaesthesia the level of LH was significantly reduced. It is suggested that the reduced level of LH, which probably resulted from a reduction in the secretion of releasing factor due to anaesthesia, failed to support oestrogen production by the Graafian follicle(s), thereby delaying the occurrence of oestrus and ovulation.

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R. Klein
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J. K. Findlay
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I. J. Clarke
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D. M. de Krester
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D. M. Robertson
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ABSTRACT

A sensitive and specific heterologous radioimmunoassay for FSH-suppressing protein (FSP or follistatin) was applied to ovine plasma. Following a logit–log dose transformation, parallel dose–response lines were observed between purified bovine 35 kDa FSP used as standard and serial dilutions of ewe plasma. Activin-A, inhibin-A and a range of other proteins showed low (<0·5%) cross-reactivity in the assay. Daily variations in the peripheral concentrations of FSP were measured across the ovine oestrous cycle. The peripheral concentrations of plasma FSP in adult ewes revealed a significant (P <0·01) increase (33%) during the luteal phase above follicular phase levels, peaking 10 days after the LH surge. FSP concentrations were determined in arterial and venous plasma from the ovary, head, kidney and liver. A significant (P <0·05) increase across the ovary was detected with no significant differences across the head, liver and kidney.

To investigate the relationship between gonadal FSP and the pituitary, ewes underwent ovariectomy and hypophysectomy. FSP levels rose (100–110%, P <0·01) during the period of surgery for both bilateral ovariectomy and sham ovariectomy, and then decreased significantly (37–44%) at 4–6 h after surgery. A further rise in plasma FSP (180–200% increase above pretreatment levels, P < 0·001) was observed 10–12 h after ovariectomy and sham ovariectomy. FSP levels then returned to preoperative levels during the following 26 h. Plasma FSP levels in long-term ovariectomized and hypophysectomized ewes were not significantly different from preoperative levels.

To determine whether the pattern of plasma FSP seen during the ovariectomy study was due to the effect of the induction of anaesthesia, ewes were treated with sodium thiopentone and halothane or 0·9% (w/v) NaCl by procedures of similar duration to that used during surgery. Both treatments resulted in an elevation of FSP levels (33–62%) over pretreatment values only at the time of induction of anaesthesia. To examine further whether this rise in plasma FSP observed after anaesthesia was due to a stress response and therefore under the control of the pituitary-adrenal axis, ewes were treated with ACTH, dexamethasone or saline only. A further group of sheep were exposed to a barking dog for 10 min. No change in FSP levels compared with pretreatment levels or saline-treated controls were noted following any of these treatments.

It was concluded that (1) FSP is present in the peripheral circulation; (2) ovariectomy contributes to plasma FSP, but no discernible contribution to circulating levels was evident from the head, liver or kidney; (3) plasma FSP levels rose significantly during the luteal phase of the ovine oestrous cycle; and (4) FSP secretion may be associated with a stress response, perhaps related to animal handling and intensive blood sampling through mechanisms that are as yet unclear.

Journal of Endocrinology (1993) 137, 433–443

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J. A. FINDLAY
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K. A. ROOKLEDGE
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ANNE BELOFF-CHAIN
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J. D. LEVER
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SUMMARY

The biochemical parameters of blood glucose, serum insulin and pancreatic insulin and glucagon were determined after single injections of the diabetogenic agent streptozotocin and in untreated animals from a strain of genetically obese mice and their lean litter-mates. Histological observations on α and β islet cells were also made in these animals by employing the phosphotungstic acid—haematoxylin and aldehyde—fuchsin staining techniques.

In untreated obese mice, endocrine hyperactivity was indicated by the presence of exceptionally large, highly vascular islets and by duct cell-islet metaplasia.

After streptozotocin injection, approximately half of the lean mice showed extensive β-cell necrosis and these animals became hyperglycaemic and hypoinsulinaemic with a much reduced pancreatic insulin over a long term. The remaining treated lean mice, whose biochemical parameters did not differ significantly from normal, showed little or no evidence of islet damage. The response of obese mice to streptozotocin treatment was less clear-cut than that of lean animals. Essentially similar histological changes were observed, though these were not consistently correlated with biochemical data. There was histological and biochemical evidence of islet cell recovery from the effects of the drug both in lean and in obese mice, and in all hyperglycaemic animals with initial severe islet damage a marked α-cell response was observed, namely an increased proportion of α cells and their random deployment throughout the islets.

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L A Salamonsen
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R J Young
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S Garcia
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J K Findlay
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Abstract

Endothelin-1 (ET-1) is present in ovine endometrium, primarily in epithelial cells, and increases around the time of implantation. We examined the cell type expressing ET-binding sites in vitro and whether ET-1 has mitogenic actions in the endometrium, alone or in synergy with other growth factors. Purified epithelial and stromal cells were prepared from luteal-phase endometrium. Specific receptors were demonstrated by binding of 125I-ET-1 and proliferative effects of ET-1 and/or other growth factors determined by uptake of [3H]thymidine by cells in serum-free culture. 125I-ET-1 bound to both epithelial (2516 ± 820 c.p.m./well) and stromal (6368 ± 1350 c.p.m./well) cells and was displaced by ET-1 (1 μmol l−1). There were no proliferative effects of ET on epithelial cells. ET-1 (10 nmol l−1) stimulated uptake of [3H]thymidine by stromal cells under serum-free conditions in 13/20 individual cell preparations, to 149 ± 13% of control (untreated=100%) with dose-dependence between the range of 1 to 100 nmol l−1. Stimulation by fetal calf serum was to 377 ± 126% of control. The effects on proliferation by other growth factors (dose; % of control ± s.e.m., number of positives/total number of cell preparations) were: IGF-I (13 nmol l−1; 182 ± 14, 4/4), epidermal growth factor (EGF; 4·8 nmol l−1; 132 ± 5%, 7/7), platelet-derived growth factor-BB (0·4 nmol l−1; 146 ± 3, 2/2) and leukaemia inhibitory factor (0·4 nmol l−1; 110 ± 2, 3/3). All stimulations except that of EGF were significant and dose-responsive but only insulin was additive with ET (350 ± 35, 5/5). ET-1 also stimulated expression of the the AP-1 cis element c-jun, this being maximal at 60 min of exposure to mitogen. ET-1, along with other growth factors has a likely paracrine role in cellular proliferation in the endometrium, possibly in association with blastocyst implantation.

Journal of Endocrinology (1997) 152, 283–290

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J. K. Findlay
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B. Doughton
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D. M. Robertson
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R. G. Forage
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ABSTRACT

Immunization of ewes against a pure recombinant preparation of the α subunit of bovine inhibin (α-bI) resulted in a three- to fourfold increase in ovulation rate, associated with antibodies in plasma recognizing pure native 31 kDa inhibin. The aim of this study was to examine the effects of this immunization on basal and GnRH-stimulated plasma concentrations of FSH and LH in ewes during the anoestrous and breeding seasons. The groups were untreated control ewes (n = 5), control ewes treated with keyhole limpet haemocyanin (KLH alone, n = 4), ewes treated with α-bI alone (n = 4) and α-bI–KLH conjugate-treated ewes (n = 3). There were no effects of immunization on basal FSH or LH in anoestrous ewes, despite the presence of antibodies recognizing 31 kDa inhibin. In the breeding season, immunization against α-bI resulted in increased basal (follicular phase, P < 0·1; luteal phase P < 0·05) and GnRH-stimulated (follicular phase only, P < 0·001) release of FSH, but not LH. The data are compatible with the hypotheses that the increase in ovulation rate in immunized ewes is due to an increase in circulating FSH concentrations and that inhibin may only have a major peripheral influence on FSH in sheep during the breeding season.

Journal of Endocrinology (1989) 120, 59–65

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L.A. Salamonsen
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S.J. Stuchbery
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C.M. O'Grady
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J.D. Godkin
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J.K. Findlay
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ABSTRACT

Ovine endometrial cells were isolated from ovariectomized oestrogen and progesterone-treated ewes and maintained in primary culture. In-vitro treatment with human interferon-α2 (Roferon, Hoffman La Roche) (5, 50 IU/ml) or purified ovine trophoblast protein 1 (oTP-1, 30 ng/ml) significantly attenuated PGF (25±17, 29±17, 28±9%±SEM of control [no in-vitro treatment = 100%] respectively, N=4 ewes) and PGE (11±4, 16±4, 16±5% of control) release from the cultured cells. Fluorography of two dimensional polyacrylamide gel electrophoretic analyses of proteins secreted by the cells following 35S-methionine incorporation, revealed that synthesis and secretion of the same "pregnancy-related" proteins was stimulated by both interferon-α2 and oTP-1. Thus, interferon-α2 (which has sequence homology with oTP-1) acts on the ovine endometrium, eliciting similar biological responses to those of oTP-1.

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L. J. Leversha
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D. M. Robertson
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F. L. de Vos
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F. J. Morgan
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M. T. W. Hearn
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R. E. H. Wettenhall
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J. K. Findlay
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H. G. Burger
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D. M. de Kretser
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ABSTRACT

Two forms of inhibin with molecular weights of 65 000 and 30 000 (65 and 30 kD) were isolated from ovine follicular fluid using a combination of gel permeation chromatography, reversed-phase high-performance liquid chromatography and preparative polyacrylamide gel electrophoresis. The 65 kD form was partially purified approximately 315-fold whilst the 30 kD form was isolated as two isoforms (29 and 30 kD) of similar biological activity and in >95% purity (1210-fold purification and 4·2% recoveries). On reduction the 30 kD form resolved into four components of 36, 31, 20–21 and 16 kD of which the 20–21 and 16 kD components were similar to the corresponding inhibin subunits isolated from porcine and bovine follicular fluid. The 36 kD component was established as a non-reducible inhibin-like material, based on its binding to antiserum raised against bovine 58 kD inhibin. The nature of the remaining non-reducible 31 kD component is unknown. Two NH2-terminal amino acid sequences (first 13 amino acids) identified in purified 30 kD inhibin were identical to the corresponding subunit amino acid sequences of bovine 31 kD inhibin.

J. Endocr. (1987) 113, 213–221

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

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