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D. M. ROBERTSON
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BERIT FRÖYSA
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There was no loss of biological activity in vitro when the 1st International Reference Preparation (IRP) for human pituitary gonadotrophins [FSH and LH/interstitial cell stimulating hormone (ICSH) for bioassay] code no. 69/104 and the 1st IRP for human pituitary LH/ICSH [for immunoassay] code no. 68/40 were stored for 1 year at −70 °C in a buffered 0·8% saline solution containing 1% bovine plasma albumin (BPA). However, storage of the 69/104 preparation at −20 °C in either 0·1 or 1% BPA, or at −70 °C in the presence of 0·1% BPA showed a small but significant decrease (∼ 10%) in activity over the same period. It is, therefore, advantageous to store these reference preparations at −70 °C in the presence of 1% BPA.

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H. B. WAYNFORTH
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D. M. ROBERTSON
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SUMMARY

Oestradiol-17β in ovarian venous blood and ovarian tissue was assayed by a competitive protein-binding method. Oestradiol was found in similar amounts in the ovarian vein blood of pregnant rats hypophysectomized on Day 12 and killed on Days 16 and 21 and in pregnant rats sham-hypophysectomized on Day 12 and killed on Day 16. The pituitary therefore plays no part in oestrogen production after mid-pregnancy until some time between Day 16 and Day 21, when it gives rise to an increased ovarian venous blood oestradiol content just before parturition, in intact sham-hypophysectomized rats. It is suggested that this increase is associated with the advent of the post-partum ovulation. The corpus luteum and the extraluteal component of the ovary in hypophysectomized rats autopsied on Days 16 and 21 and in sham-hypophysectomized rats autopsied on Day 16, contain similar amounts of oestradiol within each group. The extraluteal component contains about five times more oestradiol than corpora lutea in sham-hypophysectomized intact rats autopsied on Day 21. The ovaries of these animals also show an increased amount of oestradiol over that of the ovaries in the other three groups. It is suggested that secretion of oestradiol after mid-pregnancy in rats involves concurrently both the corpus luteum and the extraluteal component of the ovary.

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H. M. Fraser
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D. M. Robertson
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D. M. De Kretser
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ABSTRACT

Concentrations of immunoreactive inhibin in serum samples collected daily from six adult stumptailed female macaques during normal menstrual cycles were measured with a heterologous radioimmunoassay. Serum inhibin concentrations were low during the follicular phase of the cycle. After ovulation they began to rise, reaching a plateau between 8 and 11 days, before falling in parallel with the decline in luteal progesterone secretion. The dependence of the inhibin secretion by the corpus lutem on pituitary gonadotrophins was investigated by the administration of an LHRH antagonist [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10]LHRH once daily for 3 days beginning on day 8 of the luteal phase in six macaques. LHRH antagonist treatment markedly suppressed serum levels of inhibin and progesterone and these remained at the level found in the follicular phase for the remainder of the luteal phase. These results show that inhibin in the macaque is secreted into the peripheral blood almost exclusively during the luteal phase, being highest when FSH is at its nadir. Suppression of serum inhibin concentrations during the luteal phase by LHRH antagonist suggests that its secretion is integrated with the LH control of the corpus luteum.

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A. H. Torney
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D. M. Robertson
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D. M. de Kretser
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ABSTRACT

Bovine fetal gonads have been shown previously to contain inhibin bio- and immunoactivity although the ratio of these activities was markedly lower in testicular compared with ovarian extracts throughout gestation. The basis for this difference is examined in this study. Fetal testicular and ovarian high-speed supernatant preparations from bovine fetuses aged 180 to 270 days of gestation were sequentially fractionated by dye affinity chromatography, gel permeation chromatography, reversed phase highperformance liquid chromatography and preparative polyacrylamide gel electrophoresis and monitored by inhibin radioimmunoassay and in-vitro bioassay. Three immunoactive fractions were identified in testicular extracts with molecular masses of 30 kDa (Peak I), 43 kDa (Peak IIa) and 29 kDa (Peak IIb). Peak I material only was bioactive. On the basis of these characteristics, Peak I is probably 31 kDa inhibin as previously described, and Peaks IIa and lib are probably different inhibin α subunit precursor fragments.

In ovarian extracts, two bio- and immunoactive fractions were identified with molecular masses of 30 kDa (Peak I) and 29 kDa (Peak II). On the basis of size, and biological and immunological activities, the ovarian extract Peak I material is probably bovine 31 kDa inhibin, while the Peak II material is probably a novel inhibin-like protein. FSH-suppressing protein (or follistatin) bio- and immunoactivities were also identified in both testicular and ovarian extracts.

It is concluded that the low ratio of inhibin biological/immunological activity in testicular extracts is attributed to the presence of high concentrations of immunoactive α subunit precursor fragments which are low to non-detectable in ovarian extracts. These results support our previous hypothesis that, in contrast to the ovary, the inhibin α subunit is produced in excess in the fetal testis.

Journal of Endocrinology (1992) 133, 111–120

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C. L. Au
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D. M. Robertson
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D. M. de Kretser
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ABSTRACT

The hormonal control of inhibin production by adult rat testes was investigated using an in-vitro inhibin bioassay validated for the measurement of inhibin activity in charcoal-treated rat testicular extracts. The effect of hypophysectomy examined at 16 h, 3, 7 and 42 days after surgery showed a decrease in testicular inhibin content and seminiferous tubule fluid production by 7 days and a decrease in inhibin production by 42 days. Serum FSH and LH were suppressed 3 days after surgery. In 30-day chronically hypophysectomized adult rats treated for 3 days with twice daily s.c. injections of (a) human FSH (hFSH, 22 i.u./rat per day), (b) testosterone (5 mg/rat per day), (c) hFSH + testosterone (same doses as a and b), or (d) human chorionic gonadotrophin (hCG, 12 i.u./rat per day), hFSH or hFSH and testosterone stimulated an increase in testicular inhibin content but not in inhibin production or tubule fluid production. Testosterone and hCG had no effect on these parameters.

It is concluded that in vivo, FSH alone stimulates an increase in testicular inhibin content. The failure to observe an increase in inhibin production in vivo is attributed to the suppression of seminiferous tubule fluid production under the same experimental conditions.

J. Endocr. (1985) 105, 1–6

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D M Robertson
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J Sullivan
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M Watson
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N Cahir
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Abstract

In order to identify the molecular weight forms of bioactive and immunoactive inhibin in human plasma, plasma/serum was sequentially fractionated by immunoaffinity chromatography (using immobilised inhibin α subunit antiserum), reversed phase HPLC and preparative SDS-PAGE. The electroeluted gel fractions were assayed for inhibin in vitro bioactivity and immunoactivity, the latter by RIA. Initial experiments examined human follicular fluid as an inhibin-rich source. Bioactive and immunoactive fractions of 30, 35, 53, 65 and ∼120 kDa were identified in addition to bio-inactive, immunoactive fractions of 26 kDa and 32 kDa. These molecular weights correspond to those of known inhibin forms and are attributed to differing degrees of glycosylation of the inhibin α subunit and variable processing of the α and β inhibin subunits.

Fractionation of male plasma pools revealed the presence of higher molecular weight immunoactive forms (55–120 kDa) as well as 28–31 kDa forms although the molecular weight distribution of activity between pools varied. To assess if the molecular weight pattern was modified by storage and/or subsequent fractionation, protease inhibitors were added initially to plasma and fractionated as above. The molecular weight distribution of immunoactivity was largely unaffected by the treatment, indicating that minimal processing had occurred. Postmenopausal serum itself showed low to undetectable activity. The addition of recombinant human 31 kDa inhibin to postmenopausal serum resulted in a molecular weight profile of inhibin immunoactivity consistent with the presence of 31 kDa inhibin. Fractionation of a serum pool from women undergoing gonadotrophin stimulation, in which inhibin levels were elevated, showed a range of bioactive and immunoactive inhibin forms over the 30–120 kDa range. A good correspondence between activities was observed.

It is concluded that: 1. inhibin exists in plasma/serum as a range (28–120 kDa) of molecular weight forms. 2. In female serum, the majority of inhibin isoforms appear to be bioactive. 3. This fractionation procedure provides a basis for investigating the forms of inhibin in plasma and provides a means of assessing the specificity of new assay methods.

Journal of Endocrinology (1995) 144, 261–269

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B. A. Panaretto
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G. P. M. Moore
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D. M. Robertson
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Twenty-four adult Merino wethers were given mouse epidermal growth factor (mEGF) subcutaneously at doses ranging from 0·02 to 0·12 mg/kg body weight or intravenously in the dose range 010 to 0·14 mg/kg body weight for periods ranging from 3 to 48 h. Plasma concentrations of mEGF were measured by radioimmunoassay and effects of treatment on food consumption and wool growth were observed.

Plasma concentrations of the protein sustained for 15–24 h at about 20 ng mEGF/ml (or exceeding this) almost invariably caused feed rejection and casting of the fleeces. This last result clearly indicated disruption of proliferative activity among the replicating cells in wool follicles which regulate wool growth.

The inhibitory effects on appetite and wool growth of smaller doses of the protein and of plasma concentrations equal to those above which were sustained for shorter periods have also been examined.

Approximately 10% of the dose of mEGF appeared in the urine of three sheep 1 to 3 days after the start of s.c. infusions of 5 mg for 7 h.

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G. P. M. MOORE
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B. A. PANARETTO
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D. ROBERTSON
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The growth of the first hair coat in male mice was studied during administration of epidermal growth factor (EGF). Injections of 1 or 4 μg EGF/g body weight for 14 consecutive days from birth resulted in the development of curved overhairs (monotrichs), caused a retardation in rate of growth in length of hair and a reduction in hair diameter and length of follicle bulb. Growth rate partially recovered after cessation of EGF treatment. However, some of the effects produced by injections of EGF during the formation of the first coat were detected in the second and third generations of hair. Since EGF also retarded rate of body growth, we compared the effect of EGF on hair growth with that of restricting food intake in neonatal mice during the development of the first coat. Hair growth was slowed in underfed animals but the effects were less marked than those found in EGF-treated mice of similar body weights.

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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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D. M. de Kretser
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R. I. McLachlan
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D. M. Robertson
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H. G. Burger
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ABSTRACT

Serum concentrations of inhibin, FSH and LH were measured in 39 normal men and 127 men with testicular disorders resulting in infertility. The infertile men were divided into groups on the basis of their mean sperm count, FSH levels and karyotype. The mean (±s.d) serum concentrations of inhibin in the normal men was 554 ± 156 U/l and did not differ significantly from those groups with oligospermia, azoospermia or Klinefelter's syndrome. Combined analyses of all groups did not reveal any significant correlation between serum concentrations of inhibin and FSH or with any other parameter measured. Serum concentrations of FSH and LH were positively correlated, and Leydig cell dysfunction, as evidenced by increased serum LH levels, low testosterone levels or a declining testosterone/LH ratio were found with severe spermatogenic damage. The failure of serum concentrations of inhibin to correlate with those of FSH levels or the degree of testicular damage raise questions as to the clinical value of this parameter alone.

Journal of Endocrinology (1989) 120, 517–523

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