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  • Author: D. M. de Kretser x
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H. M. Fraser, D. M. Robertson and D. M. De Kretser

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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C. L. Au, D. M. Robertson and D. M. de Kretser

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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A. H. Torney, D. M. Robertson and D. M. de Kretser

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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T. Yohkaichiya, D. Irby and D. M. de Kretser

ABSTRACT

This study examines the source of inhibin in the maternal circulation of pregnant rats by measuring serum immunoactive inhibin levels following a range of experimental procedures. Ovariectomy at days 7, 13 or 19 of gestation, with maintenance of pregnancy by supplementation with progesterone and oestradiol dipropionate, led to a profound fall of serum inhibin levels in comparison with controls, demonstrating that the ovary is a major source of circulating inhibin. This conclusion was supported by the inhibition of the late rise (days 16–22) in serum inhibin in pregnant rats which were hypophysectomized on day 15 and maintained with oestrogen and progesterone supplementation. These data support the view that the rise in serum inhibin from days 16 to 22 is due to re-activation of follicular development in preparation for the post-partum oestrus. Reduction of fetal numbers by hemihysterectomy on days 7, 13 or 19 did not alter serum inhibin levels. Induction of delayed implantation by ovariectomy on day 3 and progesterone supplementation together with initiation of reimplantation by the addition of oestradiol dipropionate on day 7 or 11 did not significantly alter inhibin levels. The induction of pseudopregnancy by mating to vasectomized rats did not result in the maintenance of stable serum inhibin levels until oestrous cycles recommenced. Taken together, the studies have identified the ovary as the predominant source of circulating maternal inhibin levels throughout pregnancy in the rat.

Journal of Endocrinology (1992) 132, 469–475

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A. J. Tilbrook, D. M. de Kretser and I. J. Clarke

ABSTRACT

To determine whether Leydig cells produce inhibin in the ram, Leydig cells were stimulated by administering human chorionic gonadotrophin (hCG) or raising the levels of endogenous LH by an injection of gonadotrophin releasing hormone (GnRH). Plasma concentrations of testosterone increased in the 72 h after either a single injection (P < 0·05) or two injections (P < 0·01) of hCG. Plasma concentrations of inhibin were not significantly influenced by either one or two injections of hCG. Administration of GnRH (1 μg) caused an 11-fold increase in plasma concentrations of LH but did not influence concentrations of inhibin in either the jugular or testicular veins (pampiniform plexus). In contrast, concentrations of testosterone were increased by about fourfold in both jugular (P < 0·01) and testicular (P < 0·05) veins. The concentrations of inhibin in the testicular vein were 1·3-fold higher than in the peripheral plasma (P < 0·05) both before and following treatment with GnRH whereas the concentrations of testosterone were 18- to 21-fold greater than in peripheral concentrations.

In view of the difference in concentrations of inhibin between testicular and jugular veins, in a further experiment a sample was taken from the jugular vein, a vein located in the tunica vasculosa of the testis (testicular vein) and from a vein (spermatic vein) and lymph vessels located in the spermatic cord. The mean (± s.e.m.) concentrations of inhibin were highest in the testicular lymph (45·93±4·21 μg/l; P < 0·001) compared with the peripheral (4·14±0·31 μg/l), spermatic (8·0±1·17 μg/l) or testicular (6·4±0·82 μg/l) plasma. Plasma concentrations of inhibin were significantly higher in the spermatic vein than in the testicular vein (P < 0·05) and jugular vein (P < 0·01), and concentrations of inhibin in the testicular vein were significantly (P < 0·05) higher than in the jugular vein. There were no significant differences in the concentrations of testosterone in the spermatic vein, testicular vein or testicular lymph but the concentrations of testosterone in the peripheral plasma were significantly (P < 0·05) less than in the testicular plasma or lymph.

These results suggest that, in the ram, the Leydig cell does not respond to hCG or endogenous LH by secreting inhibin or by influencing other cells within the testis to secrete inhibin within the time-frame of these experiments. The low testicular to jugular differences in the concentration of inhibin and the high concentrations of inhibin in the testicular lymph suggest that the lymph may be an important route of secretion of inhibin from the testis in the ram.

Journal of Endocrinology (1991) 130, 107–114

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G. F. Gonzales, G. P. Risbridger and D. M. de Kretser

ABSTRACT

The effect of epidermal growth factor (EGF) on the production of immunoreactive inhibin by adult rat isolated seminiferous tubules in vitro has been investigated. EGF (0·1–1000 ng/ml) added to cultures of seminiferous tubules from adult rats caused a dose-dependent increase in inhibin content in the tubules without changing the amount secreted into the media. However, after continuous stimulation with EGF for periods in excess of 5 days, an increase in inhibin secretion was observed. In the presence of 10 and 100 ng FSH/ml, EGF (10 ng/ml) produced a further increment in the inhibin content of the tubules, but this effect was not found with FSH concentrations of 500 or 1000 ng/ml. EGF also increased the tubule content of inhibin after the addition of 100 μg dibutyryl cyclic AMP/ml but no effect of EGF was observed on the FSH- or dibutyryl cyclic AMP-induced secretion of inhibin into the medium.

The effect of EGF on inhibin content in the tubules was partially suppressed by the addition of 4β-phorbol-12β-myristate-13α-acetate (20 ng/ml). Insulin (1–100 ng/ml) decreased basal inhibin secretion without changing the inhibin content of tubules and this effect was antagonized by EGF (10 ng/ml) with insulin doses of 1–50 ng/ml whereas, at 100 ng/ml, the effect of EGF on tubule inhibin content was reversed. The addition of EDTA (2 mmol/l) resulted in an inhibition of basal and EGF-induced inhibin production. These data demonstrate a stimulatory effect of EGF on inhibin production by isolated seminiferous tubules which is inhibited by insulin and phorbol esters, both stimulators of protein kinase C activity.

Journal of Endocrinology (1989) 123, 213–219

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V. W. K. LEE and D. M. DE KRETSER

Changes in levels of LH and FSH in the circulation were examined during repeated blood sampling in untreated rats and gonadectomized male and female rats treated with oestrogen, progesterone and thyroxine. Blood depletion induced a significant increase in levels of LH in steroid-treated rats but the increase was abolished when the depleted blood volume was replaced with egg albumen. The rise in LH was less dramatic in male than in female animals. In untreated rats, levels of LH either decreased or did not change with repeated phlebotomy. In contrast, the levels of FSH either did not alter or were lowered in all situations.

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D. M. de KRETSER, R. C. ATKINS and C. A. PAULSEN

SUMMARY

Autoradiographic localization of 125I-labelled luteinizing hormone (LH) in the renal proximal convoluted tubule of rats initiated a study of the role of the kidney in the metabolism of LH. Incubation of 131I-labelled LH with rat renal homogenates for 90 min failed to destroy its immunological reactivity.

The plasma half-life of 131I-labelled LH injected i.v. was investigated in normal ewes and rams. Preliminary studies indicated that the use of a preparation containing a high proportion of 'damaged' iodinated hormone could erroneously prolong the plasma half-life. Before use, 131I-labelled LH was purified by gel filtration and column chromatography using DEAE-cellulose. The mean plasma half-life of 131I-labelled LH in normal ewes was 26·7 min and was not significantly altered after oophorectomy (31·1 min). After bilateral oophorectomy and nephrectomy, marked prolongation of the plasma half-life was observed (mean 70·7 min). In normal rams, mean plasma half-life of 131I-labelled LH was 32·0 min, after orchidectomy 44·0 min and after orchidectomy and nephrectomy 82·0 min. The plasma half-life of 131I-labelled LH in a nephrectomized ewe maintained on haemodialysis was also prolonged (67·5 min), the determination being performed 24 h after the last haemodialysis. Although the kidney does not degrade LH, the plasma half-life was significantly increased after nephrectomy. This suggests that the localization of 125I-labelled LH in the proximal convoluted tubule may represent a secretory mechanism allowing renal excretion of LH.

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A. J. Tilbrook, D. M. de Kretser and I. J. Clarke

ABSTRACT

The roles of inhibin and testosterone in the negative feedback control of the secretion of FSH were explored in experiments using castrated rams administered human recombinant inhibin A (hr-inhibin) and testosterone propionate (TP). Two experiments were conducted in the non-breeding season. In experiment 1, two groups of long-term castrated rams (wethers) were treated with an i.v. injection of either vehicle or hr-inhibin in two doses (25 and 50 μg) given 2 weeks apart. Plasma concentrations of FSH, measured by radioimmunoassay, were suppressed significantly (P<0·01) and equally by both doses of hr-inhibin with a mean (± s.e.m.) maximal suppression of FSH of 19·9 ± 2·60% occurring 6–10 h after injection. In experiment 2, hypothalamo-pituitary disconnected (HPD) wethers given 125 ng gonadotrophin-releasing hormone (GnRH) every 2 h, were treated with vehicle or 25 or 50 μg hr-inhibin before or after treatment (32 mg/day) with TP. A cross-over design was used so that each wether was treated with vehicle and hr-inhibin. Treatment with TP significantly (P<0·001) suppressed plasma concentrations of FSH by 56%. Both doses of hr-inhibin were similarly effective in significantly (P<0·05) suppressing plasma concentrations of FSH causing a mean suppression of 31·1 ± 5·60% 6–10 h after injection. The suppressive effect of hr-inhibin was significantly (P<0·05) increased when the wethers were treated with TP to a mean suppression of 50·7 ± 5·6% 6–10 h after injection. These data indicate that both inhibin and testosterone exert negative feedback control on FSH secretion in rams and that the suppressive effects of inhibin may be enhanced by testosterone. Furthermore, both inhibin and testosterone acted directly on the pituitary to suppress FSH secretion in rams. The inhibition of FSH by a direct pituitary action of testosterone in this study is at variance with our previous findings with HPD wethers during the breeding season when it was shown that testicular steroids have minimal feedback effects at this level. These discrepancies suggest that the sensitivity of the pituitary to negative feedback by testicular steroids may change with the breeding season independent of an input from the hypothalamus. Finally, the greater suppressive effects of hr-inhibin in HPD wethers in experiment 2 compared with the hypothalamo-pituitary intact wethers in experiment 1 suggests that the sensitivity of the pituitary to inhibin may be increased by limiting the GnRH stimulus to the pituitary.

Journal of Endocrinology (1993) 138, 181–189

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D. M. DE KRETSER, T. J. MARTIN and R. A. MELICK

SUMMARY

The distribution and localization of 125I-labelled bovine parathyroid hormone was studied by radioautography in adult rats. The principal site of localization was the proximal convoluted tubule of the kidney. Light and variable localization was present in the liver, but no significant amount of label was found in muscle, spleen, bone or articular cartilage. The localization of this labelled compound in the proximal convoluted tubule suggests that this region degrades 125I-labelled parathyroid hormone.