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

Summary. The concentrations of inhibin in samples of rat testicular venous and arterial blood and interstitial fluid were measured by an in-vitro bioassay using pituitary cells in culture in which the standard was an ovine testicular lymph preparation (assigned potency 1 unit/mg). Inhibin levels were undetectable ( < 2 U/ml) in both blood samples but reached a mean concentration of 120 ± 7 U/ml in testicular interstitial fluid. After unilateral efferent duct ligation the rate of inhibin accumulation in seminiferous tubules was determined by the difference in the inhibin content of the ligated and unligated testes. Additionally, the rate of seminiferous tubule fluid production was obtained from the difference in weight between the ligated and non-ligated testes. In the 24 h after efferent duct ligation there were linear increases in inhibin (18·5 ± 1·0 U/h) and in seminiferous tubule fluid production (26 ± 1 μl/h), but there were no changes in serum FSH and LH levels.

Experimental induction of bilateral cryptorchidism led to a decrease in the inhibin content of the testis after 10 days. The rate of inhibin accumulation after efferent duct ligation declined more rapidly than the inhibin content, being significantly depressed in cryptorchid testes after 3 days, suggesting that this measurement is a more sensitive index of inhibin production than the determination of testicular inhibin content.

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

Summary. The testicular inhibin content showed an initial increase in the first 2–3 days after bilateral ligation of the efferent ducts of rats, followed by a subsequent decline to levels significantly below normal by 14 days, and reached 25% of control values at 42 days. Serum concentrations of FSH and LH were significantly increased at Day 6–7 after treatment and were still elevated after 42 days. The decline in testicular inhibin content at times associated with elevated FSH concentrations is consistent with the hypothesis of inhibin being involved in the feedback control of FSH secretion.

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

Summary. Rats were given s.c. implants of high (HT) or low (LT) doses of testosterone and 10 days later hypophysectomy or sham-operation was performed. The rats were killed after 50 days. Unilateral efferent duct ligation was performed 16 h before death to measure seminiferous tubule fluid production and the increment in testicular inhibin values (inhibin production). Inhibin levels in testis cytosols were measured by a pituitary cell culture bioassay. The LT implants maintained serum testosterone at control values and decreased testicular weight whereas HT implants raised serum testosterone 3-fold and maintained testicular weight at 75–85% of pretreatment levels. In intact rats, LT implants caused no change in testicular inhibin content but decreased inhibin production; no significant changes occurred with HT implants. After hypophysectomy both values were significantly suppressed and could not be maintained by HT or LT implants. However, the HT implants partly restored inhibin production despite their inability to influence testicular inhibin content. In contrast, tubule fluid production depended mainly on intratesticular testosterone levels and occurred normally in intact or hypophysectomized rats with HT but not LT implants. These results indicate that inhibin and seminiferous tubule fluid production, both functions of the Sertoli cell, are under different hormonal control. The maintenance of inhibin production by the testis requires the support of pituitary hormones, presumably FSH, while seminiferous tubule fluid production requires testosterone, presumably through LH stimulation of Leydig cells. These findings are consistent with the hypothesis that inhibin is produced in response to trophic stimulation by FSH.

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J. K. Findlay, D. M. Robertson, and I. J. Clarke

Summary. Ovariectomized Merino ewes were used to develop an in-vivo bioassay for purified bovine inhibin of M r 31 000. Various doses (0·25, 0·5, 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 (1·0 ml 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, 1420·4690 fiducial 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 M r 31 000, which is believed to be the predominant circulating form, is biologically active when administered in vivo.

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R. S. Carson, D. M. Robertson, and J. K. Findlay

Summary. The ability of antral follicular fluid obtained from sheep follicles to inhibit 3T3 fibroblasts maintained for 48 h in concentrations of fetal calf serum optimal for cell growth was examined. Addition of pooled follicular fluid to cultures resulted in a dose-dependent and reversible inhibition of [3H]thymidine incorporation. Serum from ovariectomized ewes, fetal calf serum, bovine inhibin, oestradiol-17β, testosterone, cortisol or progesterone were without effect over a range of doses. Treatment of pooled follicular fluid with charcoal–dextran did not reduce inhibitory activity which was only partly removed by heating at 85°C. Fluid obtained from large follicles (>5 mm) was more potent as an inhibitor than was fluid obtained from smaller (<5 mm) follicles. Gel chromatography of pooled fluid resolved two peaks of inhibitory activity associated with material of M r ∼ 180 000 and <10 000 respectively. No inhibitory activity was evident in fractions of serum from ovariectomized ewes chromatographed in an identical manner. These results indicate that ovine follicular fluid contains two components able to inhibit reversibly mitosis of 3T3 fibroblasts in vitro.

Keywords: fibroblast mitosis in vitro; inhibition; ovarian follicular fluid

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Y. T. Sun, D. M. Robertson, G. Gonzales, G. P. Risbridger, and D. M. de Kretser

Summary. Intact and hypophysectomized rats were treated with graded doses of testosterone via subcutaneous Silastic implants over a 13-week period. Serum inhibin concentrations fell 50% (P < 0·001) after 2 weeks of hypophysectomy, remaining suppressed at this level for 13 weeks. The administration of testosterone to hypophysectomized rats (serum testosterone values 2–12 ng/ml; control values 5·5 ng/ml) was without effect on serum inhibin values. In contrast, administration of testosterone to intact animals for 7 weeks resulted in an initial fall (P < 0·05) in inhibin levels to 50–70% of controls then increasing to reach control levels at higher doses. Serum FSH concentrations were similarly biphasic with increasing dose of testosterone and values for these two hormones were significantly correlated (r = 0·44, P < 0·01). Segments of seminiferous tubules in culture from rats after various times of hypophysectomy showed a partly suppressed secretion of inhibin. The administration of testosterone did not modify inhibin production although inhibin production was sensitive to FSH.

It is concluded that (1) serum inhibin concentrations are partly suppressed after hypophysectomy and testosterone has no effect on serum inhibin values; and (2) the suppression of serum inhibin in intact rats treated with increasing doses of testosterone is attributable to the concomitant fall in serum FSH concentrations.

Keywords: inhibin; testosterone; hypophysectomy; rat

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D. M. Robertson, Susan Ellis, Lynda M. Foulds, J. K. Findlay, and B. M. Bindon

Summary. Pituitary content of FSH and LH, using radioreceptor assay methods, was determined in control and Booroola Merino ewes on the 3rd day of the oestrous cycle and in adult rams slaughtered in winter. Significantly more pituitary FSH (as per gland or per g wet wt) was found in the Booroola than in the control ewe. No significant differences were found in LH content although the difference in FSH/LH ratio between Booroola and control ewes was significant (P < 0·001). Pituitary FSH content was similar in the rams of the two genotypes.

A good correspondence between FSH values by the radioreceptor assay and by radioimmunoassays using anti-ovine and anti-human serum was observed for the Booroola ewes. However, radioimmunological estimates of FSH activity were significantly higher than radioreceptor estimates in control ewes and in Booroola and control rams in which the pituitary FSH values were 5–6% of that of the ewe. This overestimation is attributed to differences in specificity between methods. Fractionation of pituitary extracts by electrofocussing indicated a similar pI profile of FSH for ewes and rams of both genotypes.

It is concluded that quantitative rather than qualitative differences in pituitary FSH occur in Booroola and control Merino ewes. It is suggested that increased FSH levels contribute to the hormonal basis for the increased ovulation rate of the Booroola Merino.

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S O'Leary, M J Jasper, G M Warnes, D T Armstrong, and S A Robertson

In pigs, uterine exposure to the constituents of semen is known to increase litter size but the underlying physiological mechanisms remain undefined. Studies in rodents and humans implicate immune modulating moieties in seminal plasma as likely candidates, acting through enhancing the receptivity of the female tract. In this study, the acute and longer term effects of seminal plasma on cytokine expression and leukocyte abundance in the pig endometrium during early pregnancy have been characterised. The reproductive tracts of gonadotrophin-primed pre-pubertal gilts treated with intrauterine infusions of either pooled seminal plasma or phosphate-buffered saline (PBS) were retrieved at 34 h, or on day 5 and day 9 after treatment. Seminal plasma elicited an endometrial inflammatory infiltrate comprised of predominantly macrophages and major histocompatibility complex class II+-activated macrophages and dendritic cells. The abundance of these cells was greatest at the pre-ovulatory (34 h) time-point and their increase relative to PBS-treated tissues was maintained until day 9 after seminal plasma treatment. Seminal plasma induced the expression of the cytokines, granulocyte macrophage colony-stimulating factor, interleukin-6 and monocyte chemoattractant protein-1, and the eicosanoid-synthesising enzyme cyclo-oxygenase-2. Expression was maximal 34 h after treatment but altered expression patterns as a consequence of seminal plasma induction persisted through early pregnancy. These changes were accompanied by altered dynamics in pre-implantation embryo development with an increase in the number of embryos and in their viability after seminal plasma treatment. Together, these findings implicate factors in seminal plasma in programming the trajectory of uterine cytokine expression and leukocyte trafficking during early pregnancy and in regulating pre-implantation embryo development in the pig.

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D. M. Robertson, M. Prisk, J. W. McMaster, D. C. Irby, J. K. Findlay, and D. M. de Kretser

Summary. After a single i.v. injection of purified human recombinant inhibin A (hr-inhibin) or bovine follicular fluid (bFF) to 3-day castrated 35-day-old male rats, serum FSH concentrations fell (P < 0·05) between 4 and 8 h, returning to control concentrations by 16–24 h. Administration of graded doses of hr-inhibin (0·625–10 μg/100 g body wt) and bFF (31·3–250 μl/100 g body wt) resulted in a parallel dose-related suppression of serum FSH with a maximum suppression 50% of controls. Similar experiments in 2-day ovariectomized 85-day-old female rats also showed a dose-related suppression with a maximum suppression approximately 30% of controls. Serum LH concentrations remained unchanged in all studies with male or female rats.

The biological activity of hr-inhibin in vivo was determined for male and female rats in terms of a standard bFF preparation defined by an in-vitro bioassay based on the suppression of FSH content in rat pituitary cells in culture. In males hr-inhibin exhibited a biopotency of 407 (159:1050; fiducial limits) U/μg protein and in females the biopotency was 358 (226:565) U/μg protein. These potencies are lower than that measured in the in-vitro bioassay (1120 (1040:1210) U/μg protein) and differences between in-vivo and in-vitro systems were attributed to the use of bFF rather than a purified human inhibin preparation as standard. These results indicate that hr-inhibin behaves similarly in vivo to bFF. Furthermore, based on the large working range and relatively good precision, the female rat system provides a good basis for an inhibin in-vivo bioassay method.

Keywords: inhibin; bioassay; in vivo; in vitro; rat

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S O’Leary, M J Jasper, S A Robertson, and D T Armstrong

Seminal plasma (SP) acts to influence the uterine endometrium after mating, activating synthesis of embryotrophic cytokines and inflammatory changes that condition the tract for embryo implantation and establishing pregnancy. The objective of this study was to investigate in pigs whether the ovary might also be responsive to SP exposure. Prepubertal gilts were synchronised with exogenous gonadotrophins and received transcervical treatment with pooled boar SP or PBS; then the ovarian tissue was recovered at 34 h (preovulation) and on days 5 and 9 after treatment. The ovarian response was assessed by measuring ovulation rate, number and size of corpora lutea, ovarian leukocyte populations, progesterone production in vivo, as well as responses of retrieved granulosa cells cultured in vitro. In SP-treated gilts, leukocyte recruitment into the ovarian tissues was increased fourfold at 34 h, with macrophages comprising the most abundant cell lineage. There was no effect of SP on the number of oocytes ovulated; however, the weight of corpora lutea was increased in SP-treated gilts. SP also induced an increase in plasma progesterone content seen from day 5 to at least day 9 after treatment. In addition, granulosa cells and thecal tissue retrieved from preovulatory follicles of SP-treated gilts were more responsive in vitro to growth factor- and gonadotrophin-stimulated cell proliferation and progesterone synthesis. These results suggest that uterine exposure to SP influences immune cell trafficking in the ovary and enhances steroidogenesis in early pregnancy. The effects of SP on ovarian function potentially contribute to reproductive success in the pig.