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R. WEBB and G. E. LAMMING

Blood samples taken on alternate days through indwelling jugular venous catheters from 12 suckled cows between days 14 and 48 post partum contained significantly less prolactin than samples collected on intermediate days by jugular venepuncture. Samples taken through the catheter every 2 h for 72 h periods revealed a repetitive daily biphasic pattern of prolactin secretion with low concentrations at 09.00 and 19.00 h and high concentrations at 13.00 and 23.00 h.

In two groups of cows, one group calving at the beginning of March (increasing photoperiod) and the other calving during June (decreasing photoperiod), there was a significant negative correlation between stage of lactation and plasma prolactin concentrations in samples taken by venepuncture.

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C. J. Ashworth, I. Wilmut, A. J. Springbett and R. Webb

ABSTRACT

The effect of an inhibitor of 3β-hydroxysteroid dehydrogenase on peripheral progesterone concentration during the luteal phase of the oestrous cycle and on embryo survival was determined in sheep. Following administration of 10, 50, 100 or 250 mg epostane (4,5-epoxy-17-hydroxy-4,17,dimethyl-3-oxo-androstane-2-carbonitrile) progesterone concentrations were significantly lower than control levels 4 h after injection, from 2·5 to 22 h, 1·5 to 24 h and 1 to 24 h after injection respectively. There appeared to be no effect on peripheral oestradiol concentrations. Adrenal progesterone production was small and not influenced by epostane treatment.

Epostane was administered on day 9 of the oestrous cycle to cause a reduction in progesterone concentrations for approximately 12-18 h on day 9 only (group 1, 250 mg epostane on day 9), or a series of such reductions on 3 consecutive days (group 2, 50 mg epostane on days 9, 10 and 11) or a continuous reduction for 3 days (group 3, 250 mg epostane on days 9, 10 and 11). The proportion of ewes that were pregnant was significantly (P<0·05) lower in ewes treated to give a continuously low progesterone concentration for 3 days than in either controls or ewes in which progesterone concentration was reduced for less than 24 h (in controls and groups 1, 2 and 3 the proportion was 85, 92, 54 and 18% of ewes treated respectively). Embryo survival was not affected by administration of 250 mg epostane on days 9, 10 and 11 if luteal phase levels of progesterone were maintained by insertion of a silicone elastomer implant of the steroid. The proportion of embryos surviving was 72% in controls compared with 78% in the treated animals.

J. Endocr. (1987) 112, 205–213

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J. G. Gong, D. McBride, T. A. Bramley and R. Webb

ABSTRACT

Treatment of heifers with recombinant bovine somatotrophin (BST) significantly increases the population of small ovarian follicles and peripheral concentrations of somatotrophin, insulin-like growth factor-I (IGF-I) and insulin. To investigate the possible mechanism(s) involved in the action of BST on ovarian follicles, the effects of BST, IGF-I and insulin, given alone or in combination with either FSH or LH, on the proliferation of bovine granulosa cells in vitro were examined using a serum-free culture system. Bovine granulosa cells were obtained from antral follicles classified into three size categories according to diameter: small <5 mm; medium-sized 5–10 mm and large >10 mm. The proliferation of granulosa cells was assessed by the incorporation of [3H]thymidine into the cultured cells.

Both FSH and LH (1–1000 ng/ml) inhibited the proliferation of bovine granulosa cells obtained from all three size classes of follicles in a dose-dependent manner. BST, at doses ranging from 1 to 1000 ng/ml, had no effect on the proliferation of granulosa cells from small and medium-sized follicles, but inhibited the division of granulosa cells from large follicles in a dose-dependent manner. Treatment with either IGF-I (10–3000 ng/ml) or insulin (0·5–1000 ng/ml) stimulated, in a dose-dependent manner, the proliferation of granulosa cells obtained from all three size categories of follicles. No synergistic interaction between BST (30 ng/ml) and either FSH (50 ng/ml) or LH (5 ng/ml) was observed in granulosa cells from all three size classes of follicles. In contrast, physiological concentrations of both IGF-I (100 ng/ml) and insulin (1 ng/ml) acted in synergy with both FSH (50 ng/ml) and LH (5 ng/ml) to stimulate the proliferation of granulosa cells from small follicles, whilst no such synergistic interactions were observed in granulosa cells from medium-sized and large follicles.

It was concluded that the increase in the number of small ovarian follicles induced by BST treatment in heifers may be mediated by increased peripheral concentrations of IGF-I and/or insulin, possibly acting in synergy with gonadotrophins. Furthermore, insulin probably acts through its own receptor rather than acting via the type-I IGF receptor, as it can stimulate the proliferation of bovine granulosa cells at physiological concentrations.

Journal of Endocrinology (1993) 139, 67–75

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R. Webb, G. Baxter, D. McBride and A. S. McNeilly

ABSTRACT

Two experiments were carried out during the breeding season in ewes, first to investigate the effects of oral administration of a 3β-hydroxysteroid dehydrogenase (3β-HSD) inhibitor (epostane) on the number of corpora lutea, and secondly to investigate the mechanism through which epostane acts.

In the first experiment Dorset Horn ewes were treated orally with 25, 50, 100 or 200 mg epostane twice daily between days 10 and 15 of the oestrous cycle. All doses of epostane resulted in an increase in the number of corpora lutea per ewe, although the response was curvilinear, with the 25 mg dose showing the largest response and the 200 mg group the smallest response. Although there was no difference between groups in the number of ewes showing oestrus, the higher doses of epostane had a detrimental effect on fertility.

In the second experiment Welsh Mountain ewes were treated twice daily with 25 mg epostane from day 10 of the oestrous cycle and the ovaries were removed for analysis during either the luteal or the follicular phases. Treatment significantly increased the number of follicles >6 mm in diameter, but significantly reduced in-vitro follicular oestradiol and testosterone production. Despite a marked increase in peripheral inhibin concentrations there was no effect on in-vitro inhibin production. Epostane treatment also caused a significant reduction in peripheral FSH concentrations and an increase in mean LH concentration. The latter was due to an increase in LH pulse frequency during the luteal phase and LH pulse amplitude during the follicular phase.

These results confirm that treatment of ewes with epostane orally has a significant effect on follicular steroidogenesis and causes a significant increase in the number of corpora lutea per ewe. This effect on ovulation rate is not via an increase in peripheral FSH concentration, but may be caused by a reduction in follicular steroid activity either directly on the ovary or via an alteration in the pattern of LH secretion.

Journal of Endocrinology (1992) 134, 115–125

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J G Gong, D McBride, T A Bramley and R Webb

Abstract

Our previous studies have demonstrated that physiological concentrations of metabolic hormones, including recombinant bovine somatotrophin (BST), insulin-like growth factor-I (IGF-I) and insulin, can significantly stimulate the proliferation of bovine granulosa cells cultured under serum-free conditions. In this study we investigated the effects of these factors on bovine granulosa cell steroidogenesis using the same culture system. Bovine granulosa cells were obtained from antral follicles classified into three size classes: small, <5 mm; medium-sized, 5–10 mm and large, >10 mm in diameter.

Whilst not affecting steroidogenesis by granulosa cells from small and medium-sized follicles, BST (10–1000 ng/ml) stimulated the secretion of both oestradiol and progesterone by granulosa cells from large follicles in a dose-dependent manner. Insulin (1–1000 ng/ml) and IGF-I (10–1000 ng/ml) stimulated the secretion of oestradiol and progesterone by granulosa cells from all three size categories of follicles in a dose-dependent manner. FSH (200 ng/ml) alone increased progesterone secretion by granulosa cells from all three size classes of follicles, but had no effect on oestradiol secretion by granulosa cells. Both IGF-I (200 ng/ml) and insulin (30 ng/ml) acted in synergy with FSH (200 ng/ml) to stimulate steroidogenesis by granulosa cells from all three size categories of follicles, but no such interaction was observed between BST (50 ng/ml) and FSH (200 ng/ml).

In conclusion, BST, IGF-I and insulin significantly influence the steroidogenic activity of bovine granulosa cells cultured under serum-free conditions. However, unlike their effects on cell proliferation, the minimal effective concentrations of these factors required to stimulate granulosa cell steroidogenesis were higher than those observed in our previous studies in vivo.

Journal of Endocrinology (1994) 143, 157–164

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M. J. TAYLOR, R. WEBB, M. D. MITCHELL and J. S. ROBINSON

The concentration of progesterone in the peripheral plasma of seven sheep during late pregnancy was reduced by injection of an inhibitor of 3β-hydroxysteroid dehydrogenase activity. Concentrations of progesterone were 10·0± 1·0(s.e.m.) ng/ml (n = 6) before injection of the inhibitor, fell to 1·39 ± 0·40 ng/ml (n = 6) 30 min after injection, and remained within this lowered range for 6 h after injection. By 20–24 h and 30–35 h after injection progesterone concentrations had recovered to 4·63±0·94 and 14·07 ±4·17 ng/ml respectively (n = 6). Six out of seven ewes delivered prematurely 32·5± 2·9 h after injection. Delivery appeared to be normal, and was associated with increasing concentrations of 13,14-dihydro-15-oxo prostaglandin F in peripheral plasma. Concentrations of oestradiol-17β17β in peripheral plasma were slightly raised immediately before delivery, at which time progesterone concentrations were within the preinjection range. These data suggest that progesterone withdrawal is one mechanism that initiates increased prostaglandin F secretion in the pregnant sheep.

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DG Armstrong, CG Gutierrez, G Baxter, AL Glazyrin, GE Mann, KJ Woad, CO Hogg and R Webb

IGFs regulate gonadotrophin-stimulated proliferation and differentiation of granulosa and theca cells in vitro. However, the detailed pattern of mRNA expression of IGFs in bovine follicles remains controversial. The objectives of this study were therefore to describe the temporal and spatial pattern of expression of mRNA encoding IGF-I, IGF-II and the type 1 IGF receptor in bovine follicles in vivo. The expression of mRNA encoding IGF-II was detected in theca tissue from around the time of antrum formation up to and during the development of dominance. No IGF-II mRNA expression was detected in granulosa cells. In the majority of follicles we were unable to detect mRNA encoding IGF-I in either granulosa or theca tissue from follicles at any stage of development. Occasionally low amounts of mRNA encoding IGF-I were detected in the theca externa and connective tissue surrounding some follicles. Type 1 IGF receptor mRNA was detected in both granulosa and theca cells of preantral and antral follicles. Expression was greater in granulosa tissue compared with theca tissue. We also measured IGF-I and -II mRNA in total RNA isolated from cultured granulosa and theca cells using reverse transcriptase PCR. In contrast to the in vivo results, IGF-II mRNA was detected in both granulosa and theca tissue. IGF-I mRNA was detected in theca tissue and in very low amounts in granulosa cells. Using a specific IGF-I RIA we were unable to detect IGF-I immunoreactivity in granulosa conditioned cell culture media. Using immunohistochemistry we detected IGF-I immunoreactivity in some blood vessels within the ovarian stroma. We conclude from these results that IGF-II is the principal intrafollicular IGF ligand regulating the growth of bovine antral follicles. In preantral follicles the expression of mRNA encoding type 1 IGF receptor but absence of endogenous IGF-I or -II mRNA expression, highlights a probable endocrine mechanism for the IGF regulation of preantral follicle growth.

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H. M. Charlton, Dilys Parry, D. M. G. Halpin and R. Webb

Hypogonadal mice are deficient in LH releasing hormone (LH-RH), the releasing factor for LH and FSH, with a consequent failure of postnatal ovarian and testicular development. After intravenous injection of hypogonadal females with 125I-labelled human chorionic gonadotrophin (hCG), followed by autoradiography of semi-thin (1 μm) slices of the ovary, labelled hCG was found to be associated with interstitial cells and thecal cells with little or no labelling of granulosa cells. Labelled human FSH was associated solely with granulosa cells. Hypogonadal females, implanted for 5 days with a silicone elastomer capsule of oestrogen, showed a similar response to that of normal females with hCG labelling of the granulosa cells of the larger follicles as well as of the thecal cell layer. Furthermore, subcutaneous injection of hypogonadal females with LH-RH (50 ng), 12 times daily for 5 days, increased uterine weight and stimulated ovarian development with some large follicles binding hCG to both thecal and granulosa cells. Therefore stimulation of follicular development may possibly be associated with increased oestradiol concentrations.

In the male, after injection of 125I-labelled hCG, silver grains were associated with the interstitial cells alone in both hypogonadal and normal mice. Labelled human FSH was undetectable in semi-thin testicular sections, but the mode of injection (intravenous) may not have allowed enough labelled hormone to reach the testis in order to resolve the question as to whether the hypogonadal or normal testis can bind FSH.

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T. A. Bramley, G. S. Menzies, G. Baxter, R. Webb and A. S. McNeilly

ABSTRACT

Immunoreactive α-inhibin (ir-inhibin) was measured in luteal homogenates and subcellular fractions of ovine and porcine corpora lutea (CL) and in pig granulosa cells (GCs), using a sensitive radioimmunoassay specific for the 1–26 amino acid sequence of the N-terminus of the α chain of porcine inhibin (p1–26 α-inhibin). Inclusion of N-ethylmaleimide (N-EM) and/or EDTA in the immunoassay had no effect on the measurement of p1–26 α-inhibin peptide standards, on ir-inhibin levels in ovine follicular fluid and serum, or on ir-inhibin in subcellular fractions of pig GC. Fractionation of porcine GC homogenates on sucrose gradients demonstrated a major particular peak of ir-inhibin (buoyant density, 1·15–1·21 g/cm3) with variable activity in the cytosol. The particulate ir-inhibin peak was released into the cytosol by pretreatment of GC homogenates with the saponin, digitonin, prior to fractionation. Porcine GC extracts contained a protein (M r 45 000) which immunoblotted against p1–26 α-inhibin antibody.

In the absence of inhibitors of proteolysis, apparent ir-inhibin activity was very high in extracts of sheep and pig CL. However, inclusion of N-EM or EDTA in the radioimmunoassay significantly reduced ir-inhibin levels in porcine and ovine CL extracts in a dose-dependent manner. Measurements of peptide tracer integrity indicated that porcine luteal cytosol degraded 125I-labelled p1–26 α-inhibin peptide. Subcellular fractionation studies demonstrated high levels of apparent ir-inhibin in luteal cytosol fractions, with only minor activity peaks associated with particulate fractions; however, this material was not releasable by digitonin. Immunoblotting of detergent extracts of porcine luteal particulate fractions failed to demonstrate α-inhibin material, and immunocytochemical localization studies of α-inhibin in porcine and ovine luteal sections were negative.

Our results are consistent with the intracellular packaging/storage of a form of α-inhibin (M r similar to that of α-inhibin subunit precursor) in the porcine granulosa cell. However, luteinization of the porcine follicle was associated with a dramatic fall in ir-inhibin content, and the loss of immunostaining for α-inhibin peptides. We conclude that porcine and ovine CL contain little, if any, authentic inhibin. These studies emphasize the importance of excluding proteolytic artefacts when measuring biological peptides in luteal tissue extracts by radioimmunoassay.

Journal of Endocrinology (1992) 134, 341–352

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C G Gutiérrez, B K Campbell, D G Armstrong and R Webb

Abstract

Insulin-like growth factor-binding protein (IGFBP) extraction protocols were tested for their efficacy in removing IGFBPs from bovine plasma and bovine granulosa cell culture medium compared with standard acid exclusion chromatography. Traditional extraction methods, acidification, Sep–Pak, ethanol:acetone:acetic acid (EAA) and EAA-cryoprecipitation (EAA-C), failed to remove all the IGFBPs from both granulosa cell culture medium and plasma. However, EAA and EAA-C treatment of plasma samples did give values similar to those obtained by acid exclusion HPLC, when corrected for extraction efficiency. There was an inverse relationship between insulin-like growth factor-I (IGF-I) concentration in plasma samples, as measured using HPLC chromatography, and IGF-I concentration after EAA extraction. Furthermore, the interference caused by residual IGFBPs differed between samples taken from animals given various treatments that altered peripheral IGF-I concentrations.

As for plasma samples, EAA was the most effective extraction method for culture media, but residual IGFBPs caused an overestimation of IGF-I concentrations. In culture media, but not plasma, it was possible to block the interference of IGFBPs in the IGF-I assay, in both extracted and non-extracted culture samples, by the addition of excess IGF-II. Using this assay procedure, no IGF-I production by bovine granulosa cells was detected. This was confirmed by HPLC acid chromatography.

It is concluded that HPLC extraction is needed for the accurate measurement of peripheral IGF-I concentrations. For granulosa cell culture media it is possible to measure IGF-I concentrations in non-extracted samples if the IGFBPs are blocked by adding IGF-II. Using either this assay, or after HPLC acid chromatography, no IGF-I was detected in culture media, suggesting that IGF-I is not produced by non-luteinised bovine granulosa cells.

Journal of Endocrinology (1997) 153, 231–240