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I. A. Forsyth
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A. Turvey
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ABSTRACT

Explants of mammary glands from 60-day pregnant goats showed a mean fourfold increase in fatty acid synthesis from acetate when cultured with insulin+ cortisol. Epithelial cells increased their area by 60% but no secretory activity was induced. In 120-day pregnant goats, fatty acid synthesis and epithelial cell area were greater than at day 60 of pregnancy and were unaffected by hypophysectomy or by daily treatment with bromocriptine from day 60. Neither increased further on culture of mammary explants in insulin + cortisol.

Ovine prolactin increased fatty acid synthesis two-fold when added to insulin + cortisol in cultures of mammary tissue from goats on day 60 of pregnancy and secretory activity was induced. On day 120 of pregnancy insulin + cortisol + prolactin sustained or slightly stimulated both fatty acid synthesis and the extensive secretion present in the tissue at the start of culture. Synthesis of medium-chain fatty acids of milk-fat was also sustained by prolactin in one goat.

An atmosphere of air was found to maintain normal histological structure of the mammary gland. By contrast, in 95% oxygen, explants from goats which were 60 days pregnant showed epithelial cells filling the lumina of ducts and alveoli in 60% of explants and a poor response to prolactin.

J. Endocr. (1984) 100, 87–92

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C D Moorby
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J A Taylor
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I A Forsyth
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Abstract

Microsome fractions prepared from the mammary glands of non-pregnant, pregnant and lactating sheep have been used to study binding of 125I-labelled transforming growth factor-α (TGF-α). Binding was dependent on microsomal protein concentration, time and temperature. It showed the characteristics of an epidermal growth factor (EGF) receptor, being displaced by TGF-α and EGF, but not by insulin or IGF-I. The non-linear curve fitting program LIGAND was used to determine affinity and number of binding sites. A single class of high-affinity binding sites was found. The apparent dissociation constant (K d) was similar in all physiological states (2·43±0·27 mol/l × 10−10, n=23). Numbers of binding sites were lower in late-pregnant (20 weeks) and lactating sheep (14·07± 2·45 fmol/mg protein, n=10) than in non-pregnant, 10-or 15-week pregnant sheep (43·04±5·93 fmol/mg protein, n=13).

DNA synthesis by mammary alveolar epithelial cells cultured on collagen gels was increased twofold by TGF-a (maximum response at 10 μg/l; 1·8 nmol/l) but not by EGF. Cells derived from 15- to 20-week pregnant sheep responded significantly to TGF-α on day 3 of culture, but the response was delayed to day 4–5 of culture in cells from other physiological states. Dose–response was not significantly affected. TGF-α and IGF-I produced an additive effect on DNA synthesis. Oestradiol (10−12 to 10−9 m), a potential stimulator of the TGF-α gene, did not stimulate DNA synthesis alone, or in combination with IGF-I. It is concluded that growth factors acting via the EGF receptor play a role in ruminant mammary development, but whether they mediate oestradiol effects remains unresolved.

Journal of Endocrinology (1995) 144, 165–171

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S. J. Winder
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A. Turvey
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I. A. Forsyth
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ABSTRACT

Ovine mammary epithelial cell clumps (30–90 μm) were plated onto attached gels of rat tail collagen in serum-free medium. Synthesis of DNA by these cultures could be stimulated by insulin-like growth factor-I (IGF-I) with a median effective dose of 5 μg/l, irrespective of stage of pregnancy. The time-course of response, however, was significantly slower in cells prepared from mammary tissue of non-pregnant and early pregnant sheep compared with sheep later in pregnancy. IGF-II had approximately 10% of the potency of IGF-I in stimulating DNA synthesis. Insulin acted over a wide concentration range and produced a maximum rate of stimulation not significantly different from that produced by IGF-I. These results are consistent with actions through the type-I IGF receptor although insulin may also act through its own receptor, possibly stimulating local IGF-I production. It is concluded that IGF-I is an important mitogen for ovine mammary epithelial cells.

Journal of Endocrinology (1989) 123, 319–326

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I. A. Forsyth
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J. C. Byatt
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S. Iley
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ABSTRACT

Ten British Saanen goats were treated daily with 5 mg bromocriptine intramuscularly from week 8 of pregnancy until week 20 (day 140). By comparison with untreated control goats (n = 8), concentrations of prolactin in plasma were suppressed throughout the treatment period and remained significantly lower until 3 days prepartum, parturition occurring on day 153 ± 0·7 (mean ± s.e.m., n = 10). Growth hormone concentrations were low, but the incidence of levels exceeding 1 μg/l was increased in bromocriptine-treated goats. Plasma concentrations of placental lactogen, progesterone and oestrone sulphate were unaffected. The accumulation of pre-colostrum in the udder (lactogenesis stage I) was not affected by bromocriptine treatment in goats carrying twin fetuses, but in goats with single kids it was delayed by about 4–6 weeks to week 17 of pregnancy. Secretion could not be expressed from the udder and the concentration of α-lactalbumin in plasma remained low. Udder volume was significantly reduced in week 15–16 but not week 20–21 of pregnancy by bromocriptine treatment. Milk yields after 50 or 203 days of lactation were not significantly different from those in control goats. Placental lactogen concentrations in late pregnancy and udder volume in week 20–21 were the only variables measured which correlated with milk yield post partum. It is concluded that in vivo placental lactogen is an effective mammotrophic hormone, although less potent than prolactin as evidenced by the delay in lactogenesis stage I in bromocriptine-treated goats bearing single kids.

J. Endocr. (1985) 104, 77–85

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S. V. Smith
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I. A. Forsyth
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B. T. Donovan
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A radioimmunoassay for canine prolactin has been used to measure prolactin in the ferret. Serial dilutions of extracts of ferret pituitary glands and of ferret plasma yielded curves that were parallel with the canine prolactin standard curve. The sensitivity, accuracy, reproducibility and precision of the assay were within acceptable limits. Plasma prolactin levels increased after the administration of thyrotrophin releasing hormone (TRH) or chlorpromazine, but not after giving luteinizing hormone releasing hormone. Female ferrets, which were anoestrous, oestrous or spayed, and male ferrets had similar basal prolactin levels when sampled under sodium pentobarbitone anaesthesia. These basal levels were higher than in conscious males and the latter also showed a lesser response to TRH. Hypophysectomy significantly reduced basal prolactin levels in female ferrets by 2 h postoperatively and abolished the response to TRH.

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S. J. Winder
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S. D. Wheatley
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I. A. Forsyth
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ABSTRACT

Sucrose density centrifugation was used to prepare a partially purified membrane fraction from the mammary glands of non-pregnant, pregnant and lactating sheep. The binding of125 I-labelled insulin-like growth factor-I (IGF-I) was dependent on membrane protein concentration, pH, time and temperature. The binding showed the characteristics of a type-1 IGF receptor, being displaced by IGF-I (median effective dose (ED50) 0·55 nmol/l), less effectively by IGF-II (ED50 8·8 nmol/l) and least effectively by insulin. Glucagon, ovine prolactin and ovine placental lactogen could not displace binding. A molecular weight of 135 000 was determined by affinity cross-linking using disuccinimidyl suberate; this was consistent with the reported size of the type-1 receptor α-subunit. Scatchard analysis was used to determine binding affinity and numbers of IGF-I-binding sites. A single class of high-affinity binding sites was found in all physiological states. In non-pregnant sheep and sheep at days 40, 75 and 110–120 of pregnancy and at term, the binding affinity was similar (apparent dissociation constant (K d) 2·73 ±0·31 nmol/l, n = 22). In lactating sheep (weeks 1, 4 and 10), the binding affinity was significantly (P = 0·02) higher (K d 0·77± 0·06 nmol/l n = 9). Binding capacity was similar in non-pregnant and pregnant sheep (1005 ± 113 fmol/mg, n = 19), but fell by parturition and remained low in lactation (570±52 fmol/mg membrane protein, n = 12). The results suggest that the mammary growth of pregnancy is not regulated at the level of the type-1 IGF receptor.

Journal of Endocrinology (1993) 136, 297–304

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I A Forsyth
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J A Taylor
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G Gabai
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I R Fleet
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Abstract

125I-Labelled ovine prolactin was infused for 15 min into a pudic artery supplying one mammary gland of lactating goats (n=17). Between 0 and 4·25 h significandy more total (P<0·01) and trichloroacetic acid (TCA)-precipitable (P<0·001) radioactivity appeared in the milk of the infused compared with the non-infused gland. Gel chromatography and antibody precipitation indicated the presence of undegraded 125I-labelled prolactin in milk whey. Maximum transfer occurred 60–80 min after the end of infusion suggesting passage via a transcellular route. High plasma prolactin concentrations, resulting from infusion of cold prolactin with labelled prolactin in late lactation or from seasonally elevated prolactin at peak lactation, reduced the specific activity of infused prolactin and depressed the difference in secretion of 125I-labelled prolactin into milk of infused and non-infused glands. This suggests the operation of a competitive and saturable mechanism. Together with the increase in the milk to blood ratio of prolactin in goats given long-term (3 week) bromocriptine treatment, the results suggest that the goat mammary gland has a high avidity for prolactin especially when circulating prolactin is low. There was also evidence from TCA precipitation that prolactin may be protected from degradation in these circumstances. These mechanisms may contribute to the resistance of ruminant lactation to reduction in plasma prolactin and protect lactation from seasonal prolactin fluctuations.

Journal of Endocrinology (1995) 146, 411–420

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C. A. Carrington
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H. L. Hosick
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I. A. Forsyth
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R. Dils
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Multi-alveolar mammary structures (mammary lobules) were prepared from mammary glands of pseudopregnant rabbits by controlled digestion with collagenase and hyaluronidase. The overall rate of fatty acid synthesis and the proportion of milk-specific fatty acids (C8:0 and C10:0) synthesized by these lobules when cultured with insulin, corticosterone and prolactin were measured. Maximum response to physiological concentrations of prolactin (1·1 or 2·2 nmol/l) occurred in the presence of insulin (1·7 μmol/l) and corticosterone (0·58 μmol/l). In general, the results obtained on the effect of progesterone were negative. Though explants showed a ninefold greater response to prolactin per mg DNA than did mammary lobules, the latter have the advantage of being easily prepared for culture in large numbers. Reduction to below 500 μm diameter and culture in conditions which allow cell outgrowth onto plastic limited their response to prolactin. The probable roles of membrane damage by digesting enzymes and of tissue architecture in limiting prolactin response are discussed.

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G. Thordarson
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G. H. McDowell
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S. V. Smith
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S. Iley
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I. A. Forsyth
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ABSTRACT

Continuous intravenous infusions of saline or of a placental extract containing ovine placental lactogen were given to three non-pregnant, non-lactating ewes over periods of 36 h, 1 week apart. During saline infusion no placental lactogen was detected in jugular vein plasma, but infusion of the placental extract raised the placental lactogen concentration from undetectable to 40-50 μg/l, similar to concentrations in ewes with one fetus on day 90 of pregnancy. By comparison with the saline control period, infusion of the placental extract consistently increased both plasma concentrations and irreversible loss of non-esterified fatty acids. Plasma concentrations of glucose and urea, but not irreversible loss of these metabolites, were consistently increased. Although the placental extract was not subjected to extensive purification, it was enriched in placental lactogen and contained no detectable contamination with insulin, prolactin or growth hormone. The results are suggestive of a role for placental lactogen in modifying metabolism and acting during pregnancy to provide nutrients for fetal metabolism.

J. Endocr. (1987) 113, 277–283

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