Cloning and expression of ovine placental lactogen.

Ovine placental lactogen (oPL) is active in a wide range of GH and PRL assays, a property that it shares with human GH (hGH). In addition, oPL is one of a small number of hormones that bind the human GH receptor with high affinity. In order to compare the sequence of oPL to the sequences of other members of the GH family, full-length cDNA clones have been isolated. These clones predict that the full sequence of oPL contains 198 amino acids preceded by a 38 amino acid signal sequence. The mature oPL sequence includes six cysteine and two tryptophan residues and shows substantially more identity to bovine PL (67%) and oPL (49%) than to mouse (31%) or human (25%) PL or to oGH (28%) or (26%) hGH. Like the natural hormone, oPL expressed in mammalian tissue cells binds with high affinity to a soluble form of the recombinant hGH receptor. Thus, oPL binds to the human receptor in spite of having a sequence that is considerably divergent from hGH. Interestingly, the sequence of oPL differs from hGH at most of the amino acids recently found by mutagenesis studies to be important residues in the binding of hGH to the human receptor.

Light and electron microscopic studies of cellular localization of oPL with monoclonal and polyclonal antibodies.

Accurate knowledge of placental lactogen localization is fundamental to any hypothesis of its synthesis and secretion. We used locally generated monoclonal and polyclonal antibodies from three separate sources to localize ovine placental lactogen immunoreactivity on light and electron microscope Lowicryl K4M sections of ovine placentomes of 97-145 days of gestation, using immunogold techniques. All antibodies demonstrated that immunoreactivity was exclusively localized in the trophoectoderm binucleate cell Golgi body and granules and in granules in the syncytium derived from binucleate cell migration. No evidence was found to support a recent claim that monoclonal antibodies to oPL that were produced in Canada indicated a predominant localization of ovine placental lactogen to uninucleate trophectodermal cells.

Placental lactogen in the cow.

Placental lactogen secretion has been demonstrated in cows on days 36, 178, 182 and 270 of pregnancy by co-culture of cotyledonary tissue with mouse mammary gland explants. Bovine placental lactogen probably originated from the foetal cotyledon and showed no detectable cross-reaction in immunoassays for bovine prolactin or growth hormone. Peripheral plasma samples collected from seven primiparous heifers at 2-week intervals throughout pregnancy showed a seasonal rhythm in prolactin concentration, measured by radioimmunoassay, with high levels in the summer. Monthly samples were bioassayed for total lactogenic activity by a rabbit mammary gland organ culture method. Postive responses were obtained only when prolactin levels were high (greater than 70 ng/ml), indicating that levels of placental lactogen in the circulation are much lower in the cow than in sheep, goats or women.

Placental lactogen (chorionic mammotrophin) in the field vole, Microtus agrestis, and the bank vole, Clethrionomys glareolus.

Placental lactogen has been detected in the field vole, Microtus agrestis, and the bank vole, Clethrionomys glareolus, using a co-culture technique. In field voles this activity could be detected from about day 8 of pregnancy to shortly before term, and stimulated both mouse and vole mammary gland to secrete in vitro. Partial immunological cross-reaction was detected in a radioimmunoassay system between rat prolactin and either extracts of vole pituitaries or media on which vole pituitaries had been cultured; vole placental lactogen showed no cross-reaction with rat prolactin. One site of origin for this hormone is probably the trophoblastic giant cells.

Fatty acid synthesis by explant cultures from the mammary glands of goats on days 60 and 120 of pregnancy.

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.

Hormone concentrations, mammary development and milk yield in goats given long-term bromocriptine treatment in pregnancy.

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 microgram/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 alpha-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.

Stimulation of DNA synthesis in cultures of ovine mammary epithelial cells by insulin and insulin-like growth factors.

Ovine mammary epithelial cell clumps (30-90 microns) 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 micrograms/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.

A panel of monoclonal antibodies to ovine placental lactogen.

A panel of 11 rat monoclonal antibodies (mAbs) has been raised to ovine placental lactogen (PL). By competitive enzyme-linked immunoabsorbent assay (ELISA), confirmed by two-site ELISA, the antibodies were shown to recognize six antigenic determinants on the ovine PL molecule, two of which overlap. One antigenic determinant (designated 1) was shared by other members of the prolactin/growth hormone (GH)/PL family in ruminants, humans and rodents. The binding of (125)I-labelled ovine PL to crude receptor preparations from sheep liver (somatotrophic) or rabbit mammary gland (lactogenic) was inhibited by mAbs recognizing antigenic determinants 2-6. Both types of receptor preparation were affected similarly. In the local in vivo pigeon crop sac assay, mAbs directed against determinants 3 and 6 enhanced the biological activity of ovine PL.

A pigeon crop sac radioreceptor assay for prolactin.

Ovine prolactin, labelled with 125I by either lactoperoxidase or a mild chloramine T method, bound to receptors from the pigeon crop sac mucosa cells of prolactin-injected pigeons. Binding was demonstrated in a crude homogenate of mucosal cells removed from the crop by scraping and in a subcellular fraction in which 5'-nucleotidase activity was enhanced two- to threefold. The binding was specific, dependent on time, temperature and the concentration of receptors and had a dissociation constant of 7 X 10(-10) mol/l. The binding capacity of the crop tissue was 71 fmol/mg membrane protein. Nine purified preparations of prolactin from four species were assayed by local pigeon crop sac bioassay and by radioreceptor assay. The two methods were highly correlated (r = 0.934). The regression equation was radioreceptor assay = 1.22 bioassay--0.18 indicating a 1 : 1 correspondence between the two methods for prolactin purified from sheep, rat, horse and pig anterior pituitary glands.

Ontogeny and control of prolactin receptors in the mammary gland and liver of virgin, pregnant and lactating rats.

Prolactin receptors were identified and partially characterized in the mammary gland of the rat. The binding of 125I-labelled ovine prolactin to a subcellular particulate fraction of rat mammary gland decreased between days 30 and 100 of age. Over the same period, binding to the liver increased and there was a significant negative correlation between prolactin binding in the two tissues. Binding to the mammary gland was low during pregnancy, increased in early lactation and declined after the litters were weaned. Binding to the liver was lower during lactation than during pregnancy or the period after weaning suggesting that tissue-specific factors may operate in the control of this receptor. In virgin rats, prolactin binding by the mammary gland was increased by oestrogen. This effect was blocked by hypophysectomy and partially restored by replacement therapy with prolactin. Hypothyroidism and treatment with progesterone also reduced the response to oestrogen. The maintenance of prolactin binding by the mammary gland of lactating rats depends on the presence of the ovaries and pituitary, thyroid and adrenal glands. Examination of the ratio epithelium: stroma suggests that prolactin acts by increasing the number of epithelial cells in the mammary gland and that thyroid, adrenal and ovarian hormones modulate the number of receptors per cell.

Blood prolactin concentrations affect prolactin transfer into goat milk: implications for maintenance of lactation.

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 significantly 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.

Transforming growth factor-alpha: receptor binding and action on DNA synthesis in the sheep mammary gland.

Microsome factions prepared from the mammary glands of non-pregnant, pregnant and lactating sheep have been used to study binding of 125I-labelled transforming growth factor-alpha (TGF-alpha). 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-alpha 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 (Kd) was similar in all physiological states (2.43 +/- 0.27 mol/l x 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-alpha (maximum response at 10 micrograms/l; 1.8 nmol/l) but not by EGF. Cells derived from 15- to 20-week pregnant sheep responded significantly to TGF-alpha 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-alpha and IGF-I produced an additive effect on DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of prolactin levels in the ferret.

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.

Milk-fat synthesis by lobules prepared from rabbit mammary gland: response to insulin, corticosterone, prolactin and progesterone.

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 mumol/l) and corticosterone (0.58 mumol/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 microns 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.

Receptor binding of insulin-like growth factor-I to mammary microsomes from non-pregnant, pregnant and lactating sheep.

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 of 125I-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 alpha-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 (Kd) 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 (Kd 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.

DNA synthesis by ovine mammary alveolar epithelial cells: effects of heparin, epidermal growth factor-related peptides and interaction with stage of pregnancy.

Amphiregulin is a heparin-binding member of the epidermal growth factor (EGF) family, which we have recently shown to be expressed in sheep mammary gland. Uniquely among known EGF-like growth factors, its mitogenic activity is inhibited by soluble heparin, but heparin-like molecules on the cell surface and/or in extracellular matrix appear to be necessary for amphiregulin to exert its biological effect. In primary cultures of sheep mammary alveolar epithelial cells, heparin (1-20 mg/l) inhibited DNA synthesis in a dose-dependent manner. The extent of the inhibition was influenced by physiological state, being greater (P < 0.05) in mammary cell cultures derived from 5- to 10-week pregnant sheep (63.1 +/- 8.2%, mean +/- S.E.M., n = 8) than in cultures derived from sheep which were non-pregnant (35.8 +/- 8.3% inhibition, n = 6) or late, 20-week, pregnant (39.8 +/- 5.6%, n = 6). Both EGF and transforming growth factor-alpha (TGF-alpha) significantly (P < 0.001) increased DNA synthesis in the presence of heparin. The effect of TGF-alpha was dose-related, wholly reversing the inhibitory effect of heparin in cell cultures from non-pregnant and 20-week pregnant sheep. DNA synthesis was stimulated by amphiregulin and TGF-alpha increased the maximum response. The heparin antagonist, hexadimethrine, inhibited DNA synthesis, but, in the presence of amphiregulin, approximately equivalent concentrations of heparin overcame this inhibitory effect. In the presence of heparin, TGF-alpha showed synergistic interactions with insulin or IGF-I. The results indicate interactive effects of EGF and IGF growth factor families in sheep mammary growth.

Effects of continuous intravenous infusion of an ovine placental extract enriched in placental lactogen on plasma hormones, metabolites and metabolite biokinetics in non-pregnant sheep.

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 micrograms/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 nonesterified 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.

Placental lactogen in the goat in relation to stage of gestation, number of fetuses, metabolites, progesterone and time of day.

Placental lactogen has been measured in goats throughout pregnancy by radioreceptor assay of prolactin-like activity. Lactogenic activity, which is not prolactin, increased from less than 5 nmol/l in week 8 to 27 nmol/l by week 16. There was no further change until term. Plateau concentrations (week 16 to term) were highest in animals carrying triplets, 49.5 nmol/l. There were marked functuations in placental lactogen over a 24 h period. These short-term fluctuations were not related to changes in glucose, non-esterified fatty acids or, in two animals, progesterone. However, there was a negative correlation between mean concentrations of placental lactogen and glucose in plasma of 20 goats sampled over a 24 h period between weeks 15 and 20 of gestation. There was no difference in placental lactogen concentration from week 16 to term between goats in their first and second pregnancies although the normal period of increase in placental lactogen was delayed by some 3 weeks in goats in their second pregnancy. In hemimastectomized goats, hypophysectomy on day 60 did not affect placental lactogen but daily treatment with bromocriptine (5 mg/day) from day 60 to day 120 blocked the normal rise in concentration.

Expression of amphiregulin in the sheep mammary gland.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify, from sheep mammary gland total RNA, a 280 bp sequence of amphiregulin cDNA. Cloned and sequenced, it corresponded to the 78 amino acids of the major secreted form of amphiregulin, showing 81, 70 and 69% identity with human, rat and mouse amphiregulin, respectively. Expression of amphiregulin was detected by RT-PCR in the mammary gland at several developmental stages (fetal, lamb, early and late pregnant and lactating ewes) and in isolated myoepithelial cells. By Western blotting with an antiserum to human amphiregulin, two molecular weight forms, 27 and 51 kDa were detected in sheep mammary gland microsomal preparations, in a mammary gland extract after heparin affinity chromatography and in a medium conditioned by mammary epithelial cells. By immunocytochemistry, amphiregulin was detected in the cytoplasm and nuclei of luminal epithelial cells, myoepithelial cells and in intralobular stroma. An autocrine/paracrine role in sheep mammary growth is indicated.

Activity of progesterone and anti-progestins in a rat mammary primary cell culture system.

A primary culture system of virgin rat mammary epithelial cells, grown in a serum-free medium, was developed as a means of assaying the efficacy of compounds with known anti-progestational properties. Cells were grown in 24-well plates on hydrated collagen gels and could be cultured for at least seven days. Experiments were routinely stopped three days after overnight attachment of cells using fibronectin (4 micrograms/ml). DNA synthesis, measured by thymidine incorporation, was significantly increased by the addition of ovine prolactin (43 nM; P < 0.01) or progesterone (0.15 microM; P < 0.05) or both (P < 0.01) to the basal medium. When added to medium containing progesterone plus prolactin (complete medium), RU486 (mifepristone) and ZK98734 (lilopristone) significantly depressed DNA synthesis in a dose-dependent manner using doses ranging from 0.015 microM to 15 microM. Maximum inhibition was achieved at 15 microM for both compounds. DNA synthesis was 24.5 +/- 2.6% (mean +/- SEM, n = 4) and 32.0 +/- 2.2% (n = 3) of that in complete medium for RU486 and ZK98734, respectively (both P < 0.001). There was no inhibitory effect of either compound in basal medium or basal medium plus prolactin, indicating the absence of toxicity and that the inhibitory effect is specific for a progesterone-mediated process.