Intravenous administration of cholecystokinin (CCK) stimulates prolactin and growth hormone release in the pig.

Blood samples were taken from prepubertal pigs (n = 7) surgically prepared with venous catheters before, and at intervals during the 30 min period after, administration of physiological saline (vehicle) or sulphated cholecystokinin octapeptide (CCK) (0.3, 0.7 and 1.3 mu g/kg). Plasma concentrations of prolactin and growth hormone were measured by radioimmunoassay. Statistically significant dose-related increases in prolactin occurred mainly in the first 10-min period following CCK administration. The peptide also stimulated growth hormone secretion although in a less consistent manner, with the increases tending to occur over a longer time period. Together with earlier reports showing that CCK induces cortisol release in pigs, these results suggest that the peptide's effect on anterior pituitary function could be indicative of a stress response.

Milk whey proteins in plasma of sows: variation with physiological state.

The whey proteins alpha-lactalbumin and beta-lactoglobulin have been investigated as potential markers of mammary development in sows by measuring their concentrations in plasma. The whey proteins were isolated from porcine milk by gel filtration, ion-exchange and hydrophobic interaction chromatography, characterized by several criteria and used to raise antibodies. Specific radioimmunoassays were set up for porcine alpha-lactalbumin and beta-lactoglobulin and validated for use in porcine blood and milk. Plasma levels of the whey proteins were measured in sows that were pregnant, suckling litters post partum, weaned abruptly at birth or were pregnant but mastectomized. Both whey proteins showed similar patterns in plasma post partum, falling from a maximum 1 d after parturition to values < 0.02% those in milk by day 4-5 post partum in suckling sows and showing a transient peak associated with early involution before declining to very low concentrations in non-suckling sows. alpha-Lactalbumin was first detected in the last week prepartum, rising markedly in the 3 d before parturition, correlated with rising prolactin (r = 0.986) and falling progesterone (r = -0.998). beta-Lactoglobulin rose much earlier from 5 weeks prepartum, at the time when lobulo-alveolar mammary development is occurring, and correlated (r = 0.929) with oestradiol-17 beta. In mastectomized sows, concentrations of whey proteins in plasma were reduced by 90% or more when compared with intact animals, though following a similar pattern. This study shows that whey protein concentrations in plasma vary with physiological state and reflect aspects of the development of the mammary gland. The very different profiles for alpha-lactalbumin and beta-lactoglobulin prepartum indicate that they are differently controlled.

Hormonal induction of alpha-lactalbumin and beta-lactoglobulin in cultured mammary explants from pregnant pigs.

Mammary tissue from pigs on days 60, 80, 90, 100 and 100+ (days 106-111) of pregnancy has been cultured in vitro as explants. The total accumulation in tissue and culture medium of the whey proteins alpha-lactalbumin and beta-lactoglobulin has been measured using specific radioimmunoassays. The control, uncultured tissue showed progressive morphological development from sparse, non-secretory epithelial tissue on day 60 to full lobulo-alveolar development with some accumulated secretion from day 100. In uncultured explants beta-lactoglobulin could be detected consistently from day 90 (13 +/- 12 ng/micrograms DNA, n = 4) and alpha-lactalbumin from day 100 (1.3 +/- 0.5 ng/micrograms DNA, n = 11). At all stages of pregnancy, both whey proteins increased markedly during the period of culture (up to 7 d). Stimulation of alpha-lactalbumin appeared to be primarily under prolactin control. Prolactin increased alpha-lactalbumin accumulation to a similar extent alone, or in the presence of insulin and/or corticosterone. The response to prolactin was dose-dependent over the range 0.4-20 nM (10-500 ng/ml). Porcine prolactin was more potent than ovine prolactin. There was no effect of porcine growth hormone and no synergism detected between prolactin and tri-iodothyronine. By contrast, no specific hormonal requirements were established for accumulation of beta-lactoglobulin, which appeared to increase in vitro if tissue remained viable in various combinations of insulin, corticosterone and prolactin. It was not stimulated by growth hormone. There was some indication of a prolactin-sensitive component in longer term cultures after day 4.

The effect of insulin and relaxin upon mitosis (in vitro) in mammary tissue from pregnant and lactating pigs.

The rate of cellular proliferation in the mammary glands of pigs during late gestation and lactation was assessed by measuring the incorporation of 3H-thymidine (T1) into the DNA of mammary gland explants in vitro. The T1 showed a linear response over the first 9 hr in vitro, and was not affected by the addition of 500 ng insulin/ml medium. From day 100 to parturition the T1 rose, reached a peak at 2 d after parturition and declined during lactation to the lowest levels seen at day 21 of lactation. The inclusion of 0-1000 ng relaxin/ml medium on T1 at 24-72 hr in vitro had no effect in stimulating T1 in mammary tissue explants taken from either pregnant or lactating pigs.

Progesterone receptor in the mammary tissue of pregnant and lactating gilts and the effect of tamoxifen treatment during late gestation.

An isotope exchange assay using [3H]progesterone was used to examine progesterone receptor moieties in cytosolic extracts obtained from mammary tissue of gilts over the course of pregnancy and lactation, and during treatment of pregnant gilts with tamoxifen. Scatchard analysis was used to determine the concentrations and dissociation constants of progesterone receptors. The concentration of progesterone receptor was high at the onset of pregnancy (1394 fmol/mg DNA), fell to a nadir at 45 days (36 fmol/mg DNA), increased to a second maximum at 75 days (1232 fmol/mg DNA) and declined thereafter till parturition: the dissociation constant (Kd) of progesterone for its receptor remained stable during pregnancy with a mean Kd of 0.78 nmol/l. Progesterone receptors were not identifiable at day 21 of lactation. Treatment of pregnant gilts with tamoxifen (100 mg or 1.0 g/gilt per day orally identical to 0.70 or 7.0 mg/kg per day) did not affect the development of mammary structures or the ability to lactate at parturition; however, mammary progesterone receptor content in tamoxifen-treated animals tended to be lower than the controls at day 90 of pregnancy (15.7 +/- 1.56 vs 27.0 +/- 3.75 fmol/mg protein respectively). The results show that a temporal relationship exists between oestrogen concentrations in the circulation of pregnant gilts with progesterone receptor content in mammary tissue.

Effect of oestradiol benzoate and tamoxifen on the growth of and induction of progesterone receptors in the uterus and mammary gland of immature pigs.

Intramuscular injection of oestradiol benzoate (0.1, 1 or 10 micrograms/kg per day) and tamoxifen (0.1 or 1 mg/kg per day) to 6-week-old immature pigs for 7 days induced a dose-dependent increase in the wet weight of the uterus and in the total content of uterine DNA, RNA and protein. Both compounds also stimulated a dose-dependent increase in the concentration of progesterone receptors in uterine cytosolic extracts (in terms of either fmol/mg DNA or fmol/g uterus). The concurrent administration of tamoxifen with oestradiol benzoate provoked significant (P less than 0.05) increases in total uterine protein and in the concentration of progesterone receptors (P less than 0.01) compared with treatment with oestradiol benzoate alone. Hence tamoxifen is an oestrogen agonist in the uterus of immature pigs. The effects of oestradiol benzoate and tamoxifen on mammary growth in immature pigs were assessed by image analysis of mammary sections across the gland (in a ventro-dorsal direction through the teat). Oestradiol benzoate at 10 micrograms/kg per day stimulated a fourfold increase in mammary duct area (P less than 0.01), and tamoxifen, at doses of 0.1 or 1 mg/kg per day, stimulated a threefold increase (P less than 0.05). Tamoxifen partially inhibited (P less than 0.05) the effect of oestradiol benzoate. The concentration of progesterone receptors was found to be very heterogeneous in cytosol extracts of mammary tissue of immature pigs and independent of treatment with oestradiol benzoate and/or tamoxifen.

Intracerebral administration of insulin-like growth factor I decreases circulating growth hormone levels in the fetal pig.

Radioimmunoassayable IGF-I levels were measured in the cerebrospinal fluid and plasma of pig fetuses at 94 days gestational age. Mean plasma IGF-I levels were 128.5 +/- 5.8 micrograms/l while the concentration in the cerebrospinal fluid was 25.8 +/- 4.4 micrograms/l. The effect of intracerebroventricular administration of IGF-I on circulating GH levels was also studied in pig fetuses in utero. Eighteen pig fetuses were fitted with indwelling carotid artery and jugular vein catheters. Nine fetuses were given 1500 ng of pure IGF-I in 100 microliters 0.9% saline by direct injection into a right lateral ventricle. Nine further fetuses (controls) were similarly given 100 microliters of saline without IGF-I. GH levels in the control fetuses were approximately 200 micrograms/l and showed marked fluctuations with episodic intervals of about 40 min. By contrast, in the IGF-I-treated fetuses, GH levels were dramatically lowered by 20 min after IGF administration and remained low throughout the 4-h study. The episodic variations in GH were abolished and levels remained fairly constant at ca. 40 micrograms/l. From these results we surmise that the low levels of IGF-I in the fetus may contribute to their high GH levels. At this stage it is not possible to identify whether the IGF-I inhibition is a direct effect on the pituitary or is mediated by increased somatostatin, decreased GHRH or both.

Differences in growth hormone and prolactin secretion associated with environmental temperature and energy intake.

The combined effects of environmental temperature and level of energy intake on plasma concentrations of growth hormone (GH) and prolactin (PRL) have been investigated in 14 week old pigs acclimated to 35 or 10 degrees C on a high (H) or low (L) energy intake (H = 2L). Measurements were made at 15 min intervals between 08.00 and 18.00 hours, after feeding at 17.00 hours on the previous day. Mean values of GH were greater in pigs on the L than H intake and there was a tendency for values to be higher at 35 than 10 degrees C. However, there was wide individual variation within each treatment group and the differences were not statistically significant. Mean PRL concentrations were greater at 35 than 10 degrees C (P less than 0.05). It is concluded that circulating levels of plasma GH do not have a major role in maintaining the differences in growth and morphology of young pigs kept in widely different environmental conditions. However, these differences could be related at least in part to the GH-like properties of PRL.

Role of the ovaries in inducing mammogenesis in pregnant pigs.

Ovariectomy, but not removal of the corpora lutea, of gilts at mid-pregnancy delayed the onset of lobulo-alveolar development in the mammary glands. Lactogenesis at the time of parturition was delayed by both ovariectomy and removal of the corpora lutea.

Control of binucleate cell migration in the placenta of sheep and goats.

In the ruminant placenta 15-20% of the trophectodermal epithelium consists of granulated binucleate cells (BNC). In the sheep the granules contain ovine placental lactogen (oPL). These cells migrate from the trophectoderm to form fetomaternal hybrid tissue from implantation to term. The number of BNC, their percentage migration and the potential secretory activity of the syncytium they form were estimated by semiquantitative transmission electron-microscopical techniques after several surgical techniques and hormone or drug infusions. BNC numbers decrease normally just before parturition, and this fall could be eliminated by fetal hypophysectomy or induced early by administration of tetracosactrin to intact or hypophysectomized fetuses. If only one twin was treated with tetracosactrin the placenta of the untreated twin did not show the fall in BNC numbers found in the other unless it died in utero some time before sampling. This indicates fetal control of BNC number and migration. However, fetal catheterization, hypophysectomy, stalk section, adrenalectomy, infusion of mouse epidermal growth factor or bromocriptine had little or no effect on binucleate cell numbers or migration percentages. Maternal carunclectomy, ovariectomy, or epostane or bromocriptine administration also had no consistent significant effect. Previous reports of degeneration of BNC structure plus a decrease in their number (with bromocriptine) or an increase in migration frequency (after adrenalectomy or stalk section) have not been confirmed by this study. The BNC migration delivers the oPL-containing BNC granules close to the maternal circulation but the variation in migration seems only loosely correlated with the reported maternal oPL concentrations. The results indicate that BNC migration is independent of the hormonal milieu, but that BNC production is greatly modified by the hormonal changes just before parturition, with cortisol production by the fetus a possible primary cause.

Simultaneous determinations of uterine blood flow and plasma concentrations of placental lactogen in late-pregnant goats.

Blood flow in the middle uterine artery was measured with electromagnetic blood flow probes, and placental lactogen in jugular and uterine venous plasma was estimated as total lactogenic activity using a radioreceptor assay. There was no circadian variation in uterine arterial blood flow in late pregnancy (Days 105-124) and the pattern of blood flow varied between goats. Blood flow was quite stable for periods of up to 40 min although at other times a rapid fall (by up to 90%) was followed by a gradual recovery. These spontaneous changes lasting up to 30 min could not be consistently related to postural or behavioural changes. Acute decreases of about the same duration could also be induced by administration of adrenaline. In the short-term there was no association between uterine blood flow and total lactogenic activity in the peripheral circulation during spontaneous or adrenaline-induced depression of blood flow. More limited short-term observations on total lactogenic activity in the uterine vein also failed to show a relationship with blood flow in the uterine artery.

The luteotrophic complex in hysterectomized and pregnant goats.

The pituitary hormones responsible for the maintenance of progesterone secretion by the corpus luteum have been determined by replacement therapy in hysterectomized-hypophysectomized goats and in goats hypophysectomized on day 40 of pregnancy. Progesterone concentrations were 7.2 +/- 0.81 ng/ml plasma (mean +/- S.E. of mean) in intact goats during the luteal phase of the cycle and were 5.9 +/- 0.56 ng/ml plasma after hysterectomy. After hypophysectomy of hysterectomized goats, treatment with 25 or 150 i.u. prolactin/day failed to maintain the corpora lutea, and progesterone concentrations fell to below 1.0 ng/ml plasma. Treatment with 1.0 mg luteinizing hormone (LH)/day after hypophysectomy maintained progesterone concentrations at 2.5 +/- 0.48 ng/ml and treatment with 1.0 mg LH and 150 i.u. prolactin/day resulted in increased peripheral concentrations of 12.9 +/- 1.66 ng progesterone/ml plasma. In goats hypophysectomized on day 40 of pregnancy treatment with 1.0 mg LH/day or 1.0 mg LH and 150 i.u. prolactin/day resulted in maintenance of the pregnancy and maintenance of high peripheral concentrations of 15.0 +/- 1.77 ng progesterone/ml plasma, with no difference between the two treatments. The results show that LH is the pituitary hormone primarily responsible for the maintenance of progesterone secretion by the corpora lutea in the goat and that prolactin is synergistic with LH in stimulating this function. The difference in the pituitary luteotrophic complex between non-pregnant and pregnant animals may be explained if placental lactogen can substitute for prolactin in the luteotrophic complex of pregnant goats.

A design for flexible intestinal cannulas.

A design for a cannula made by fixing silicone rubber tubing to rigid internal and external flanges is described. These cannulas have been used to fit simple duodenal, simple ileal and abomasal fistulas in adult lactating cattle for periods up to 1 year and have caused minimal tissue reaction at the site of fistulation.

A note on the absence of acth-like activity secreted by the goat placenta in vivo and on the independence of fetal endocrine development of the maternal hypophysis.

In the goat, maternal hypophysectomy on Day 60 of gestation or treatment with 5 mg bromocriptine/day between Days 60 and 120 of gestation did not affect fetal body weights or the weights of the fetal adrenals, thyroid, pituitary gland or gonads when examined at 120 days gestation. The maternal adrenal cortex regressed after hypophysectomy of the pregnant goat.

Mammary growth during pregnancy in hypophysectomized or bromocriptine-treated goats.

Mammogenesis in primiparous hypophysectomized goats has been assessed between days 60 and 120 of gestation and compared with that found in untreated goats and goats treated with 5 mg bromocriptine/day. There were fivefold increases in the weight of lobulo-alveolar tissue in the hypophysectomized and bromocriptine-treated goats and a tenfold increase in the untreated goats. Histological examination of the mammary glands at 120 days showed normal structure, and determinations of lactose, lactose synthetase, cytosol enzymes, protein, DNA and RNA indicated qualitatively normal initiation of milk synthetic capabilities in both the hypophysectomized and bromocriptine-treated goats. Bromocriptine treatment lowered the plasma concentration of placental lactogen as well as that of prolactin. The results indicate that placental lactogen has important mammogenic effects during pregnancy.

The maintenance of pregnancy in hypophysectomized goats.

Pregnancy in goats is maintained after hypophysectomy at about Day 90 of gestation by injection of 20 mg progesterone or 1 mg LH/day, but not by 5 mg prolactin/day.

The effect of anterior cervical ganglionectomy on the seasonal variation in prolactin concentration in goats.

Bilateral anterior cervical sympathetic ganglionectomy in castrated male goats had no effect upon the subsequent seasonal variation in the concentration of prolactin (Prl) in plasma when surgery was performed in summer. After ganglionectomy in winter, however, the subsequent rise in Prl concentration associated with season was accelerated.

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.