Tissue-specific variation in expression of prolactin receptor gene subtypes in hypergravity-exposed rats.

Prolactin (PRL) effects are mediated by membrane receptors (PRLR) of which the long (PRLR-L) and short form (PRLR-S) predominate. Our objective was to compare the distribution pattern of PRLR-L and PRLR-S transcripts and their ratio in adipose (AD), liver (LV), mammary (MG) and pituitary (PG) tissues of stationary (SC, n = 8) and hypergravity (HG, n = 8) exposed periparturient rats. Pregnant rats were exposed to 2 g force from day 11 of gestation (G11) through post partum day 1 (P1). PRLR-L mRNA expression compared to PRLR-S was greater (P < 0.001) in AD, MG and PG but was lower (P < 0.001) in LV in both HG and SC animals at P1. The ratio of PRLR-L/PRLR-S mRNA in the AD, LV, MG and PG was not different between HG and SC rats. In summary, these data reveal that the hypergravity-induced downregulation of PRLR is not directly triggered by deranged distribution of PRLR isoforms.

Exogenous TGF-beta1 promotes stromal development in the heifer mammary gland.

The objectives of this study were to determine the local effects of transforming growth factor-beta1 (TGF-beta1) on mammary epithelial and stromal cell proliferation and expression of the TGF-beta1 responsive genes c-myc and fibronectin. A single slow-release plastic pellet containing 5 microg of TGF-beta1 and 20 mg of BSA was implanted in the parenchyma of the right rear quarter of the mammary gland of 9-mo-old prepubertal heifers. A control pellet containing 20 mg of BSA was implanted in the left rear quarter of each heifer. All heifers were treated with bromodeoxyuridine (BrdU) at 4, 12.5, and 22 h after the pellets were implanted to label proliferating cells. Two hours after the last BrdU injection, the animals were euthanatized, and their mammary glands were recovered. Proliferation of mammary stromal cells was significantly higher in TGF-beta1-treated quarters than in BSA-treated, control quarters (3.5 vs. 1.8% BrdU-positive cells). This result coincided with a lack of significant effect of TGF-beta1 on proliferation of mammary epithelial cells and apoptosis. By quantitative reverse transcriptase-polymerase chain reaction, we found that c-myc gene expression was unchanged after TGF-beta1 treatment, but fibronectin gene expression was increased 3-fold in TGF-beta1-treated quarters compared with BSA-treated, control quarters. Thus, we concluded that TGF-beta1 selectively acts on the stromal compartment of the bovine mammary gland by increasing cell proliferation and gene expression of the extracellular matrix protein fibronectin.

Association between gravitational force and tissue metabolism in periparturient rats.

Recently, interest in mammalian reproduction and offspring survival in altered gravity has been growing. Because successful lactation is critical for mammalian neonate survival, we have been studying the effect of gravity metabolism. We have shown an exponential relationship between glucose metabolic rate in mammary tissue of periparturient rats and an increase in gravity load. In this study we showed that changes in mammary metabolic rate due to gravity force were accompanied by a decrease in glucose metabolism in adipose tissue and by a reduced size of adipocytes. We assume that these changes are likely due to changes in prolactin or leptin levels related to altered gravity load.

Effects of hypergravity on mammary metabolic function: gravity acts as a continuum.

Mammary metabolic activity in pregnant rats is significantly increased in response to spaceflight. To determine whether changes in mammary metabolism are related to gravity load, we exposed pregnant rats to hypergravity and measured mammary metabolic activity. From days 11-20 of gestation (G), animals were centrifuged (20 rpm; 1.5, 1.75, or 2.0 x gravity) or were maintained at 1 G. On G20, five rats from each group were removed from the centrifuge and euthanized. The remaining dams (n = 5/treatment) were housed at 1 G until parturition. After 2 h of nursing by the pups, the postpartum dams were euthanized (G22). Glucose oxidation to CO2 and incorporation into lipids was measured. Mammary glands from dams euthanized on G20 revealed a strong negative correlation between metabolic rate and increased G load. Approximately 98% of the variation in glucose oxidation and 94% of the variation in glucose incorporation into lipids can be accounted for by differences in G load. Differences in metabolic activity disappeared in the postpartum dams. When we combined previous data from the microgravity with hypergravity environments and plotted the ratio of mammary metabolic rate vs. G load, there was a significant exponential relationship (r2 = 0.99). These data demonstrate a remarkable continuum of response across the microgravity and hypergravity environments and support the concept that gravitational load influences mammary tissue metabolism.

The effects of spaceflight on mammary metabolism in pregnant rats.

The effects of spaceflight on mammary metabolism of 10 pregnant rats was measured on Day 20 of pregnancy and after parturition. Rats were flown on the space shuttle from Day 11 through Day 20 of pregnancy. After their return to earth, glucose oxidation to carbon dioxide increased 43% (P < 0.05), and incorporation into fatty acids increased 300% (P < 0.005) compared to controls. It is unclear whether the enhanced glucose use is due to spaceflight or a response to landing. Casein mRNA and gross histology were not altered at Day 20 of pregnancy. Six rats gave birth (on Day 22 to 23 of pregnancy) and mammary metabolic activity was measured immediately postpartum. The earlier effects of spaceflight were no longer apparent. There was also no difference in expression of beta-casein mRNA. It is clear from these studies that spaceflight does not impair the normal development of the mammary gland, its ability to use glucose, nor the ability to express mRNA for a major milk protein.

Progesterone stimulates DNA synthesis and lobulo-alveolar development in mammary glands in ovariectomized mice.

The objective of this study was to determine whether sustained progesterone (P) use in the absence of estrogen could influence mammary development in mice. Three-week-old intact or ovariectomized mice were primed with subcutaneous (s.c.) cholesterol (C), estrogen (E), P, or estrogen and progesterone (E/P) together. Nine days after priming, mammary glands were removed and incubated as a whole organ in media supplemented with various combinations of lactogenic hormones. After 5 days in whole organ culture, glands were removed and end buds, alveolar buds and lobulo-alveoli were quantified. Glands from mice primed with C or E developed significantly less lobulo-alveoli than glands from mice primed with P or E/P. While the development was greater in animals treated with E/P compared to those treated with P, it was clear that P in the absence of E could still induce lobulo-alveolar development. We have shown in this paper that P, in the absence of E, can stimulate cell proliferation during priming. Subsequently, the P primed glands can differentiate in response to lactogenic hormones.

Effects of steroid hormone treatment on mammary development in prepubertal heifers.

The objective of this study was to determine the effects of steroid hormone implantation in heifer calves on the ability of mammary tissue to develop subsequently in organ culture. Twenty-four calves were paired by date of birth and assigned to groups (eight calves/group). At 4, 7, or 10 mo of age, calves were implanted subcutaneously (s.c.) with pellets containing cholesterol or cholesterol, 17 beta-estradiol, and progesterone for 9 or 18 d. The calves were euthanized and uteri and mammary glands were removed and weighed. Slices of mammary parenchymal tissue were incubated for 5 d at 37 degrees C in a 50% O2, 5% CO2 humidified atmosphere in Waymouth's 752/liter medium supplemented with insulin (5.0 micrograms/ml) or lactogenic hormone complex insulin (5.0 micrograms/ml), aldosterone (0.1 microgram/ml), hydrocortisone (0.1 microgram/ml), and prolactin (1.0 microgram/ml) in the presence or absence of epidermal growth factor (EGF) (0.06 microgram/ml) to promote lobulo-alveolar development. Tissue sections were stained and mounted on slides for morphologic and histologic analysis or prepared to evaluate expression of beta-casein mRNA. There were no morphologic differences in slices from calf mammary tissues despite age, steroid hormone priming, or hormones used in tissue culture. The 4-mo-old calves required steroid priming followed by incubation of the tissue slices with the lactogenic complex with or without epidermal growth factor to induce cytological changes associated with lactogenesis but did not express beta-casein mRNA. At 7 mo of age, steroid hormone priming was not necessary for induction of alveolar formation and secretion. Incubation of the tissue slices from 7-mo-old calves with the lactogenic complex was sufficient to induce alveolar formation and secretion. However, beta-casein mRNA was not expressed. At 10 mo of age, exposure of tissue from calves to the lactogenic hormones caused histologic changes reminiscent of the ability to secrete milk regardless of hormone priming. However, estrogen and progesterone priming was necessary before incubation of the tissue slices with the lactogenic hormones to induce beta-casein mRNA expression. When epidermal growth factor was added to the lactogenic hormone complex, beta-casein mRNA expression decreased. These data support the concept that there is a sequential development of responsiveness of the mammary gland to various hormones. By 10 mo of age, prepubertal heifers reach a stage of maturity where steroid hormone priming followed by incubation of tissue slices with the lactogenic hormones is sufficient to induce both structural and functional differentiation.

Negative effects of a high level of nutrient intake on mammary gland development of prepubertal goats.

Ten French Alpine kids were used to determine whether mammary development was altered by level of nutrient intake during the prepubertal period. At 6 wk of age, kids were paired on the basis of BW and assigned to an ad libitum or restricted diet of milk from Jersey cows. Kids on the restricted diet were fed 70% of their pair mate's milk intake for 4 wk and then 50% for 9 wk. Low quality grass hay was available for all kids. The ADG was greater for kids fed for ad libitum intake. Kids fed for ad libitum intake had larger mammary glands and increased adipose deposition. Measurement of hydroxyproline indicated that connective tissue increased with gland size. Kids on the restricted diet had more DNA and protein per gram of mammary gland, indicating greater secretory development. Goats can be used as a model to study the effect of level of nutrient intake on hormones and growth factors that regulate mammary gland development.

Characterization of binding of transforming growth factor-beta 1 to bovine mammary membranes.

This study developed a procedure to measure binding of transforming growth factor-beta 1 to bovine mammary membranes. Mammary membranes were incubated with 3 M MgCl2 to remove endogenously bound transforming growth factor-beta 1. Binding was optimized by incubation of 200 micrograms of membrane protein with 125I-labeled transforming growth factor-beta 1 in 25 mM Tris, 10 mM CaCl2, and 1.0% BSA in a total volume of .5 ml in the presence or absence of unlabeled transforming growth factor-beta 1. The reaction equilibrated in 2 h at 4 degrees C. Specific binding was linear from 142 to 1140 micrograms of membrane protein. The reaction was specific for the beta transforming growth factors; transforming growth factor-beta 1, transforming growth factor-beta 2, and transforming growth factor-beta 3 could complete effectively with 125I-labeled transforming growth factor-beta 1 for the receptor. The growth factors, epidermal growth factor, IGF-I, or transforming growth factor-alpha did not compete effectively with 125I-labeled transforming growth factor-beta 1 for binding to bovine mammary membranes. Scatchard analysis showed that the number of receptors averaged 251 pmol/mg of membrane protein and the affinity was 8.7 x 10(-11) M. Binding to mammary membranes was higher during the prepubertal and pubertal periods than during lactation. Binding to mammary membranes during early lactation averaged 24% of the binding observed during other physiological states.(ABSTRACT TRUNCATED AT 250 WORDS)

The determination of the post-test likelihood for coronary disease using Bayes Theorem.

This report describes a method of using Bayes Theorem for determining the post-test likelihood for coronary disease from the exercise test. The pre-test likelihood is determined from the age, sex, and type of chest pain. Discriminant analysis of the amount of S-T depression, maximum pressure rate product and sex of the patient is then used to determine the sensitivity and specificity of the patient's exercise test for coronary artery disease. The post-test likelihood for coronary artery disease can then be calculated using Bayes Theorem. The discriminant function was estimated from a training set of 174 patients. This was then applied to 113 new patients who had both exercise tests and coronary arteriograms. In this new set, 47 patients had a post-test likelihood of 90% or greater for coronary disease. Only one of these patients had normal coronary arteriograms, a predictive accuracy of 98%. Of the 25 patients with a post-test likelihood of 10% or less for coronary disease, four had multivessel disease and four had single vessel disease. The predictive accuracy for the absence of coronary disease was 68%. The predictive accuracy for the exclusion of multivessel disease was 84%. Eight of 10 patients with left main disease had a post-test likelihood for coronary disease of greater than 90%.