Increasing evidence suggests that GH is important in normal mammary gland development. To investigate this further, we studied the distribution and levels of growth hormone receptor (GHR) and GH-binding protein (GHBP) in the mouse mammary gland. At three weeks of age, the epithelial component of the right fourth inguinal mammary gland of female mice was removed. These animals were then either maintained as virgins until they were killed or they were mated. One group of the mated mice was killed on day 18 of pregnancy and the remaining mated animals were allowed to carry their pups until term and were killed on day 6 of lactation. At the time of death, both the intact left and the de-epithelialized right mammary glands were collected from all three groups. Some of the intact glands served as a source of epithelial cells, free of stroma. The mRNA levels for GHR and GHBP were measured in intact glands, epithelia-cleared fat pads, and isolated mammary epithelial cells. GHR and GHBP mRNAs were expressed in both the mammary epithelium and stroma. However, the levels of both GHR and GHBP mRNAs were significantly higher in the stroma as compared with the epithelium component. This increase for both mRNAs was from 3- to 12-fold at each physiological state examined. In the intact gland, both GHR and GHBP transcripts were highest in virgins, declined during late pregnancy, and the lowest levels were found in the lactating gland. GHBP and GHR protein concentrations were also assessed in intact glands and epithelia-free fat pads. Similar to the mRNAs, GHR and GHBP protein levels (means+/-s.e.m.) in intact glands were highest in virgin mice (0.891+/-0.15 pmoles/mg protein and 0.136+/-0.26 pmoles/mg protein respectively), declined during late pregnancy (0. 354+/-0.111 pmoles/mg protein and 0.178+/-0.039 pmoles/mg protein respectively), and were lowest during lactation (0.096+0.037 pmoles/mg protein and 0.017+0.006 pmoles/mg protein respectively). Immunocytochemistry utilizing specific antisera against mouse (m) GHR and mGHBP revealed that the two proteins are localized to both the stroma and parenchyma of mouse mammary glands, with similar patterns of immunostaining throughout the different physiological stages analyzed. GHR immunolocalized to the plasma membrane and cytosol of mammary epithelial cells and adipocytes, whereas the GHBP immunostaining was nuclear and cytosolic. In conclusion, we report here that GHR and GHBP mRNAs and proteins are expressed in both the epithelium and the stroma of mammary glands of virgin, pregnant, and lactating mice. In intact glands, GHR and GHBP proteins, as well as their transcripts are higher in abundance in virgin relative to lactating mice. At all physiological stages, GHR and GHBP mRNA levels are higher in the stroma compared with the parenchyma. These findings indicate that the actions of GH in the mammary gland are both direct through its binding to the epithelia, and indirect by binding to the stroma and stimulation of IGF-I production which, in turn, affects mammary epithelial development.

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

ABSTRACT

The ability of mouse placental lactogen (mPL), mouse prolactin (mPRL), mouse GH (mGH) and ovine prolactin (oPRL) to stimulate synthesis of α-lactalbumin was tested in a primary culture of mouse mammary gland epithelial cells. Mammary tissue was obtained from 10-day pregnant Swiss Webster mice, enzymatically dissociated and the cells were cultured on floating collagen gels for 5 days. The basic culture medium consisted of Nutrient Mixture F12/Dulbecco's Modified Eagle's Medium (1:1, v/v), containing 10 mg insulin/1, 5 mg cortisol/l, 10 μg epidermal growth factor/l, 5 g bovine serum albumin/l and 50 mg gentamycin/l. Mouse PL, mPRL, mGH and oPRL were added to the basic medium in concentrations from l μg/l to l mg/l. Accumulation of α-lactalbumin in the culture medium was measured. For that purpose, mouse α-lactalbumin was purified from mammary tissue obtained from lactating Swiss Webster mice and a radioimmunoassay was developed. Mouse PL, mPRL and oPRL stimulated a dose-dependent increase in α-lactalbumin secretion. Mouse GH also caused a significant, but dose-independent, increase in α-lactalbumin secretion. Mouse PL showed the greatest activity in stimulating α-lactalbumin secretion. It was concluded that mPL is an important lactogenic hormone in the latter half of pregnancy in the mouse, when circulating mPRL concentrations are low.

J. Endocr. (1986) 109, 263–274

The importance of prolactin (PRL) in regulating growth and differentiation of the mammary gland is well known. However, it is not well established whether PRL acts solely on the mammary epithelia or if it can also directly affect the mammary stroma. To determine where PRL could exert its effects within the mammary gland, we investigated the levels of expression and the localization of the PRL receptor (PRLR) in the epithelia and stroma of the rat mammary gland at different physiological stages. For these studies, we isolated parenchymal-free 'cleared' fat pads and intact mammary glands from virgin, 18-day-pregnant and 6-day-lactating rats. In addition, intact mammary tissues were enzymatically digested to obtain epithelial cells, free of stroma. The mammary tissues, intact gland, stroma and isolated epithelia, were then used for immunocytochemistry, protein extraction and isolation of total RNA. PRLR protein was detected in tissues using specific polyclonal antisera (PRLR-l) by immunocytochemistry and Western blot analysis. Messenger RNA for PRLR was measured by ribonuclease protection assay. Immunocytochemistry and Western blots with the PRLR-1 antisera detected PRLR in wild-type rat and mouse tissues, whereas the receptor protein was absent in tissues from PRLR gene-deficient mice. PRLR was found to be present both in the epithelia and stroma of mammary glands from virgin, pregnant and lactating rats, as determined by immunocytochemistry and Western blotting. Western blots revealed the predominance of three bands migrating at 88, 90 and 92 kDa in each of the rat mammary samples. These represent the long form of the PRLR. During pregnancy and lactation, PRLR protein increased in the epithelial compartment of the mammary gland but did not change within the stromal compartment at any physiological stage examined. We also found PRLR mRNA in both the epithelia and stroma of the mammary gland. Again, the stroma contained lower levels of PRLR mRNA compared with the epithelia at all physiological stages examined. Also, the PRLR mRNA levels within the stroma did not change significantly during pregnancy or lactation, whereas PRLR mRNA within the epithelia increased twofold during pregnancy and fourfold during lactation when compared with virgin rats. We conclude from this study that PRLR is expressed both in the stromal and epithelial compartment of the mammary gland. This finding suggests PRL may have a direct affect on the mammary stroma and by that route affect mammary gland development.

Abstract

GH-binding protein (GHBP) or GH receptor is present in numerous extrahepatic tissues in the rodent. From mid- to late gestation in the mouse, the maternal serum concentration of GHBP increases 30- to 50-fold. We have investigated whether the placenta might synthesize GHBP and potentially contribute to this increase. RNA was isolated from placentas and subjected to Northern analysis using a cDNA probe to the shared region of GHBP and GH receptor-encoding mRNAs. From day 8 to day 18 of gestation, the GHBP-encoding mRNA (1·4 kb) increased 45-fold in quantity. The GH receptor-encoding mRNA (4·2 kb) increased sixfold by day 14 and then remained steady until day 18. These changes which were not co-ordinated parallel reported changes in the steady-state concentrations of 1·4 and 4·2 kb mRNAs in maternal liver, suggesting shared regulatory factors. Extracts of freshly isolated trophoblasts were assayed for GHBP with a radioimmunoassay specific for GHBP with a hydrophilic carboxyl terminus. The cytosolic content of immunoreactive GHBP increased fourfold from mid- to late gestation. Trophoblasts were isolated from placentas and cultured for 2 days on collagen gels in defined medium. Cultured cells were at least 90% viable and secreted mouse placental lactogen-II (mPL-II). Immunocytochemistry was carried out simultaneously on cells cultured from day 7 to day 17 of gestation using a monoclonal antibody (MAb 4·3), which recognizes the hydrophilic C-terminus of GHBP. Cell-localized GHBP was present in trophoblasts cultured for 2 days, but GHBP was not detectable by radioimmunoassay or by immunoprecipitation in concentrated culture media from cultures treated with 100 ng mouse GH/ml or 100 ng mPL-II/ml or from untreated cultures. RNA was isolated from cells cultured in an identical manner to those analysed by immunocytochemistry. Three GH receptor/GHBP mRNA species of 8, 4·2 and 1·4 kb were observed. The quantity of 4·2 and 1·4 kb mRNAs did not change significantly in cultures from day 7 to day 15 of gestation but, in cultures from day 17 of gestation, the amount of 1·4 kb mRNA dropped significantly, while that of the 4·2 kb mRNA remained unchanged. GHBP- and GH receptor-encoding mRNAs are not co-ordinately regulated in vivo or in vitro. Although mPL-II was secreted into the medium by cultured trophoblasts, secretion of GHBP could not be detected. The culture medium may not contain the specific factors required for secretion of placental GHBP, or placental GHBP may not be destined for secretion.

The results show that GHBP (as distinct from GH receptor) is expressed by the placenta in vivo and trophoblasts in vitro. From mid-gestation onwards, GHBP mRNA increases dramatically in vivo and the cytosolic content of GHBP in freshly isolated trophoblasts increases. This suggests an important local regulatory role for placental GHBP during gestation.

Journal of Endocrinology (1994) 140, 125–135