Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia.

The factors that regulate pancreatic beta cell proliferation are not well defined. In order to explore the role of murine placental lactogen (PL)-I (mPL-I) in islet mass regulation in vivo, we developed transgenic mice in which mPL-I is targeted to the beta cell using the rat insulin II promoter. Rat insulin II-mPL-I mice displayed both fasting and postprandial hypoglycemia (71 and 105 mg/dl, respectively) as compared with normal mice (92 and 129 mg/dl; p < 0.00005 for both). Plasma insulin concentrations were inappropriately elevated, and insulin content in the pancreas was increased 2-fold. Glucose-stimulated insulin secretion by perifused islets was indistinguishable from controls at 7.5, 15, and 20 mm glucose. Beta cell proliferation rates were twice normal (p = 0. 0005). This hyperplasia, together with a 20% increase in beta cell size, resulted in a 2-fold increase in islet mass (p = 0.0005) and a 1.45-fold increase in islet number (p = 0.0012). In mice, murine PL-I is a potent islet mitogen, is capable of increasing islet mass, and is associated with hypoglycemia over the long term. It can be targeted to the beta cell using standard gene targeting techniques. Potential exists for beta cell engineering using this strategy.

Opposite effects of transforming growth factor alpha and epidermal growth factor on mouse placental lactogen I secretion.

This study was undertaken to determine whether transforming growth factor alpha (TGF-alpha) regulates the production of mouse placental lactogen I (mPL-I) and mPL-II in a manner that is similar to that of epidermal growth factor (EGF), which was previously shown to stimulate mPL-I secretion and inhibit mPL-II secretion. In contrast to the activity of EGF, human (h) and rat (r) TGF-alpha (each at 100 ng/ml) inhibited secretion of mPL-I by placental cells isolated from mice on day 7 of pregnancy. Maximum inhibition of mPL-I secretion occurred on the third day of a 5-day culture period and ranged between 37% and 56% in multiple trials. Incubation of cells with hTGF-alpha and EGF was not followed by a change in the mPL-I concentration of the medium, suggesting the peptides antagonized each other's effects. hTGF-alpha and rTGF-alpha inhibited secretion of mPL-II; maximum inhibition ranged between 62% and 84% in multiple trials. The lowest concentrations of hTGF-alpha that affected mPL-I and mPL-II secretion were 10 ng/ml and 1 ng/ml, respectively. EGF and hTGF-alpha bound to the same receptors on placental cells, as assessed by cross-linking, and both peptides stimulated receptor phosphorylation, as assessed by Western blot analysis. There are three types of mPL-containing cells in placental cultures: cells that contain only mPL-I, cells that contain only mPL-II, and cells that contain both mPLs. The percentage of each type of mPL-containing cell in the culture was determined by immunostaining. hTGF-alpha affected the differentiation of the subpopulations of PL-containing cells in a manner that differed from that of EGF. The data suggest that TGF-alpha and EGF do not regulate the production of mPL-I and mPL-II in a similar manner.

Co-localization of placental lactogen-I, placental lactogen-II, and proliferin in the mouse placenta at midpregnancy.

This study was undertaken to determine whether mouse placental lactogen (mPL)-I, mPL-II, and proliferin (PLF) are expressed by the same population of placental giant cells at midpregnancy. Tissue sections from Day 9 of pregnancy were analyzed by double immunofluorescence staining. Sections were stained for PLF by use of a rhodamine-conjugated second antibody, and for mPL-I or mPL-II by use of a fluorescein-conjugated second antibody. All three proteins were present in most of the same giant cells. The distribution of mPL-I and PLF among giant cells in vitro was also examined. When placental cells from Day 7 of pregnancy were cultured for 5 days, > 90% of the cells that immunostained for mPL-I also immunostained for PLF on the first 3 days of culture. Thereafter, the percentage of cells that contained both proteins declined rapidly while the percentage that contained only PLF increased, suggesting continued differentiation of the cells in vitro. These data demonstrate that the same trophoblast giant cells express mPL-I, mPL-II, and PLF simultaneously at midpregnancy, suggesting that their gestational profiles in maternal blood during this period result at least partly from changes in gene expression in one population of cells and not from differentiation of several subsets of giant cells, each expressing only one member of the gene family.

Mouse placental cells secrete immunoreactive growth hormone-releasing factor.

The initial objective of this study was to establish a placental cell culture system in which the secretion of mouse growth hormone-releasing factor (mGHRF) could be examined during a several-day period. To determine when during pregnancy placental cells begin to express mGHRF, Northern blot analysis was carried out on total RNA from placentas collected on Days 6, 9, 11, 13, 15, 17, and 18 of pregnancy. Mouse GHRF mRNA could be detected as early as Day 11 of pregnancy. Its steady-state levels increased to maximum values on Days 15-17 and then declined slightly on Day 18. Placentas from Day 12 of pregnancy were selected for cell culture. The basal zone and labyrinth were dispersed in collagenase, and the cells were fractionated on a Percoll gradient. Two bands of cells were selected for further study. Both released significant amounts of immunoreactive mGHRF during a 5-day culture period. Effects of prolonged exposure of the cells to 8-bromo-cAMP and to agents that elevate intracellular cAMP concentration were then examined. Treatment of the cells with 0.5 mM 8-bromo-cAMP resulted in a significant decrease in the mGHRF concentration of the medium by the second day of culture. Mouse GHRF secretion was also inhibited by treatment of the cells with 100 ng/ml cholera toxin or 0.1 mM forskolin. The effect of 8-bromo-cAMP was concentration-dependent, with 0.1 mM being the lowest concentration that was active. 8-Bromo-cAMP treatment also reduced the steady-state level of mGHRF mRNA in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective inhibition of mouse placental lactogen II secretion by tumour necrosis factor-alpha.

The placental members of the prolactin-GH-placental lactogen (PL) gene family of the mouse include mPL-I, mPL-II, proliferin (PLF) and proliferin-related protein (PRP). The aim of the present study was to assess the effects of tumour necrosis factor-alpha (TNF-alpha) on the secretion of these proteins in primary cultures of placental cells from days 7, 9 and 12 of pregnancy. The effects of epidermal growth factor (EGF) on the secretion of PLF and PRP were also determined. EGF has previously been shown to stimulate mPL-I and inhibit mPL-II secretion. Incubation of placental cells from day 7 of pregnancy for 5 days with 10 nmol human (h)TNF-alpha/l did not affect the mPL-II concentration of the medium, but similar treatment of cells from days 9 or 12 of pregnancy resulted in a significant reduction in the mPL-II concentration of the medium by the second or third day of culture. The intracellular concentration of mPL-II, the number of cells that released mPL-II as assessed by reverse haemolytic plaque assay, and steady-state levels of mPL-II mRNA as assessed by Northern analysis were also reduced by hTNF-alpha treatment. The lowest concentration of hTNF-alpha that significantly inhibited mPL-II secretion by cells from day 12 of pregnancy was 0.01 nmol/l. hTNF-alpha treatment did not affect the secretion of mPL-I, PLF or PRP, as assessed by the concentrations of these proteins in the medium during a 5-day incubation. Incubation of the cells with 20 ng EGF/ml also did not affect the PLF or PRP concentration of the medium during 5 days of culture. To determine whether the effect of hTNF-alpha on mPL-II secretion was mediated by interleukin-6 (IL-6), the IL-6 concentration of the medium of control and hTNF-alpha-treated cells was determined. Bioactive and immunoreactive IL-6 could not be detected in medium from either treatment group. The presence of binding sites for hTNF-alpha was assessed in cells from day 12 of pregnancy. Scatchard analysis detected a single class of binding sites having a Kd of 1.61 +/- 0.34 nmol/l, with about 1350 sites per cell. The results of this study demonstrate that hTNF-alpha inhibits the secretion of mPL-II by placental cells from days 9 and 12 of pregnancy, suggesting that TNF-alpha may be one of the factors that regulate the production of this hormone in vivo.

Expression of growth hormone-binding protein with a hydrophilic carboxyl terminus by the mouse placenta: studies in vivo and in vitro.

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.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 6 inhibits mouse placental lactogen II but not mouse placental lactogen I secretion in vitro.

The mouse placenta produces several polypeptides belonging to the prolactin-growth hormone gene family, including mouse placental lactogen (mPL) I and mPL-II. The present study was undertaken to determine whether the secretion of mPL-I and mPL-II is regulated by interleukin 6 (IL-6), which is present in the placenta and has previously been reported to stimulate the secretion of pituitary members of this gene family. Effects of human and mouse IL-6 on mPL-I and mPL-II secretion were examined in primary cultures of placental cells from days 7, 9, and 12 of pregnancy. IL-6 caused a dose-dependent reduction in the mPL-II concentration in the medium of cells from days 9 and 12 of pregnancy but did not affect the mPL-II concentration in the medium of cells from day 7 of pregnancy or the mPL-I concentration in the medium of cells from days 7 or 9 of pregnancy. The lowest concentration of human IL-6 that significantly inhibited mPL-II secretion was 250 pM. The effect of IL-6 on the mPL-II concentration in the medium was due primarily to inhibition of mPL-II synthesis, which resulted at least partly from a decrease in the steady-state level of mPL-II mRNA. These data raise the possibility that IL-6 may regulate mPL-II production after midpregnancy in vivo.

Effect of homologous placental lactogens, prolactins, and growth hormones on islet B-cell division and insulin secretion in rat, mouse, and human islets: implication for placental lactogen regulation of islet function during pregnancy.

Up-regulation of maternal islet function is essential to accommodate the increased demand for insulin during pregnancy. Previously, we suggested that lactogenic activity regulates islet function during pregnancy. However, this hypothesis was based on the effect of homologous PRLs on islets, since the homologous placental lactogens (or islets) were unavailable. In this study we examine the direct effects of homologous placental lactogens (PL), PRL, and GH on insulin secretion and B-cell division in rat, mouse, and human islets in vitro. Neonatal rat islets were cultured for 8 days in the presence of 0-1000 ng/ml rat PL-I (rPL-I), rPRL, or rGH. Media were changed daily, and the insulin concentration was determined. rPL-I and rPRL (500 ng/ml) treatment resulted in a 2-fold increase in insulin secretion. rGH (1000 ng/ml) elicited a 30% increase in insulin secretion. Similarly, cell replication, as indicated by BrdU incorporation into B-cells, was increased 4-fold in the presence of rPL-I and rPRL. The ED50 for insulin secretion and 5'-bromo-2'-deoxyuridine (BrdU) incorporation was 70 ng/ml for rPL-I and 150 ng/ml for rPRL. Similarly, in adult rat islets, insulin secretion was increased 1.6-fold, and B-cell replication increased 3-fold in the presence of the lactogenic hormones. Neonatal mouse islets were cultured for 8 days in the presence of 500 ng/ml mouse (m) PL-I, mPL-II, mPRL, or mGH. mPL-I, mPL-II, and mPRL treatment resulted in a 2-fold increase in insulin secretion. mGH elicited a 30% increase in insulin secretion. BrdU incorporation into B-cells was increased 3-fold in the presence of mPL-I and mPRL and 2-fold in the presence of mPL-II. Adult human islets were cultured for 8 days in the presence of 1 microgram/ml human (h) PL, hPRL, or hGH. For human islets isolated from six pancreata obtained from females, hPL (138 +/- 10%), hPRL (133 +/- 9%), and hGH (117 +/- 3%) significantly increased insulin secretion compared to that from control islets. This study compares the direct effects among homologous PLs, PRLs, and GHs on insulin secretion and B-cell division in rat, mouse, and human islets. The results indicate that placental lactogen directly regulates islet function in several species and is probably the principal hormone responsible for the increased islet function observed during normal pregnancy.

Regulation of the hepatic growth hormone receptor and serum growth hormone-binding protein during pregnancy in the mouse: effects of litter size.

The regulation of hepatic GH receptor (GHR) and serum GH-binding protein (GHBP) during pregnancy in the mouse was investigated by manipulating the number of conceptuses carried by the dam. Animals carrying 1-4 conceptuses had significantly lower amounts of hepatic GHR (GH-binding activity) than mice carrying 10-13 conceptuses on days 9 and 13 of pregnancy. There was no significant difference in hepatic GHR on day 17 of pregnancy between animals carrying 1-4 and 10-13 conceptuses. Animals carrying 1-4 conceptuses had significantly lower concentrations of GHBP in serum than animals carrying 10-13 conceptuses on days 9, 13, and 17 of pregnancy. The relative amounts of liver GHR- and GHBP-encoding messages in animals with low and high conceptus numbers were investigated by Northern analysis. There were higher levels of both messages in animals carrying 10-13 conceptuses than in mice carrying 1-4 conceptuses. On day 13 of pregnancy, animals carrying 10-13 conceptuses had significantly higher levels of GHBP-encoding message than animals carrying 1-4 conceptuses. Total hepatic mass was not significantly different between animals with low and high conceptus numbers. No significant difference was found in GHBP concentration between blood from the uterine vein and the trunk in 17-day pregnant animals. Mouse placental lactogen-I (mPL-I), mPL-II, GH, and corticosterone concentrations were measured by RIA and related to hepatic GHR activity and serum GHBP concentration. Hepatic GHR activity and serum GHBP concentration were significantly correlated with each other on days 9, 13, and 17 of pregnancy. Hepatic GHR activity was significantly correlated with mPL-I and mPL-II on day 9 of pregnancy. GHBP concentration was significantly correlated with mPL-I and mPL-II on day 9 of pregnancy and with mPL-II and GH on day 13 of pregnancy. Data are consistent with the hypothesis that mPL-I, mPL-II, and GH may affect hepatic expression of the GHR/GHBP gene during pregnancy in the mouse.

Epidermal growth factor stimulates mouse placental lactogen I but inhibits mouse placental lactogen II secretion in vitro.

This study was undertaken to determine whether epidermal growth factor (EGF) regulates the secretion of mouse placental lactogen (mPL)-I and mPL-II. Primary cell cultures were prepared from placentas from days 7, 9, and 11 of pregnancy and cultured for up to 5 days. Addition of EGF (20 ng/ml) to the medium resulted in significant stimulation of mPL-I secretion by the second day of culture in cells from days 7 and 9 of pregnancy and significant inhibition of mPL-II secretion by the third or fourth day of culture in cells from days 7, 9, and 11. Dose-response studies carried out with cells from day 7 of pregnancy demonstrated that the minimum concentration of EGF that stimulated mPL-I secretion and inhibited mPL-II secretion was 1.0 ng/ml. EGF did not affect the DNA content of the cells or cell viability, assessed by trypan blue exclusion, nor did it have a general effect on protein synthesis. There are three types of PL-containing giant cells in mouse placental cell cultures: cells that contain either mPL-I or mPL-II and cells that contain both hormones. Immunocytochemical analysis and the reverse hemolytic plaque assay indicated that EGF treatment was accompanied by a significant increase in the number of cells that produce mPL-I, but among the PL cells that contained mPL-I, there was no change in the fraction of cells that contained only mPL-I or the fraction that contained both mPL-I and mPL-II. In contrast, EGF treatment did affect the distribution of mPL-II among PL cells. In control cultures, about 75% of the cells that contained mPL-II also contained mPL-I, but in EGF-treated cultures, all of the cells that contained mPL-II also contained mPL-I. These data suggest that EGF regulates mPL-I and mPL-II secretion at least partly by regulating PL cell differentiation.

Production of mouse placental lactogen-I and placental lactogen-II by the same giant cell.

In previous studies, mouse placental lactogen I (mPL-I) and mPL-II were localized to trophoblast giant cells in the placenta at midpregnancy. The present study was undertaken to determine whether mPL-I and mPL-II are produced by two distinct populations of giant cells or by the same cells. A heterogeneous population of cells that included trophoblast giant cells was obtained by enzymatic dispersion and Percoll gradient centrifugation of placentas from days 7 and 9 of pregnancy. Cells from day 7 of pregnancy were cultured in serum-free medium for 5 days, and cells that contained mPL-I, mPL-II, or both mPL-I and mPL-II were identified by double-staining immunocytochemistry. The percentage of PL cells that contained both mPL-I and mPL-II increased from about 30% on the first day of culture to about 90% on the third, and then declined to zero by day 5. Between 50% and 60% of the PL cells contained only mPL-I on the first 2 days of culture, and then the percentage of PL cells containing only mPL-I declined. The percentage of cells that contained only mPL-II was low for 3 days (<10%) and then increased to about 80% of the PL-containing cells by day 5. Cells from day 9 of pregnancy were analyzed for the release of mPL-I and/or mPL-II by sequential reverse hemolytic plaque assay. Cells that released only one of the PLs, as well as those that released both PLs, were identified. A shift was present in the type of PL released by the cells when they were followed for two consecutive days of culture. On day 1, most of the plaque-forming cells released only mPL-I, but by day 2, the fraction of plaque-forming cells that released only mPL-I declined whereas the fraction that released only mPL-II increased. Cells that released only mPL-I on the first day of culture and both mPL-I and mPL-II or only mPL-II on the second day of culture were observed. These data suggest that under these culture conditions, PL cells follow a pathway in which they initially produce only mPL-I, then both mPL-I and mPL-II, and finally only mPL-II. In vivo, there is a shift at midpregnancy in the type of PL that is produced by the mouse placenta, and these data suggest that this shift results, at least partly, from a change in gene expression in one population of giant cells.

Mammary gland differentiation in hypophysectomized, pregnant mice treated with corticosterone and thyroxine.

Swiss Webster mice were hypophysectomized or sham-operated on Day 11 of pregnancy. The animals were fitted s.c. with osmotic minipumps containing either corticosterone (B) dissolved in Molecusol (Pharmatec, Alachua, FL) or the vehicle alone immediately after they were hypophysectomized. Animals in some of the experimental groups also received thyroxine (T4) in their drinking water. The mice were killed on Day 18 of gestation, and mammary tissue was homogenized and extracted for assessment of DNA, RNA, alpha-lactalbumin, and alpha-casein. Serum was assayed for placental lactogen-I (PL-I), and placental lactogen-II (PL-II), B, and T4. The concentration of PL-II in serum was elevated in the hypophysectomized mice, whereas the PL-I concentration did not differ among experimental groups. Hypophysectomy decreased both T4 and B concentrations in serum, and administration of these hormones restored their serum concentrations to normal or, in some cases, somewhat higher than normal levels. Hypophysectomy reduced the total RNA content and RNA/DNA ratio of the mammary gland, but treatment with B alone or with B and T4 restored RNA levels to those of sham-operated animals. T4 alone was ineffective in restoring RNA levels. Sham-operated animals that received hormonal treatment (B and T4) had the highest levels of RNA in the mammary tissue. Hypophysectomized animals had reduced content and concentration of alpha-lactalbumin in the mammary gland as compared to all other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the growth hormone receptor and growth hormone-binding protein during pregnancy in the mouse.

A 20-fold increase in the relative expression of the hepatic GH-binding protein (GHBP)-encoding message between nonpregnant and 17-day pregnant mice was found. The hepatic GH receptor (GHR)-encoding message increased 8-fold between nonpregnant and pregnant mice. The increase in both messages began on day 9 of pregnancy. The steady state level of the GHBP-encoding message continued to increase steadily until day 17 of pregnancy; however, by day 13 of pregnancy, the steady state level of the GHR-encoding message reached a plateau that continued to day 17. The ratio between the GHBP- and GHR-encoding messages gradually increased during the second half of pregnancy, reaching a maximum on day 17. There was a 10- to 16-fold increase in GH-binding capacity in liver microsomes and a 30- to 50-fold increase in serum GH-binding capacity between nonpregnant and late pregnant mice. The increase in hepatic GH-binding capacity began on day 9 of pregnancy and reached a plateau on day 11, which was maintained until the end of gestation. The increase in serum GH-binding capacity began on day 9 of pregnancy and continued to increase until day 17. No significant change in mouse (m) GHR (mGHR) or mGHBP affinity constants were observed between nonpregnant and pregnant mice; however, the mGHR had a 20-fold greater affinity for mGH than did the mGHBP. The serum GH concentration increased in the second half of pregnancy. The GHBP-bound and the free fractions of GH during pregnancy were predicted. While the bound fraction of GH is predicted to parallel the total GH concentration measured by RIA, the concentration of free mouse GH remains unchanged during pregnancy.

Modulation of mouse placental lactogen-I secretion in vitro: effects of progesterone and mouse placental lactogen-II.

The primary objective of this study was to develop a cell culture system for assessing effects of putative secretagogues on mouse PL-I (mPL-I) secretion. Trophoblast from days 7 to 11 of pregnancy was dispersed in collagenase, and the cells were fractionated on a Percoll gradient and plated on collagen gels in serum-free medium. Cells from days 7-9 of pregnancy yielded five bands on Percoll gradients and those from days 10 and 11 yielded six. mPL-I was present in four of the bands of cells from each day of pregnancy. Cells from day 7 of pregnancy that banded at a density of 1.044 g/ml secreted the largest amount of mPL-I during 5 days of culture. The mPL-I concentration of the medium of these cells increased for the first 3 or 4 days of culture and then declined on the fifth day. mPL-II could not be detected in the medium until the third or fourth day of culture, and its concentration increased thereafter. Cell viability was about 90% at the time of plating, remained at about 80% between days 1 and 4, and then declined on day 5. The cell type that produced mPL-I was identified with the reverse hemolytic plaque assay and by staining with anti-mPL-I antiserum. Both methods indicated that mPL-I was produced by giant cells. The ability of the cells to respond to putative secretagogues was examined using mPL-II and progesterone. mPL-II, at concentrations ranging between 10 ng/ml and 10 micrograms/ml, had no effect on the mPL-I concentration of the medium when it was present for up to 3 days of culture, which suggests that mPL-II does not inhibit mPL-I secretion in vitro. Incubation of the cells in the presence of 100-1000 ng/ml progesterone caused a dose- and time-dependent reduction in the mPL-I concentration of the medium and a decrease in the number of cells that stained with anti-mPL-I antiserum. The effect of progesterone on both endpoints was not apparent until the second day of treatment. These data suggest that progesterone inhibits mPL-I secretion at least in part by inhibiting the differentiation of mPL-I-producing giant cells. The fact that the mPL-I-producing cells responded to progesterone indicates that this culture system will be useful in assessing effects of putative secretagogues on mPL-I secretion.

Localization of placental lactogen-I in trophoblast giant cells of the mouse placenta.

The purpose of this investigation was to identify the cellular origin of placental lactogen-I (PL-I) expression in the mouse placenta and to cytologically define the transition from PL-I to PL-II expression during gestation. PL-I mRNA expression was assessed by in situ hybridization, and expression of PL-I and PL-II protein was determined by immunocytochemical analysis. PL-I mRNA and protein were localized to trophoblast giant cells. Trophoblast giant cells ceased producing PL-I at midgestation and began expressing PL-II. PL-I immunoreactivity was present in trophoblast giant cells on Days 9 and 10 of gestation but was not detectable in trophoblast giant cells on Day 11 of gestation. Immunoreactive PL-II-producing giant cells were detected first on Day 10 of gestation, continuing on Day 11 of gestation. Expression of PL-I and PL-II signals a significant functional transition in trophoblast giant cells of the developing mouse placenta.

Recombinant mouse placental lactogen-I binds to lactogen receptors in mouse liver and ovary: partial characterization of the ovarian receptor.

The binding of recombinant mouse placental lactogen-I (mPL-Ir) to liver and ovarian membranes was investigated in virgin and pregnant mice. Competitive binding assays demonstrated that mPL-Ir, mouse placental lactogen-II (mPL-II), and mouse PRL (mPRL) bind to the same receptors in ovarian membranes. The relative abilities of the three hormones to displace [125I]iodo-mPL-Ir from the ovarian lactogen receptors was mPL-II greater than mPL-Ir much greater than mPRL. Scatchard analysis of mPL-Ir binding to ovarian membranes from day 10 pregnant mice showed a Ka of 2.0 x 10(9) M-1 and a binding capacity of 3.2 x 10(-14) mol/mg membrane protein. The specific binding of [125I]iodo-mPL-Ir to ovarian membrane preparations was significantly higher on day 17 than on day 10 of gestation. Dissociation of endogenous hormones with 4 M MgCl2 increased the binding of [125I]iodo-mPL-Ir to ovarian membranes but not to liver membranes. Affinity cross-linking of [125I]iodo-mPL-Ir to liver and ovarian membranes resulted in the specific labeling of proteins with receptor mol wt (Mr) of 44K and 40K (nonreduced) and 50K and 42K (reduced), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lactogen receptors from liver and ovary appeared structurally homologous, producing fragments with similar Mr when treated with proteolytic enzymes and undergoing similar reductions in Mr when treated with glycolytic enzymes. The ability of mPLs to bind specifically and with high affinity to receptors in mouse ovarian membranes suggests that these hormones may regulate ovarian function during gestation.

Mouse placental lactogen-I: RIA and gestational profile in maternal serum.

A RIA for mouse placental lactogen-I (mPL-I) was developed using recombinant mPL-I as the standard, radioligand, and antigen for antiserum production. Displacement curves for dilutions of serum and placental extracts from pregnant mice were parallel to the recombinant mPL-I standard curve. Serum from male and nonpregnant female mice and high concentrations of mouse PRL, GH, PL-II, proliferin, and proliferin-related protein did not cross-react in the assay. mPL-I appeared in maternal serum on day 6 of pregnancy. Its concentration remained low until day 8 and them increased to a very large peak on days 9-11 (maximum concentration, approximately 8 micrograms/ml). The mPL-I concentration declined after day 11, but the hormone could be detected at low concentration in maternal serum until the end of pregnancy. On day 10 of pregnancy, the mPL-I concentration of maternal serum was correlated with litter size. Fractionation of serum from 10-day pregnant mice by size exclusion chromatography indicated the absence of high mol wt forms of mPL-I in the circulation.

Ethanol consumption during pregnancy in mice: effects on hormone concentrations.

Hormonal and metabolic responses to ethanol consumption were studied in pregnant Swiss Webster mice and their fetuses. On day 10 of pregnancy, mice were assigned to one of three liquid diet groups: 36% ethanol-derived calories fed ad libitum (ETOH); control liquid diet fed ad libitum (control); control liquid diet administered in the same amount as that consumed by the ETOH group on the same day of pregnancy [pair-fed controls (PF)]. Dams were killed on days 14-18 of pregnancy, and fetuses were collected on days 16-18. Serum mouse placental lactogen-II (mPL-II), prolactin (mPRL), growth hormone (mGH), insulin, and glucose concentrations were determined for each group. Ethanol consumption resulted in a significant reduction in fetal weight on day 18 of pregnancy. The maternal serum mPRL concentration was reduced in ETOH mice on days 16 and 17 of pregnancy when compared with control mice, but when compared with PF mice, it was significantly reduced only on day 16. Maternal and fetal serum insulin concentrations were reduced in ETOH mice when compared with control mice, but it is unclear whether this was a specific effect of ethanol consumption or due to reduced food intake. Maternal serum glucose concentrations were reduced in the ETOH mice but not as severely as in the PF mice. Ethanol consumption had no effect on maternal or fetal serum mPL-II and mGH concentrations. These data confirm that ethanol consumption during pregnancy results in fetal growth retardation, but they do not indicate any major effects of ethanol on mPL-II, mPRL, mGH, and insulin concentrations in either the mother or fetus.

Mammary gland development and alpha-lactalbumin production in hypophysectomized, pregnant mice.

Swiss Webster mice were hypophysectomized on Day 10 of pregnancy and the effect of the ablation on mammary gland development was estimated by measuring the total weight and the DNA, RNA, and alpha-lactalbumin contents and concentrations of the mammary gland on Days 14 and 18 of gestation. Although a significant increase in mammary gland weight occurred in the hypophysectomized animals between Days 10 and 18 of pregnancy, the mammary gland weight of the hypophysectomized mice was significantly reduced when compared with intact and sham-operated mice on both Days 14 and 18 of pregnancy. The total RNA content of the mammary gland was also reduced in the hypophysectomized mice, although it increased significantly from Day 10 to Day 18. The alpha-lactalbumin content of the mammary gland increased only slightly between Days 10 and 14 of gestation in the intact and sham-operated mice, but a large increase was found on Day 18 in both groups. There was, on the other hand, no increment in the alpha-lactalbumin content of the mammary gland in the hypophysectomized mice either on Day 14 or 18 of gestation. The DNA content of the mammary gland was not affected by hypophysectomy when estimated on Days 14 and 18 of pregnancy. The effects of hypophysectomy on the concentrations of mouse placental lactogen II (mPL-II), progesterone, corticosterone, and thyroxine in the maternal serum were also assessed. The concentration of mPL-II was significantly elevated in the hypophysectomized mice, whereas the circulating concentrations of both corticosterone and thyroxine were greatly reduced. The serum progesterone concentration was not significantly altered by hypophysectomy. (ABSTRACT TRUNCATED AT 250 WORDS)

Biological, immunological, and binding properties of recombinant mouse placental lactogen-I.

Mouse placental lactogen-I (mPL-I) cDNA was inserted into a eukaryotic expression vector and introduced into Chinese hamster ovary cells. Cell lines that secrete high concentrations of mPL-I were isolated, and this glycoprotein was purified from the cell culture-conditioned medium. Recombinant mPL-I (mPL-Ir) is very similar to placental mPL-I (mPL-Ip) in its recognition by polyclonal antisera raised against either mPL-Ip or mPL-Ir, in displacing [125I]iodo-mPL-II from binding sites on mouse liver microsomal membranes, and in stimulating the synthesis of alpha-lactalbumin in primary cultures of mouse mammary epithelial cells. Structural comparison of mPL-Ir and mPL-Ip by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that mPL-Ir comprises several proteins with mol wt ranging from 34.5-38K, while mPL-Ip consists of a similar set of proteins with mol wt ranging from 36.5-42K. Treatment of the two proteins with neuraminidase resulted in similar 2-4K decreases in mol wt. Treatment of mPL-Ip with peptide:N-glycosidase-F to remove asparagine-linked oligosaccharide chains resulted in the formation of 28K and 29K mol wt species, while treatment of mPL-Ir with the same enzyme yielded 28K and 28.5K mol wt products.