Influence of Obstructive Cholestasis and Sex Hormones on the Ratio of mRNA of Two Alternative Prolactin Receptor Isoforms in Rat Hepatocytes

The effects of obstructive cholestasis and sex hormones on the total content of prolactin receptor mRNA and ratio of mRNAs of its short and long isoforms have been studied in rat hepatocytes. Obstructive cholestasis caused insignificant changes in total content of prolactin receptor mRNA, but the proportion of mRNA of the long isoform increased. Comparison of prolactin receptor mRNA levels in gonadectomized and intact animals revealed opposite effects of male and female sex hormones on total mRNA, but both groups of these hormones increased the proportion of prolactin receptor short form mRNA. Changes in ratio of mRNA of receptor isoforms found in rat hepatocytes under obstructive cholestasis did not depend on levels of sex hormones. Obstructive cholestasis and sex hormones are suggested to regulate the content of long and short prolactin receptor isoforms in hepatocytes independently.     

A new member of the prolactin-growth hormone gene family expressed in mouse placenta.

Mouse placenta has been found to contain an mRNA that encodes a previously unidentified member of the prolactin-growth hormone family. This 1.1-kb mRNA (designated PRP mRNA) was detected as a cDNA clone that hydridized to a cDNA clone of mouse proliferin, a recently described growth-associated placental protein related to prolactin. PRP mRNA levels are highest in the fetal part of the placenta and peak at day 12 of gestation, decreasing gradually until term. The 972-bp sequence of PRP mRNA, determined from two cDNA clones, encodes a protein of 244 amino acid residues that has a hydrophobic leader sequence. The protein encoded by PRP mRNA has significant homology to all of the members of the prolactin family, yet is different from each of them; it also differs from mouse placental lactogen. Nucleotide sequence homology is most extensive between PRP and proliferin mRNAs, particularly at their 5' ends, where they share 92 of the first 97 nucleotides.     

A growth-related mRNA in cultured mouse cells encodes a placental calcium binding protein.

We have characterized an mRNA that increases in abundance after serum stimulation of quiescent mouse fibroblasts. This mRNA, designated 18A2, encodes a predicted polypeptide of 101 amino acids with homology to known calcium binding proteins. A variety of mouse tissues express the 18A2 mRNA, with the highest levels detected in the non-pregnant uterus and in the placenta. The concentration of 18A2 mRNA in total placental RNA decreases from day 8 to day 10 of pregnancy, and is below detection throughout the latter half of gestation. In serum-stimulated fibroblasts, the increase in 18A2 mRNA is dependent on protein synthesis. The 18A2 mRNA is similar in size, serum-inducibility, and sequence to the 2A9 mRNA (1), but these mRNAs are derived from distinct genes. This suggests that the mouse genome harbors a family of serum-inducible genes encoding proteins predicted to bind calcium.     

Mouse placental prostaglandins are associated with uterine activation and the timing of birth

We explored a potential mechanism linking placental prostaglandins (PGs) with a fall in plasma progesterone and increased expression of uterine activation proteins in the mouse. PG endoperoxide H synthase 2 (PGHS-2) mRNA expression increased in placenta in late gestation in association with an 8-fold increase in PGF(2alpha) concentration, reaching a peak on Gestational Day (GD) 18. This peak coincided with the final descent in plasma progesterone and birth on GD 19.3 +/- 0.2. Implantation of a progesterone-releasing pellet in intact pregnant dams on GD 16 delayed birth at term until GD 20.9 +/- 0.4 and inhibited the GD 18 increase in placental PGF(2alpha) levels in conjunction with a delayed fall in plasma progesterone that reached its lowest level 1 day after term birth. The mRNA levels of uterine activation proteins, connexin-43 (CX-43), oxytocin receptor, PGF(2alpha) receptor (FP), and PGHS-2, and the concentration of uterine PGF(2alpha) all increased at normal term birth. At progesterone-delayed term birth on GD 19.3, even though tissue PGF(2alpha) concentrations were at the same high levels observed at normal term birth, CX-43 and FP mRNA levels were lower than those at normal term birth, thereby possibly contributing to the delay of birth. These data are consistent with the hypotheses that fetal placental PGs affect the timing of birth by hastening luteolysis, that uterine activation initiates labor, and that birth may be delayed by blocking or decreasing the expression of two of the uterine activation proteins.     

Effect of a high maternal dietary intake during mid-gestation on components of the utero-placental insulin-like growth factor (IGF) system in adolescent sheep with retarded placental development

The aim of the present study was to investigate the effects of administering a high plane diet during early to mid-gestation on the uterine and placental insulin-like growth factor (IGF) system and on systemic IGF-I concentrations in pregnant adolescent ewes with restricted placental growth. Embryos recovered from superovulated ewes inseminated by a single sire were transferred in singleton to the uterus of adolescent recipients. After transfer ewes were offered a high (H) or moderate (M) amount of a complete diet calculated to promote rapid or normal maternal growth rates, respectively. Five ewes from each group were switched from either M to H or H to M diets at day 52 of gestation. Maternal and fetal blood samples and placental tissues were collected from all animals at day 104. Ewes on the high plane diet from mid-gestation (HH, MH groups) had restricted placental mass (P < 0.01) and tended to have smaller fetuses. This was associated with increased maternal plasma IGF-I concentrations (P < 0.001). The pattern of expression of components of the IGF system in the uterus and placenta was studied by in situ hybridization. IGF-I mRNA concentrations were below the limit of detection. IGF-II mRNA expression was high in the fetal mesoderm and present in maternal stroma, but was not influenced by nutritional treatment. In contrast, IGF binding protein 1 (IGFBP-1) mRNA expression was higher (P < 0.05) and IGFBP-3 mRNA expression was lower (P < 0.05) in the endometrial glands of ewes in HH and MH groups. In the fetal trophoblast, IGFBP-3 mRNA expression was higher in the MH group. Type 1 IGF receptor expression was increased (P < 0. 01) in the luminal epithelium of the HM group and IGFBP-2 mRNA expression was highest in the placentome capsule of ewes in the HH group. Together, these results indicate that reprogramming of the uterine and placental IGF axis by maternal nutrition could contribute to placental growth retardation in growing adolescent sheep.     

The regulation of IGFs and IGFBPs by prolactin in primary culture of fetal rat hepatocytes is influenced by maternal malnutrition

During perinatal development, the regulation of IGF system appears to be growth hormone (GH) independent. By using highly purified primary fetal hepatocytes, we investigated the role of prolactin (PRL) in the regulation of IGF system and hepatocyte proliferation. We also analyzed the consequence of a maternal low-protein (LP) diet on the regulation of IGF, IGF-binding protein (IGFBP), and hepatocyte proliferation by prolactin. Pregnant Wistar rats were fed a control (C) diet (20% protein) or isocaloric (LP; 8%) diet throughout gestation. On day 21.5, fetal hepatocytes were cultured for 4 days and incubated with rat prolactin. In the C hepatocytes, PRL at 100 ng/ml decreased the abundance of IGFBP-1 and IGFBP-2 by 50 (P < 0.05) and 60% (P < 0.01), respectively. It also reduced by 70% the level of IGF-II mRNA (P < 0.01). By contrast, PRL failed to modulate IGFBP-1 and IGFBP-2 production by LP hepatocytes, and this was associated with reduced abundance of the short form of PRL receptor (P < 0.05). PRL had no effect on either the proliferation or the IGF-I production by C and LP hepatocytes, although it reduced the expression of IGF-II. These results suggest that prolactin influences hepatocyte proliferation in vitro by inhibiting IGFBP-1, IGFBP-2, and IGF-II levels, which may coincide with the decline of IGF-II observed in rodents during late gestation in vivo. On the other hand, maternal LP diet induces a resistance of fetal hepatocytes to PRL.     

Temporal expression of tissue inhibitors of metalloproteinases in mouse reproductive tissues during gestation

Tissue inhibitors of metalloproteinases (TIMPs) appear to play an important regulatory role in tissue remodelling and invasion by malignant cells. Since pregnancy involves morphological changes in existing maternal tissues, as well as a strictly controlled invasion by fetal trophoblasts, we have examined the temporal expression of TIMP-1, TIMP-2, and specific metalloproteinases in the mouse uterus, decidua, placenta, amnion, and ovaries throughout gestation by examining mRNA levels on northern and slot blots. Maximal levels of TIMP-1 mRNA were observed from day 6 to day 10 in the uterus, decidua, and placenta. In clear contrast to the early burst of TIMP-1 mRNA accumulation, the level of TIMP-2 mRNA increased steadily throughout gestation in the uterus, decidua, and amnion, while in the placenta it showed a sevenfold increase after day 14. In amnion, TIMP-1 was induced specifically on day 18. Interestingly, the normally high level of TIMP-1 mRNA seen in the ovaries of virgin mice was low during gestation, until day 18 and postpartum, when a sixfold increase over the levels in virgin ovary was observed. In contrast, ovarian TIMP-2 mRNA showed a marginal increase during gestation. The temporal pattern of 72 kDa gelatinase type A followed that of TIMP-2 in the decidua and ovary. Stromelysin-2 mRNA was detected at term only in ovary and decidua. Our data show that the temporal accumulation of TIMP-1 and TIMP-2 mRNA is precisely coordinated in each of the tissue compartments and is independently regulated during the in vivo remodelling of reproductive tissues in gestation. The peak of TIMP-1 mRNA levels in the uterus, decidua, and placenta at midgestation is associated with the most invasive period of embryo development. © 1993 Wiley-Liss, Inc.     

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Temporal and spatial expression patterns of a muscarinic acetylcholine receptor (Acr60C) and an octopamine/tyramine receptor (Octyr) were determined inDrosophila melanogaster using quantitative Northern analysis andin situ hybridization to tissue sections. Expression of mRNA encoding both of these G-protein coupled receptors peaks initially in 18 to 21 hour embryos following the formation of the mature larval nervous system. Levels of mRNA then decline during larval stages, rising to a second peak in 3 to 4-day-old pupae after a period of major nervous system reorganization. The muscarinic acetylcholine receptor mRNA is expressed throughout the cortical regions of the central nervous system in adults and embryos. Particularly high levels of expression of Acr60C are observed in cell bodies adjacent to the antennal lobes, suggesting a major role for this muscarinic receptor in the processing of olfactory information. In contrast, the octopamine/tyramine receptor mRNA is distributed diffusely throughout the adult brain, with patches of signal concentrated in the cortex of the dorsal protocerebrum near the mushroom bodies. These patches may represent individual cells expressingOctyr receptors.     

Gestational regulation of granulocyte-colony stimulating factor receptor expression in the human placenta

A number of cytokines and their receptors are abundantly expressed at the materno-fetal interface and are thought to have a function in the regulation of placentation. Granulocyte-colony stimulating factor (G-CSF) is expressed by stromal cells in both placental tissue and maternal decidua throughout placentation. In this study, we examined the expression of placental G-CSF receptor (G-CSFR) mRNA and protein throughout gestation by ribonuclease protection assays, Western blotting, and immunohistochemistry. The major placental form of G-CSFR mRNA, corresponding to a membrane-bound form of the protein, was present in first-trimester placental tissues; levels decreased in second- and were highest in third-trimester placental tissues. Two placental G-CSFR molecules, 120 kDa and 150 kDa, were detected in first- and third-, but not second-, trimester tissues. The level of the 150-kDa G-CSFR was greater in the third- than in first-trimester samples. These differences were irrespective of whether or not the patients had received prostaglandin E1 analogues, prostaglandin E1 analogues and oxytocin, oxytocin alone, or mifepristone before labor. We demonstrated by immunohistochemistry that interstitial cytotrophoblast in first- and second-trimester decidual tissue and cytotrophoblast in term fetal membranes express G-CSFR. These data demonstrate that the expression of specific forms of placental G-CSFR is strictly cell type- and developmental stage-specific, and they suggest that G-CSFR may be important in decidual invasion of cytotrophoblast and in trophoblast function during placentation.     

Peroxidase activity in the mouse uterus, placenta and fetus during pregnancy

Changes in peroxidase activity during pregnancy were examined in CD-1 mice. Peroxidase activity was measured with guaiacol as the substrate in uterine extracts of nonpregnant mice and in uterine, placental, and fetal extracts of pregnant mice on days 9, 12, 14, 16, and 18 of gestation. Uterine peroxidase activity in nonpregnant mice was high, but declined logarithmically to only 0.2% by day 18 of pregnancy. In contrast to this decline, a concomitant 50-fold logarithmic increase in fetal peroxidase activity was observed between day 12 and 18. Activity in placental extracts did not change significantly throughout the gestational period examined. These results suggest that membrane bound peroxidase in mouse uterus and fetus undergoes major shifts during pregnancy.     

Increase of the trophoblast giant cells with prolactin-releasing peptide (PrRP) receptor expression in p53-null mice

Trophoblast giant cells in the mouse placentas are polyploid cells that form as a result of endoreduplication. The giant cells form the outermost layer of the extraembryonic compartment and produce a number of pregnancy-specific hormones, including prolactin family members. Here we demonstrate that trophoblast giant cells are increased, and display upregulation of prolactin releasing peptide (PrRP) receptor in the p53-null (p53(-/-)) embryonic placentas. At day 13.5 of gestation, the weight of p53(-/-) placentas was less than that of both wild-type and p53(+/-) placentas. In p53(-/-) placentas, the spongiotrophoblast layer was significantly decreased in thickness, and the trophoblast giant cells were observed not only in the outer layer of placentas but in both the spongiotrophoblast layer and the labyrinthine layer. The giant cells spread over the spongiotrophoblast and labyrinthine layer in p53(-/-) placentas displayed more intensive expression of immunoreactive PrRP receptor than in wild-type placentas. Previous studies indicated that the association between PrRP and PrRP receptor physiologically involves in the expression and secretion of the peptide hormones, including prolactin and growth hormones. These results suggest that p53 may regulate the differentiation of trophoblast giant cells, and may control the physiological PrRP stimuli in mouse placentas.     

Lactogenic response of mouse mammary explants from different days of pregnancy to placental lactogen and pituitary prolactin

Summary The lactogenic response of mouse mammary gland explants to human placental lactogen (hPL) and ovine pituitary prolactin (oPRL) was examined on days 10 to 18 of pregnancy by measuring³H-amino acid incorporation into calcium-rennin precipitable casein. To determine the lactogenic response of the explants, the mean slopes of dose-response curves were calculated for each hormone treatment. Slope means of dose-response curves for oPRL and hPL did not differ from each other on any day of pregnancy examined. A triphasic pattern of response was suggested when slope means of dose-response curves for both hormones were plotted as a function of day of gestation. Peak responses were observed on days 10, 13 and 17–18. Combinations of oPRL and hPL, in ratios of oPRL:hPL=2∶1 and oPRL:hPL=1∶2, also produced a triphasic pattern of sensitivity very similar to that produced by either hormone alone. These results suggest that mouse mammary explants may be more sensitive to oPRL and hPL on days 10, 13 and 17–18 of pregnancy. This work was supported by grants from the National Science Foundation (76-01928) and the National Institutes of Health (5 S06RR08132-0251) to F. Talamantes.     

Uteroplacental restriction in the rat impairs fetal growth in association with alterations in placental growth factors including PTHrP

During pregnancy, parathyroid hormone-related protein (PTHrP) is one of many growth factors that play important roles to promote fetal growth and development, including stimulation of placental calcium transport. Angiotensin II, acting through the AT(1a) receptor, is also known to promote placental growth. We examined the effects of bilateral uterine artery and vein ligation (restriction), which mimics placental insufficiency in humans, on growth, intrauterine PTHrP, placental AT(1a), and pup calcium. Growth restriction was surgically induced on day 18 of pregnancy in Wistar-Kyoto female rats by uterine vessel ligation. Uteroplacental insufficiency reduced fetal body weight by 15% and litter size (P < 0.001) compared with the control rats with no effect on placental weight or amniotic fluid volume. Uteroplacental insufficiency reduced placental PTHrP content by 46%, with increases in PTHrP (by 2.6-fold), parathyroid hormone (PTH)/PTHrP receptor (by 11.6-fold), and AT(1a) (by 1.7-fold) relative mRNA in placenta following restriction compared with results in control (P < 0.05). There were no alterations in uterine PTHrP and PTH/PTHrP receptor mRNA expression. Maternal and fetal plasma PTHrP and calcium concentrations were unchanged. Although fetal total body calcium was not altered, placental restriction altered perinatal calcium homeostasis, as evidenced by lower pup total body calcium after birth (P < 0.05). The increased uterine and amniotic fluid PTHrP (P < 0.05) may be an attempt to compensate for the induced impaired placental function. The present study demonstrates that uteroplacental insufficiency alters intrauterine PTHrP, placental AT(1a) expression, and perinatal calcium in association with a reduction in fetal growth. Uteroplacental insufficiency may provide an important model for exploring the early origins of adult diseases.     

Expression of galectin-1 mRNA in the mouse uterus is under the control of ovarian steroids during blastocyst implantation

Galectin-1 is a member of β-galactoside-binding lectins expressed in a variety of mammalian tissues. We report here that galectin-1 mRNA is abundantly expressed in the mouse reproductive organs such as the uterus and ovary. Uterine expression of galectin-1 mRNA is specifically regulated in the embryonic implantation process. Its expression increased at a high level on the fifth day post coitum (dpc 5) when embryos hatched into the endometrial epithelial cells. In the absence of embryos, however, galectin-1 expression in the mouse uterus decreased on dpc 5. In the delayed implantation mice, galectin-1 mRNA level was augmented by the termination of the delay of implantation. Ovarian steroids progesterone and estrogen differentially regulated galectin-1 mRNA level in uterine tissues. Treatment with RU486, a progesterone receptor antagonist, blocked progesterone-induced galectin-1 mRNA level in uterine tissues of ovariectomized mouse. ICI182780, a pure estrogen receptor antagonist, clearly blocked the estrogen effect. Taken together, galectin-1 gene expression in the uterine tissues was regulated by ovarian steroids and this regulation correlated with the implantation process. Mol. Reprod. Dev. 48:261–266, 1997. © 1997 Wiley-Liss, Inc.     

Prolactin receptors in rat cholangiocytes: Regulation of level and isoform ratio is sex independent

The presence of prolactin receptor and peculiarities of its isoform expression in bile duct cells (cholangiocytes) differentially isolated from rat liver under different conditions were investigated in the present study. Normal cholangiocytes express prolactin receptor at relatively low level comparable to those of some prolactin-dependent tissues. Long receptor isoform is predominant in cholangiocytes but not in hepatocytes. The prolactin receptor level increases significantly under obstructive cholestasis due to evaluation of long and appearance of short isoforms. In rat cholangiocytes, unlike other tissues, the main positive regulators of prolactin receptor expression are cholestasis-induced factors instead of sex hormone and prolactin levels. Long isoform is predominant and induced primarily by cholestasis-induced factors. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 2, pp. 229–236.