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Introduction The placenta is a consummate endocrine organ as it secretes many biologically active peptide hormones at various times throughout the whole pregnancy. Unlike regulated secretion from other endocrine tissues, secretion from the
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Department of Obstetrics and Gynaecology, Translational Proteomics, Mercy Perinatal Research Centre, Mercy Hospital for Women, University of Melbourne, 4th Floor, 163 Studley Road, Heidelberg 3084, Victoria, Australia
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Introduction The later part of normal human pregnancy is characterised by maternal hyperinsulinaemia and a progressive decline in insulin sensitivity, an adaptation required for adequate transport of nutrients between mother and foetus ( Buchanan
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Prolactin receptor (PRL-R) expression in the brain is increased in lactating rats compared with non-pregnant animals. The aim of the present study was to determine the time-course of changes in PRL-R mRNA levels during pregnancy and/or lactation, and to determine relative levels of the two forms (short and/or long form) of receptor mRNA in specific brain regions. Brains were collected from female rats on dioestrus, days 7, 14 or 21 of pregnancy, day 7 of lactation or day 7 post-weaning. Frozen, coronal sections were cut (300 microm) and specific hypothalamic nuclei and the choroid plexus were microdissected using a punch technique. Total RNA was extracted and reverse transcribed, then first strand cDNA was amplified using quantitative real-time PCR. Results showed an up-regulation of long-form PRL-R mRNA in the choroid plexus by day 7 of pregnancy compared with dioestrus, which further increased on days 14 and 21 of pregnancy and day 7 of lactation, and then decreased to dioestrous levels on day 7 post-weaning. Short-form PRL-R mRNA levels increased on day 14 of pregnancy relative to dioestrus, increased further on day 7 of lactation and decreased on day 7 post-weaning. Changes in mRNA were reflected in increased levels of PRL-R immunoreactivity in the choroid plexus during pregnancy and lactation, compared with dioestrus. In the arcuate nucleus, long-form PRL-R mRNA was increased during pregnancy. In contrast to earlier work, no significant changes in short- or long-form PRL-R mRNA expression were detected in several other hypothalamic nuclei, suggesting that changes in hypothalamic mRNA levels may not be as marked as previously thought. The up-regulation of PRL-R mRNA and protein expression in the choroid plexus during pregnancy and lactation suggest a possible mechanism whereby increasing levels of peripheral prolactin during pregnancy may have access to the central nervous system. Together with expression of long-form PRL-R mRNA in specific hypothalamic nuclei, these results support a role for prolactin in regulating neuroendocrine and behavioural adaptations in the maternal brain.
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ABSTRACT
Within the human utero-placental unit only decidualized stromal cells express mRNA for prolactin. However, it is not clear if the level of prolactin production is related to the number of decidualized cells or the capacity of individual decidual cells to synthesize prolactin, either or both of which parameters may change during pregnancy. In the present study, prolactin production at different stages of human pregnancy was examined using quantitative in situ hybridization to assess decidual prolactin mRNA abundance, immunocytochemistry to examine the prolactin content inside decidual cells and RIA to measure decidual prolactin output into amniotic fluid. Throughout pregnancy the proportion of stromal cells showing positive immunostaining and mRNA for prolactin increased. There was a parallel increase in decidual cell size which was correlated with an increase in prolactin gene expression and intensity of immuno-staining for prolactin in individual decidual cells. These changes in decidual cells were consistent with the changes in the concentration of prolactin in amniotic fluid. These results suggest that there is a close link between the level of prolactin gene expression and production of prolactin by individual decidual cells, which in turn is directly related to the process of decidualization that continues throughout human pregnancy.
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ABSTRACT
Progesterone-associated endometrial protein (PAEP) has been isolated from human decidualized endometrium. In-situ hybridization histochemistry was employed to determine the cellular localization of PAEP mRNA in decidua during pregnancy. PAEP mRNA was found to be expressed in the glandular epithelium of decidua spongiosa throughout pregnancy. Substantial variations in the amount of PAEP mRNA during the course of pregnancy were observed, and it was most abundant at the end of the first trimester. We also found that the PAEP gene was expressed in endometriosis and in a borderline endometrioid adenoma. As in decidual tissues, PAEP mRNA in endometriosis was abundant in the glandular compartment.
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ABSTRACT
Previous studies from our laboratory have suggested that post-receptor events at the level of β-adrenergic receptor—adenylate cyclase interaction could be altered in myometrium by steroid hormones or pregnancy. In this study, we have addressed this question by performing a direct evaluation of rat myometrial Gs proteins at various stages of pregnancy or 24 h after administration of progesterone. In the 50 000 g myometrial plasma membrane fraction, in the presence of 32 P-labelled NAD, cholera toxin ribosylated three predominant proteins with apparent molecular masses of 42, 47 and 55 kDa. Western blot analysis using the RM/1 antibody recognized the 42 and 47 kDa cholera toxin ADP-ribosylated bands but not the 55 kDa band. Thus, the 42 and 47 kDa immunoreactive bands were interpreted as being the small (Gsα-S) and large (Gsα-L) forms of Gs respectively. With a more purified myometrial plasma membrane fraction (105 000 g ) an additional minor band of 44 kDa could be observed with both techniques. Treatment of late pregnant rats with 5 mg progesterone resulted in a significant increase in both Gsα subunits: +25% and +30% after ADP-ribosylation, +50% and +60% after Western blot analysis for Gsα-L and Gsα-S respectively. Pretreatment with the antiprogestin RU 486 completely suppressed the effect of progesterone, suggesting that the expression of Gsα subunits may be under the control of progesterone. However, changes in the myometrial content of Gs in progesterone-treated rats were not associated with concomitant variations in the steady-state levels of mRNA as demonstrated by Northern blot analysis. These data suggest a post-translational regulation of Gs expression by progesterone. Amounts of ADP-ribosylated Gs showed characteristic changes during the course of pregnancy with a fourfold or threefold increase (P<0·05) on day 15 versus day 12 or delivery respectively. During pregnancy, or after progesterone administration, myometrial alterations of Gs strongly correlated (r=0·913, P<0·01) with the cholera toxin-stimulated adenylate cyclase activity. These findings provide evidence that changes in myometrial amounts of functional Gs i) are controlled by the hormonal status of pregnancy and progesterone and ii) play an important role in the transduction pattern of adenylate cyclase activity during the course of pregnancy.
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ABSTRACT
Passive immunization with a mouse monoclonal antibody against progesterone, designated DB3, blocks pregnancy in several species. We have previously reported that DB3 localizes in the mouse uterine epithelium shortly before normal implantation. This phenomenon is pregnancy dependent and specific for the progesterone antibody. In this study we demonstrate that DB3 is present in the lumen of the uterus 36 h after an i.p. injection; this correlates with the time of maximum antibody reaction on the uterine epithelium. Incubation of DB3 with free progesterone, progesterone-hemisuccinate or progesterone—bovine serum albumin before administration prevented its localization on the epithelium, indicating that the localization requires free progesterone-binding sites and thus probably depends upon progesterone binding. In addition, studies in vitro show that DB3 can effectively bind to progesterone carried by high-affinity progesterone-binding protein purified from coypu plasma. We suggest that specific targeting of DB3 may be through progesterone associated with a progesterone-binding molecule on the membrane of the uterine epithelia. This may be an important part of the mechanism of antibody action against implantation.
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ABSTRACT
We have shown previously that expression of 46 and 54 kDa human myometrial Gαs protein isoforms is increased during gestation and then subsequently decreased during labour. These proteins appear to be coded for by Gαs-Small (with a serine residue at position 72) and Gαs-Large (with a serine residue at position 87) mRNA splice variants respectively. In the study presented here we have used a Gαs cDNA template to generate [32P]cytidine cRNA ribo-probes for use in RNase protection assays, so as to measure total myometrial Gαs mRNA levels in relation to the pattern of expression of Gαs mRNA splice variants during pregnancy and labour. We report that total levels of human myometrial Gαs mRNA remain similar in non-pregnant and pregnant women but are substantially reduced during parturition. Our data also provide strong evidence that alternative splicing of Gαs precursor mRNA has a primary role in regulating expression of Gαs protein isoforms during pregnancy and labour. The inclusion of an additional serine codon in Gαs mRNAs during pregnancy involves a switch in alternative splicing pathways. We speculate that this switch may be due to a change in specificity of splicing factors that are modulated during pregnancy and labour.
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In the mouse, GH-binding protein (GHBP) and GH receptor (GHR) are encoded by a single gene via alternative splicing. We previously demonstrated that the steady-state levels of the GHR and GHBP mRNAs are significantly elevated in mouse liver during pregnancy. Hepatic GHR and GHBP mRNAs are associated primarily with one of two different 5' untranslated regions (5' UTRs), designated 5' UTR Liver1 (L1) and Liver2 (L2). Distinct promoters associated with each of these 5' UTRs have recently been characterized. In the present study, we have investigated the role of transcriptional activation in the pregnancy-induced upregulation of GHR and GHBP mRNAs in liver. We also report on the relative contribution of the 5' UTR L1 and 5' UTR L2 promoters to the hepatic expression of the GHR/GHBP gene in the liver. Our approach was to compare, by ribonuclease protection assay (RPA), GHR/GHBP transcript levels in hepatic nuclear and total cellular RNA samples from virgin and late-pregnant mice. In these RPAs we utilized riboprobes that were complementary to the coding region of GHR/GHBP transcripts, as well as to the two noncoding, alternative first exons 5' UTR L1 and L2. When employing the coding region probe, RPAs revealed that the gestational increase in the levels of nuclear GHR/GHBP transcripts were statistically comparable with the increase in GHR/GHBP transcript levels in total cellular RNA. This finding suggests that enhanced transcriptional activity, rather than increased cytoplasmic half-life, is responsible for the upregulation of GHR/GHBP RNA in the pregnant liver. In RPAs utilizing the noncoding region probes, both nuclear and total cellular GHR/GHBP transcripts associated with 5' UTR L1 were significantly upregulated in late-pregnant as compared with virgin mice. In contrast, the levels of both nuclear and total GHR/GHBP transcripts associated with 5' UTR L2 were comparable between nonpregnant and pregnant animals. Moreover, 5' UTR L2-containing transcripts were present at levels that were only 3-5% of the 5' UTR L1-associated transcripts in the late-pregnant liver. Thus, we conclude that the gestational upregulation of the GHR/GHBP gene in the mouse liver can be ascribed to the significantly enhanced transcriptional activity of the 5' UTR L1 promoter.
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Human labour is associated with increased prostaglandin synthesis within the fetal membranes. We have studied the expression of the two isoforms of the central prostaglandin synthetic enzyme, cyclo-oxygenase (COX-1 and COX-2), in human fetal membranes throughout pregnancy, at mRNA, protein and activity levels. COX-1 mRNA expression was low in human amnion and chorion-decidua and did not change with gestational age. COX-2 mRNA expression in fetal membranes increased with gestational age, with significant up-regulation prior to the onset of labour and in association with labour. Protein concentrations of COX-1 did not change, whilst concentrations of COX-2 increased from the first to the third trimester. COX activity increased with gestational age and in association with labour, although prostaglandin production in fetal membranes collected after labour was reduced, suggesting reduced substrate supply. These data suggest that it is up-regulation of COX-2, rather than of COX-1, which mediates increased prostaglandin synthesis within the fetal membranes at term. Much of the increase in COX-2 expression precedes the onset of labour, suggesting that it is a cause, rather than a consequence, of labour.
