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During pregnancy, the uterus shows marked morphological and physiological changes under the regulation of ovarian steroid. To elucidate the molecular cues of these changes, we tried to identify the transcripts differentially expressed in the pregnant rat uterus by using the suppression subtractive hybridization method. Seven independent clones were isolated and one of the up-regulated genes was secreted frizzled-related protein 4 (sFRP4). sFRP4 contains a Wnt-binding domain and belongs to the secreted frizzled protein family whose members are assumed to function as modulators of the Wnt signal. The expression level of sFRP4 mRNA reached a peak in the pregnant uterus on day 12, when uterine decidualization was almost complete in the rat. In situ hybridization histochemistry revealed that sFRP4 transcripts were observed in the decidual cells. In addition, proliferating cell nuclear antigen (PCNA)-positive cells were shown to be overlapped in decidua, suggesting that sFRP4 mRNA expression was accompanied by the late phase of decidual cell proliferation. Moreover, sFRP4 and estrogen receptor-alpha transcripts were co-localized. Furthermore, we analyzed the regulation of sFRP4 by estrogen using 17 beta-estradiol-treated ovariectomized rats. sFRP4 mRNA was detected in the uterus at 48 h after estrogen treatment, especially in endometrial stroma where PCNA-positive cells were also observed. The results in this study led us to the notion that sFRP4 mRNA may be up-regulated after estrogen treatment in the late phase of uterine cell proliferation.
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ABSTRACT
Relative levels of rat ovarian α inhibin (αI) and βA inhibin (βAI) mRNAs were measured during pregnancy by dot-blot hybridization of ovarian poly(A+) RNA. Follicular patterns of αI and βAI expression in contralateral ovaries from the same rats were also studied by hybridization histochemistry. Oligodeoxynucleotide probes specific for porcine αI and βAI were synthesized, 32P end-labelled and used as hybridization probes on dot-blots of ovarian RNA and frozen sections of ovarian tissue from pregnant rats. During pregnancy, levels of αI and βAI mRNAs remained fairly constant from day 7 after mating until parturition and then fell within 16 h post partum. In all ovaries observed, expression of inhibin genes was located in granulosa cells of healthy antral follicles. In general, the strongest signals for αI and βAI mRNAs were obtained in large follicles, with weaker signals in smaller follicles. Follicular patterns of αI and βAI expression during pregnancy were often dissimilar when βI and βAI were compared over a range of follicles. Considerable βI mRNA was detectable in some follicles in which βAI was reduced or undetectable, despite strong signals for both αI and βAI in an adjacent follicle. Essentially, αI mRNA levels were relatively consistent between groups of follicles, whereas βAI levels varied considerably. βAI mRNA was never observed in a follicle in the absence of αI mRNA, indicating that activin production in any follicle occurs in the presence of αI mRNA. Similar patterns of expression were observed in ovaries from pregnant mice. We have shown that expression of αI and βAI inhibin genes is not regulated uniformly within follicles of pregnant rat and mouse ovaries.
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In the mouse, the attachment reaction between the blastocyst trophectoderm and the receptive uterine luminal epithelium occurs at 2200-2300 h on day 4 of pregnancy and is rapidly followed by transformation of stromal cells into decidual cells (decidual cell reaction). This process can also be induced experimentally (deciduoma) by intraluminal oil infusion in the uterus on day 4 of pseudopregnancy. The decidual cell reaction is associated with up- and down-regulation of many genes in a cell-specific manner. Using mRNA differential display, we identified cyclin D3 as one of the genes that is upregulated in the uterus at the sites of blastocyst apposition during the attachment reaction. The levels of expression were low in the morning of days 1-4 as determined by Northern hybridization. In situ hybridization analysis showed that on days 1 and 2, signals were primarily localized in uterine epithelial cells, while signals were detected in both the stromal and epithelial cells on days 3 and 4. In contrast, with the initiation and progression of decidualization on days 5, 6 and 7, the levels of cyclin D3 mRNA were remarkably upregulated in stromal cells both at the mesometrial and the antimesometrial poles. However, on day 8, signals were primarily localized in stromal cells at the mesometrial decidual bed. Implanting blastocysts on these days also expressed cyclin D3 mRNA. In the progesterone-treated delayed implanting mice, the uterine levels of cyclin D3 mRNA were modest at the sites of blastocyst apposition, but were upregulated with the onset of implantation by estradiol-17beta. However, the decidual expression of cyclin D3 mRNA was not dependent on the presence of blastocysts, since increased expression also occurred in experimentally induced deciduoma in the absence of blastocysts. The importance of cyclin D3 in decidualization was further examined in Hoxa-10-deficient mice which show defective decidualization. The expression of cyclin D3 mRNA in Hoxa-10(-/-) uteri on day 5 was severely compromised after application of a deciduogenic stimulus on day 4 of pseudopregnancy. Collectively, the results suggest that cyclin D3 could be important for the process of decidualization.
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ABSTRACT
A synthetic 45-mer oligonucleotide corresponding to part of the ovine endometrial oxytocin receptor cDNA was hybridized to sections of ovine uterus collected from 40 ewes at different stages during the oestrous cycle, the first 3 weeks of pregnancy and seasonal anoestrus. The quantity of oxytocin receptor mRNA was measured as the optical density (OD) value on autoradiographs using image analysis. Message first appeared in the luminal epithelium on days 14–15 of the cycle, increasing to a peak OD of 0·48 at oestrus and decreasing again between days 2 and 5. Oxytocin receptor mRNA in the superficial glands, deep glands and caruncular stroma increased between day 15 and oestrus to peak OD values of 0·17, 0·11 and 0·11 respectively, declining again by day 2 and reaching basal values (OD<0·015) by day 5. Hybridization to the myometrium tended to rise from a mean OD value of 0·01 on days 2–15 to a peak of 0·03±0·01 (mean±s.e.m.) on days 0–1, but the change was not significant. In pregnant ewes there was no detectable oxytocin receptor mRNA on days 14–15 in any region, but hybridization to the luminal epithelium was present in two of three ewes on day 21. In anoestrous ewes oxytocin receptor mRNA concentrations in all areas of the endometrium were approximately half those measured at oestrus.
Optical density readings for oxytocin receptor mRNA in the various uterine compartments were compared with measurements of oxytocin receptors in the same regions as assessed by binding studies using the 125I-labelled oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2 9]-vasotocin (125I-labelled OTA). In the endometrium, receptor mRNA and 125I-labelled OTA binding patterns changed in parallel, and both sets of measurements were significantly correlated (P<0·01). In the myometrium, a significant increase in 125I-labelled OTA binding occurred at oestrus; this was not accompanied by a similar increase in oxytocin receptor mRNA hybridization.
This study helps to confirm that the previously identified cDNA clone is derived from the ovine oxytocin receptor, as patterns of oxytocin receptor mRNA expression in the endometrium closely resembled those of oxytocin binding. Maximum expression and binding both occurred at oestrus, suggesting that regulation of the oxytocin receptor gene in the uterus occurs principally at the transcriptional, rather than at the translational, level. Failure to detect a significant increase in myometrial mRNA expression at oestrus may indicate that the endometrial and myometrial oxytocin receptors are of different isoforms.
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It is suggested that corticotrophin-releasing hormone (CRH) is involved in parturition. We have previously reported the presence of the CRH receptor subtype 1 (CRH R1) in human uterine myocytes. The aim of the present study was to investigate whether expression of the CRH R1 in myometrial tissue changes in pregnancy and labour. We used a quantitative competitive PCR method to measure the mRNA levels of this receptor in non-pregnant and in term pregnant myometrium before and at different stages of labour. The levels of mRNA for the housekeeping gene for glucocerebrosidase (GCB) were also determined. The results were expressed as a ratio of CRH R1 and GCB mRNA levels. We have found that in pregnancy the CRH R1 is down-regulated from a ratio of 0.093+/-0.011 in non-pregnant myometrium to 0.012+/-0.005 (P<0.001) in term non-labouring myometrium. No significant changes were observed in the CRH R1:GCB ratio in tissues sampled within 13 h (0.013+/-0.004) from the start of labour. In summary, normalised levels of CRH R1 are down-regulated in pregnancy and do not change during labour. We speculate that our results do not support a direct role for the CRH R1 receptor in myometrial stimulation.
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ABSTRACT
This study characterized changes in levels of mRNA and protein for endometrial oestrogen receptors (ERs) and progesterone receptors (PRs) during luteolysis and maternal recognition of pregnancy. For cyclic and pregnant ewes, endometrium was collected on days 10, 12, 14, or 16 post-oestrus (4 ewes/day for each status) for the measurement of ER and PR mRNA and protein. The amount of receptor mRNA is expressed in relative units above background, measured from radiographs of dot-blot hybridization of total endometrial RNA with ER and PR cDNAs. At hysterectomy, jugular vein blood samples were collected and assayed for progesterone, total corpus luteum weight was recorded and, in vitro, endometrial oxytocin-stimulated inositol phosphate formation was estimated. In pregnant ewes, plasma progesterone increased gradually between days 10 and 16 (P<0·01), corpus luteum weight was stable at approximately 08 g and oxytocin did not stimulate endometrial formation of inositol phosphates in vitro. In contrast, in cyclic ewes, plasma progesterone decreased from day 10 to day 16 (P<0·01), corpus luteum weight decreased after day 14 to approximately 0·48 g (P=0·05) and oxytocin stimulated an increase of approximately 1300% in the endometrial formation of inositol phosphates on day 16. cDNAs specifically hybridized with 1·6 and 31 kb transcripts for PR mRNA and a 6·5 kb transcript for ER mRNA. In cyclic ewes, the amount of PR mRNA increased from day 10 to maximum levels on days 14–16. The number of PRs decreased from day 10 (225 pmol/mg DNA) to day 12 (0·98 pmol/mg DNA) and then increased from day 14 to day 16 (2·8 pmol/mg DNA). In pregnant ewes, PR mRNA levels were greatest on days 10–12 and decreased by approximately 50% by day 16. In contrast, the number of PRs was relatively unchanged from day 10 to day 16 (1·53 to 103 pmol/mg DNA). In cyclic ewes, the amount of ER mRNA was lowest at day 10 and increased fivefold by day 16. The number of ERs remained relatively unchanged from day 10 to day 14 (607 pmol/mg DNA) and increased by day 16 (1612 pmol/mg DNA). In pregnant ewes, ER mRNA decreased by approximately 80% from day 12 to day 16. Similarly, the number of ERs decreased from day 10 to day 16 (5·41 to 205 pmol/mg DNA). Correlations between ER mRNA and PR mRNA (r=0·68), ERs and PRs (r = 0·50) and ER mRNA and ERs (r=0·50) were high (P<0·01). PR mRNA and PRs, PR mRNA and ERs, and ER mRNA and PRs were not correlated (P>0·1). Pregnancy had the apparent effect of stabilizing the number of endometrial PRs and inhibiting ER production by decreasing both the amount of ER mRNA and ER protein.
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We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) suppresses luteal steroidogenesis in the pregnant rat. We further demonstrated that the peripheral-type benzodiazepine receptor (PBR) and the steroidogenic acute regulatory protein (StAR) play key roles in cholesterol transport leading to steroidogenesis. The purpose of this study was to understand the cellular and molecular mechanisms involved in the suppression of luteal steroidogenesis leading to a fall in serum progesterone levels in GnRH-Ag-treated rats during early pregnancy. Pregnant rats were treated individually starting on day 8 of pregnancy with 5 microgram/day GnRH-Ag using an osmotic minipump. Sham-operated control rats received no treatment. At 0, 4, 8 and 24 h after initiation of the treatment, rats were killed and corpora lutea (CL) were removed for PBR mRNA, protein and radioligand binding analyses, immunoblot 1-D gel analysis of StAR, P450 scc and 3beta-hydroxysteroid dehydrogenase as well as 2-D gel analysis of StAR. The treatment decreased the luteal PBR mRNA expression at all time periods starting at 4 h compared with that in corresponding sham controls. GnRH-Ag also reduced, in the CL, the PBR protein/ligand binding, the StAR protein and P450 scc protein and its activity as early as 8 h after the treatment and they remained low compared with those in corresponding sham controls. The data from 2-D gel studies suggest that the majority of the decrease in StAR protein appears to be in the phosphorylated forms of StAR. Thus, we have demonstrated, for the first time, the presence of PBR and StAR in the pregnant rat CL and that the coordinated suppression of these proteins involved in the mitochondrial cholesterol transport along with P450 scc by GnRH-Ag leads to reduced ovarian steroidogenesis.
Department of Internal Medicine, Affiliated Hospital of Qingdao Medical College, Qingdao University, Qingdao 266003, China
Department of Physiology, Faculty of Medicine, University of Fukui, Fukui 910-1193, Japan
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Department of Internal Medicine, Affiliated Hospital of Qingdao Medical College, Qingdao University, Qingdao 266003, China
Department of Physiology, Faculty of Medicine, University of Fukui, Fukui 910-1193, Japan
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Department of Internal Medicine, Affiliated Hospital of Qingdao Medical College, Qingdao University, Qingdao 266003, China
Department of Physiology, Faculty of Medicine, University of Fukui, Fukui 910-1193, Japan
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Department of Internal Medicine, Affiliated Hospital of Qingdao Medical College, Qingdao University, Qingdao 266003, China
Department of Physiology, Faculty of Medicine, University of Fukui, Fukui 910-1193, Japan
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Department of Internal Medicine, Affiliated Hospital of Qingdao Medical College, Qingdao University, Qingdao 266003, China
Department of Physiology, Faculty of Medicine, University of Fukui, Fukui 910-1193, Japan
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some physiological actions, especially in the regulation of feeding and sleeping. The mid and later stages of pregnancy are characterized by an increase in energy demand ( Ota & Yokoyama 1967 ). In lactating rats, we found a greater number and
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corpus luteum were sufficient to maintain pregnancy in rabbits that were ovariectomized 18 h following copulation ( Allen & Corner 1929 , Corner & Allen 1929 ). With the isolation, bioassay and official naming of progesterone, the first chapter of the
Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, California, USA
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Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, California, USA
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Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, California, USA
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( Fisher 2015 ). In addition, more recent studies have shown that pregnancy complications, particularly those leading to aberrations in fetal growth, have a long-term effect, contributing to metabolic programming of the offspring and thus increasing the
