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University of Michigan Geriatrics Center, Ann Arbor, Michigan, USA
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pregnancy-associated plasma protein-A-1-deficient mice (PKO mice) all show a reduction in mTORC1 activity ( Dominick et al. 2015 , 2017 ). Lower mTOR function diminishes cap-dependent mRNA translation; despite this, slow-aging endocrine mutant mice show
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Nuclear factor kappa B (NFkappaB) is an important intracellular conveyor of extracellular signals and modulates a number of gene responses. Due to the potential significance of NFkappaB in regulating ovarian gene expression, we examined in the rat: (i) whether NFkappaB is activated and developmentally regulated in the corpus luteum (CL) throughout pregnancy; (ii) the proteins forming the NFkappaB complex in luteal cells; and (iii) the role of this transcription factor in luteal function. Western analysis and immunohistochemistry revealed that p65 and p50 were highly expressed throughout pregnancy and were located in both the nucleus and cytoplasm of luteal cells. In addition, because NFkappaB is maintained in the cytoplasm bound to IkappaB, whose phosphorylation allows NFkappaB translocation to the nucleus, we studied the developmental expression of phosphorylated and nonphosphorylated forms of IkappaBalpha. Western analysis revealed that IkappaBalpha was present and phosphorylated throughout pregnancy in the CL whereas by protein/DNA array and electromobility shift assays we found that luteal nuclear extracts bind to an NFkappaB consensus sequence, and that the binding activity decreased along pregnancy. The specific binding was supershifted only by an anti-p65 antibody and not by antibodies against p50, p52, cRel, or RelB. Using day 4 postpartum ovaries, we found higher NFkappaB binding activity in the newly formed CL than in old CL of pregnancy. Furthermore, NFkappaB DNA binding activity was enhanced by prolactin in luteinized granulosa cells. In our first functional study, blockade of NFkappaB/p65 binding to DNA with the sesquiterpene lactone helenalin in luteinized granulosa cells correlated with induction of cell death in a dose-dependent manner. In a second functional study, overexpression of NFkappaB/p65 in luteal cells resulted in inhibition of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD) promoter activity as well as endogenous 20alphaHSD mRNA expression. In summary, we have shown that: (i) NFkappaB is expressed within the CL, primary luteinized granulosa cells, and a rat luteal cell line; (ii) NFkappaB activation within the CL is developmentally regulated in pregnancy, depends on the age of the gland, and can be upregulated by prolactin; (iii) inhibition of NFkappaB/p65 binding to an NFkappaB DNA consensus sequence correlates with induction of cell death in ovarian luteinized granulosa cells; and (iv) overexpression of NFkappaB in luteal cells inhibits 20alphaHSD gene expression. The results further support a role for NFkappaB as a survival factor in the CL.
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ABSTRACT
Material with the immunochemical characteristics of human chorionic gonadotrophin (hCG) is produced by bladder tumour cells in vitro and in vivo. In order to characterize this material further, media were collected from 17 cell cultures (three choriocarcinomas, seven bladder carcinomas and seven 'normal' urothelium). The hCG-like material was compared with pregnancy hCG and purified α- and β-subunits by specific radioimmunoassays. Media were also submitted to affinity chromatography and the fractions further analysed by SDS-PAGE and Western blotting. It was shown that both the neoplastic and normal urothelium produced only free β-subunit-like material. This urothelial 'β-hCG' has the same molecular weight and electrophoretic mobility as that present in the intact hCG of pregnancy.
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ABSTRACT
While the fetal pituitary synthesizes and releases prolactin, it is also produced within the utero-placental unit during pregnancy in women and has been localized in the amnion, chorion and decidua. However, it is not clear whether prolactin is synthesized within all these non-fetal pituitary tissues. We have investigated prolactin production and its gene expression using tissue culture, immunocytochemistry and in-situ hybridization techniques. Prolactin was immunolocalized not only in the decidua but also in amnion and trophoblast cells. In contrast, the in-situ hybridization results showed that silver grains, formed by specific hybridization of a prolactin cDNA probe to prolactin mRNA, were confined to decidual cells of early and term pregnancy. The results from tissue cultures correlated well with those of in-situ hybridization, that is that only the decidua made detectable prolactin, while it was undetectable in the culture medium from trophoblast tissue, irrespective of the stage of pregnancy. This study, for the first time, establishes that only decidualized cells are involved in biosynthesis of prolactin; other prolactin-containing cells in the amnion and trophoblast appear to sequester prolactin, possibly via receptors, suggesting that prolactin may play an important paracrine role within the amnion and syncitio- and cytotrophoblast of the utero-placental unit.
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ABSTRACT
Relaxin is a polypeptide hormone that has a variety of physiological effects both on remodelling of collagen and on uterine contractility. These are most apparent during pregnancy. The sequences of relaxin cDNAs derived from ovaries of late-pregnant random-bred Swiss mice have been established. Multiple subclones obtained from three independent polymerase chain reaction experiments were found to encode relaxins which were identical except at position 11 in the A chain (Ile or Val). All mouse relaxin cDNAs expressed in the ovary during pregnancy had an extra tyrosine inserted prior to the final A chain cysteine residue, a result confirmed by direct sequencing of relaxin peptides. Whilst this tyrosine insertion must have local effects on the folding of the A chain, structure—activity studies will clarify whether it perturbs functional interaction with the relaxin receptor. We have shown that there is a single relaxin gene in the mouse genome, and that expression during pregnancy occurs in the ovary but is not detectable in the placenta, uterus or fetus.
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ABSTRACT
The aim of this study was to establish the gestational- and labour-associated variation in the relative abundance of prostaglandin synthase-1 (PGHS-1) and prostaglandin synthase-2 (PGHS-2) mRNA in ovine placenta (cotyledons). Cotyledons were collected from non-labouring ewes at 40–145 days of gestation (n=25) and from ewes in active labour (145–147 days, n=5). The relative abundance of PGHS-1 and PGHS-2 mRNA transcripts was determined by Northern blot analysis and laser densitometry, using a 2·3 kb sheep and a 1·2kb mouse cDNA probe respectively. Data were expressed as a ratio of PGHS transcript hybridization/18S rRNA hybridization. During pregnancy, the relative abundance of PGHS-2 mRNA increased sevenfold, from 0·19±0·04 at 40–85 days (n=5) to 1·39±0·05 at 140–145 days (n=4) (P<0·01). PGHS-1 mRNA relative abundance did not change significantly (P>0·05) during gestation. Neither PGHS-1 nor PGHS-2 mRNA relative abundance changed significantly in association with labour onset at term (n=5) when compared with the relative abundance observed at 140–145 days (n=4) (P>0·05). The data obtained in this study are consistent with the hypothesis that PGHS-1 is constitutively expressed in ovine placenta during pregnancy and at the time of labour, and that PGHS-2 is induced during the second half of pregnancy. It remains to be established to what extent these two isozymes contribute to the net prostaglandin-forming capacity of the ovine placenta, particularly at the time of labour.
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ABSTRACT
The effects of pregnancy or progesterone dominance on the β-adrenergic responsiveness of the uterus were studied in myometrial membranes from mid-and late-pregnant rats (day 15 and on the 16th h of day 22 of pregnancy respectively) or 24 h after administration of progesterone. Levels of the high (RH)- and low (RL)-affinity states of the β-adrenergic receptor were determined by competition experiments between 125I-labelled cyanopindolol binding and the selective β-agonist isoproterenol. The ratio K L/K H (respective dissociation constants) was determined since it also reflects the degree of formation of the high-affinity state of the β-adrenergic receptor. From day 15 to the 10th h of day 22 of pregnancy, two distinct affinity states were apparent: 80–55% RH (K H=0·31–0·21 μm) and 45–20% RL (K L=14–5 μm) with a ratio of K L/K H of 55–34. In the last 6 h before birth, β-adrenergic receptors underwent uncoupling which was paralleled by decreased responsiveness of myometrial adenylate cyclase to isoproterenol (maximum velocity (V max)=17±3 vs 44±3 fmol cyclic AMP/10 min per mg protein on day 15). At this stage of pregnancy, previous exposure to progesterone resulted in a 1·8-fold increase in 125I-labelled cyanopindolol-binding sites (Bmax) and the reappearance of the high-affinity state (67% RH, K H=0·19±0·04 (s.e.m.) μm, ratio K L/K H=81·1 ± 16·9). These results were reversed in the presence of the antiprogestin RU486 (100% RL, K L=24·6±4·1 μm, 41% reduction of Bmax). Moreover, after progesterone, adenylate cyclase activity was strongly stimulated by isoproterenol (V max=60±12 fmol cyclic AMP/10 min per mg protein vs 17±3 in controls). The data suggest (1) that progesterone may exert a permissive effect on β-adrenergic responsiveness of the pregnant rat myometrium and (2) that at term, both a desensitization mechanism involving uncoupling of β-adrenergic receptors and a decrease in activation of adenylate cyclase lead to a loss of myometrial response to β-agonists.
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ABSTRACT
A radioimmunoassay was developed and validated for the major glycoprotein (gp58) of the mouse mammary tumour virus (MMTV). Using this assay, the expression of gp58 during pregnancy and lactation was found to parallel that for MMTV RNA. In particular, there was a very rapid induction in late pregnancy and a decline in late lactation, although some residual expression persisted well into involution. In cultures of normal mouse mammary tissue, induction of gp58 occurred after a 24-h lag period and began to reach a plateau after 3 days. Both the insulin and prolactin dose—response curves for gp58 resembled those for MMTV RNA; in contrast, the effects of steroid hormones on gp58 and MMTV RNA were disparate. Although progesterone stimulated the RNA, it only slightly increased gp58 levels; however, the presence of cortisol greatly augmented this stimulation, despite the inability of cortisol to induce RNA at physiological concentrations. These results suggest that insulin, prolactin and progesterone act primarily at the level of RNA accumulation in normal mammary epithelium, while cortisol affects some more distal event.
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To elucidate the physiological importance of endothelin-1 (ET-1) in mouse uterus, we investigated quantitative changes in ET-1 mRNA levels in the uterus during the estrous cycle, pregnancy and post-parturient period by use of the real-time PCR technique and we examined the cellular distribution of the ET-1 peptide by use of immunohistochemical techniques. Low and constant mRNA levels were observed in the uterus from cyclic or pregnant mice. However, a significant increase in mRNA levels was found immediately after parturition (day 0 postpartum) which then decreased gradually to a basal level at day 14 postpartum. Discernible immunopositivity was found in myometrial cells as well as in endometrial epithelial cells in the post-parturient uterus. Myometrial cells showed the strongest staining at day 0 postpartum, and some large cells in the myometrial layers, intensely positive for ET-1, were characterized as mast cells. These findings suggest the possibility that in mouse uterus ET-1 may play a role in recovery from the uterine changes caused by pregnancy and parturition.
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ABSTRACT
We have examined the levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 17α-hydroxylase cytochrome P-450 (P-45017α ), aromatase cytochrome P-450 (P-450AROM) and 3β-hydroxy-steroid dehydrogenase (3β-HSD) in rat ovaries throughout the oestrous cycle, during pregnancy and in immature animals treated with pregnant mare serum gonadotrophin (PMSG). Total or poly(A)+-enriched RNA was prepared from adult rat ovaries throughout the oestrous cycle, from immature rat ovaries 24 and 48 h after treatment and from adult rat ovaries on days 10, 14, 17 and 21 of gestation. Expression of the mRNA species was examined by Northern analysis using specific [32P]cDNA probes. During the oestrous cycle P-450scc mRNA of ∼1·9 kb was detected at low levels, while 3β-HSD mRNA of 1·7 kb was in relatively high abundance throughout the oestrous cycle. While P-45017α mRNA of 1·9 kb and P-450AROM of 2·7, 2·2 and 1·7 kb were highly abundant during dioestrus, pro-oestrus and oestrus, the levels of these mRNA species decreased markedly to be nearly undetectable during metoestrus. During pregnancy there was considerably more variation in the expression of the mRNA species examined. Expression of P-450scc mRNA was at low, but detectable, levels until day 14, thereafter expression increased to high levels (day 14–21 of gestation). Levels of P-45017α mRNA on day 10 of gestation were lower than at pro-oestrus during the oestrous cycle and decreased further on days 14 and 17. Expression of 3β-HSD was decreased on day 10, but on days 14, 17 and 21 of gestation high mRNA levels were detectable. Ovarian expression of the three P-450AROM species was dramatically increased between days 14 and 17 of pregnancy, but declined by day 21. In immature rats, P-450scc mRNA was detected at low levels in unstimulated animals and increased markedly after treatment with PMSG, while subsequent treatment with human chorionic gonadotrophin (hCG) had a minimal effect on expression. Expression of P-45017α mRNA was high in unstimulated immature and PMSG-treated rats, but diminished after treatment with hCG. All three P-450AROM mRNA species were undetectable in ovaries from unstimulated immature animals; however, induction of all three was observed in PMSG-treated rats, but this expression decreased to undetectable levels upon subsequent administration of hCG. Further RNA blot analysis utilizing a 3′ portion of the P-450AROM cDNA as probe, which contains an intronic segment instead of the steroid-binding region, revealed that the two mRNA species of lower molecular weight hybridized to this probe. In contrast, only the largest aromatase mRNA band hybridized to a probe specific for the steroid-binding region. These findings indicate that a majority of the P-450AROM mRNA species in rat ovarian tissue during the oestrous cycle and throughout pregnancy represents alternatively spliced products, which presumably lack the ability to encode for aromatase activity. These results indicate that the pattern of steroid secretion in the rat can be explained, in part, by the differential expression of mRNA species encoding the various key steroidogenic enzymes throughout the ovarian cycle, during pregnancy and following administration of gonadotrophins.
