Lauber K, Blumenthal SG, Waibel M, Wesselborg S. Narula J, Haider N, Arbustini E, Chandrashekhar Y. Oxford University Press is a department of the University of Oxford. Genetically engineered mice allow functional assessment of newly identified genes in vivo and provide insights on … 4). Condon JC, Hardy DB, Kovaric K, Mendelson CR. Molecular Regulation of Parturition: A Myometrial Perspective. The remaining whole uterine tissue was washed in 1× PBS and flash frozen for subsequent protein and mRNA analysis. Buki A, Okonkwo DO, Wang KK, Povlishock JT. Red indicates upregulated messenger RNA (mRNA) expression and green indicates downregulated mRNA expression from baseline averages . The association between 17-hydroxyprogesterone caproate use and postpartum hemorrhage. Elevated levels of active caspase 3 were observed in P4-treated mice compared with untreated mice, as determined by Western blot analysis of cytoplasmic extracts of uterine tissue isolated from pregnant mice across gestation, treated with (E11–E18) and without P4 (Fig. In this study, we examined the role that P4 plays in regulating the levels of, and actions mediated by, uterine caspase 3. Our data also predict that active caspase 3 would render the uterine myocyte less likely to contract at earlier gestational time points. We propose that decreased progesterone action during the final days of pregnancy controls the timing of the onset of uterine contractions by removing the anticontractile action of the apoptotic protein caspase 3 locally in the pregnant myometrium. 2018 Jun;18(2):235-243. doi: 10.4314/ahs.v18i2.6. Similar studies in the rat also document increased contractile potential between laboring and nonlaboring myometrial samples [24], and between nonpregnant and pregnant term uteri [25]. Accordingly, we have carried out Affymetrix GeneChip analysis of progesterone effects on gene expression in immortalized human myometrial cells cultured from a patient near the end of pregnancy. Progesterone and several progesterone metabolites are capable of inhibiting uterine contractility. Miller ES, Sakowicz A, Roy A, Liu LY, Yee LM. Shynlova O, Tsui P, Dorogin A, Chow M, Lye SJ. Accompanying the P4-mediated increase in active uterine caspase 3, we also observed an increase in the levels of smooth muscle α- and γ-actin fragmentation (Fig. Considering the conclusion in this research it seems that the combination of the mechanical effect of the pessary by embracing the cervix, keeping the cervical mucus, bending it in a way that the pressure is towards the anterior cervical wall together with the progestative effect which increases the … Progesterone maintains pregnancy by enhancing uterine quiescence . Effects of Progesterone Treatment on Expression of Genes Involved in Uterine Quiescence By Melvyn S. Soloff, Yow-Jiun Jeng, Michael G. Izban, Mala Sinha, Bruce A. Uterine quiescence during pregnancy is essential for the normal development of the fetus. Messenger RNA from uteri of three different gestational series of mice was analyzed for the precursor of caspase 3, pro-caspase 3, using oligonucleotides that primed with equal efficiency. Data are presented as mean ± SD ROD for smooth muscle α- and γ-actin caspase 3-cleaved fragments. Caspase 3 cleavage of both smooth muscle α- and γ-actin results in the release of a 17-kDa and a 25-kDa fragment as a result of a predicted caspase 3-specific consensus site, DXXD [20], found between 156 amino acid (aa) and 158 aa in both smooth muscle α- and γ-actin. 2). Lei K, Chen L, Cryar BJ, Hua R, Sooranna SR, Brosens JJ, Bennett PR, Johnson MR. J Clin Endocrinol Metab. To implant the 8-day, time-release P4 pellet, mice were lightly anesthetized with halothane, and a small incision was made at the nape of the neck before pellet implantation, which was performed using 8 mM trocar supplied by the hormone pellet manufacturer (Innovative Research of America). We sought to identify the consequences of the protease actions of active caspase 3 on components of the uterine contractile architecture, and to determine the molecular mechanisms that regulate active caspase 3 depletion from the pregnant uterus at term. Throughout most of pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) transcriptional activity, whereas spontaneous labor is initiated/facilitated by a concerted series of biochemical events that activate inflammatory pathways and have a negative impact on PR function. Following implantation, more than 95 percent of gestation is spent in Phase 0, uterine quiescence. Elevation in the appearance of caspase 3 cleaved smooth muscle α-actin fragments was observed at E15–19 in the P4-treated animals as compared to control animals, ranging from 4-fold on E15, 1.5-fold on E16, and 2-fold on E17–E19. Messenger RNA from uteri of three different gestational series of mice was analyzed for uterine smooth muscle α- and γ-actin using oligonucleotides that primed with equal efficiency. miRNA. An important action of progesterone during pregnancy is to maintain the uterus in a quiescent state and thereby prevent preterm labor. Abstract. Antibodies raised against the N terminus of both smooth muscle α- and γ-actin identify the 17-kDa fragment and the full-length 42-kDa parent actin. Furthermore, new evidence continues to emerge implicating novel mechanisms that regulate uterine activity during normal and preterm birth. Each value is the mean ± standard error (SE) of triplicate determinations. Renthal NE, Chen CC, Williams KC, Gerard RD, Prange-Kiel J, Mendelson CR. 3). Am J Physiol Endocrinol Metab. doi: 10.1016/j.ejogrb.2009.02.044. Primary antibodies were diluted in 5% milk-1× TBS-T buffer, followed by incubation with horseradish peroxidase-conjugated secondary antibodies diluted in 5% milk-1× TBS-T buffer. Immunoblot analysis of hER-α in immortalized human myometrial cells (HM6) and Amphopack 293 (293) cells. Progesterone function is further reduced by increased expression of CAPs, estrogens, and estrogen receptor-α . The observed decline in circulating progesterone levels in the progesterone-treated animals at E19 and E19IL was expected, as our P4 pellet was designed to release hormone for 8 days in order to permit a decline in circulating P4 levels after E18 similar to that seen at term in the pregnant control animals. Clipboard, Search History, and several other advanced features are temporarily unavailable. Am J Obstet Gynecol MFM. Participation of progesterone in the regulation of uterine quiescence and motility during pregnancy and labor . Reorganization of the γ-actin network has previously been described in the pregnant rat uterus. As can be seen in Figure 2, A and B, both smooth muscle α- and γ-actin displayed elevated levels of caspase 3-specific 17 kDa cleaved fragments, resulting in decreased levels of full-length parent actins at E18. Progesterone, E, and hCG particularly prolong the quiescent periods of the uterus by reducing the frequency of uterine contractions as well as the power of the myoelectrical activity. Progesterone-mediated regulators of uterine quiescence. All data are representative of at least three individual experiments performed in triplicate. These include the possibility of electrophysiological changes in myometrial cells. Fuchs AR, Fuchs F, Husslein P, Soloff MS. Frydman R, Lelaidier C, Baton-Saint-Mleux C, Fernandez H, Vial M, Bourget P. Garfield RE, Sims SM, Kannan MS, Daniel EE. An important action of progesterone during pregnancy is to maintain the uterus in a quiescent state and thereby prevent preterm labor. Laugwitz KL, Moretti A, Weig HJ, Gillitzer A, Pinkernell K, Ott T, Pragst I, Stadele C, Seyfarth M, Schomig A, Ungerer M. Ruetten H, Badorff C, Ihling C, Zeiher AM, Dimmeler S. Lauber K, Bohn E, Krober SM, Xiao YJ, Blumenthal SG, Lindemann RK, Marini P, Wiedig C, Zobywalski A, Baksh S, Xu Y, Autenrieth IBet al. miR-200 family and targets, ZEB1 and ZEB2, modulate uterine quiescence and contractility during pregnancy and labor. … Neither treatment effected expression of recently discovered mediators of uterine quiescence: stat 5b, zeb 1, zeb 2, and 20-alpha-hydroxysteroid dehydrogenase . Progesterone serum levels increase from approximately 25 ng/mL during the midluteal phase to 150 ng/mL at the end of pregnancy. The animal studies were performed in this manner in order to assess the effect of progesterone in vivo on uterine caspase 3 activity without the complications of excessive uterine stretch as a result of delayed labor. NIH In this study, we tested the central hypothesis that progesterone maintains uterine quiescence through regulation of active uterine caspase 3. The role of progesterone in later pregnancy, however, is less clear. Values are expressed as the mean ± standard error (SE) of triplicate samples. Uterine samples were collected from three different gestational series of pregnant mice, ranging from E12 to E19, with or without progesterone treatment. | Epub 2011 Mar 30. pregnancy, uterine quiescence is maintained by elevated circu-lating progesterone (P 4) acting via the progesterone receptor (PR). The final group is the progesterone receptor antagonists, of which mifepristone is the most widely used (see Chapter 40). 6). Progesterone acts through the progesterone receptor to direct physiological adaption of the uterus in preparation and completion of pregnancy. This normally occurs at approximately day 14 of the menstrual cycle and it stimulates the release of an egg from the ovary and the formation of the corpus luteum. However, similar to the pro-caspase mRNA data, caspase 3 activity levels declined toward term. | Recent studies have demonstrated that the contractile state of cardiac, skeletal, and smooth muscle types is controlled by caspase 3 activity [12–14]. In fertile women, directed unilateral transport of immotile sperm-like material through the genital tract to the side bearing the dominant follicle can be assessed using hysterosalping… With the exception of MYLK, E2 + P4 treatment caused a significant (. In this study, we tested the central hypothesis that progesterone maintains uterine quiescence through regulation of active uterine caspase 3. Lack or disorders for maintaining uterine quiescence are progesterone and/or its related metabolites (such as 5 a-pregnane-3,20-dione [5 -DHP], relaxin and parathyroid hormone related peptide [PTHrP]) (Holtan et al . We executed our animal studies in this manner to determine the in vivo effects of elevated P4 on caspase 3 activity and expression, independent of excessive stretch associated with a P4-mediated delay in the onset of labor, which may modify or interfere with uterine caspase 3 signaling. ACTA2 and ACTG2 shall hereafter be referred to as mouse smooth muscle α-actin and mouse smooth muscle γ-actin, respectively. These are particularly useful for termination of early pregnancy, when uterine quiescence is dependent principally on progesterone. Williams KC, Renthal NE, Gerard RD, Mendelson CR. 7). The uterine horn was cleared of all embryonic material and maternal decidua. Condon JC, Jeyasuria P, Faust JM, Wilson JW, Mendelson CR. Epub 2009 Mar 18. Progesterone binds to its nuclear receptor PR and activates genomic pathways that maintain uterine quiescence. During pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) activity, but labor is facilitated by a series of events that impair PR function. Proc Natl Acad Sci U S A. In most mammalian species, but not the human, a precipitous decline in circulating levels of progesterone (P4) heralds the onset of labor [4]. Heat map analysis of differentially regulated genes showing intersample variability of triplicate replicates and lack of differences between 3 doses of progesterone. Various tocolytics are used to suppress uterine contractility in patients in preterm labor. The P4 pellets were designed to release 1.25 mg P4 each day over an 8-day period. Two major isoforms, PR-A and PR-B, exist in humans. [16], high caspase 3 levels were demonstrated in the pregnant rat uterus at midgestation, and were observed to decline at term. *Significantly different (P < 0.005). The intraassay coefficient of variation was 6.4%. Data are presented as mean ± SEM. 2B). Genome-wide transcriptome and cistrome analyses have uncovered new members and novel modifiers of the progesterone signaling pathway. Progesterone, by its maintenance of uterine quiescence through anti-inflammatory effects, has been shown to decrease the risk of PTB in women with the aforemen-tioned risk factors [11, 12]. In the human and the mouse, a decrease in uterine progesterone receptor (PR) action has also been associated with the onset of labor [6–11]. Symbols a, b, and c indicate significant differences (. 7B) in response to elevated P4 levels. Described briefly here, we pulverized the myometrial tissue in liquid nitrogen, and homogenized in ice-cold buffer containing 10 mM Hepes (pH 7.5), 10 mM MgCl2, 5 mM KCL, and 0.1% Triton X-100. The Role of Placental Hormones in Mediating Maternal Adaptations to Support Pregnancy and Lactation. Adequate uterine contractility is involved in the transport of the semen and gametes and in successful embryo implantation, whereas inadequate uterine contractility may lead to ectopic pregnancies, miscarriages, retrograde bleeding and endometriosis (Leyendecker et al., 2004; Bulletti and de Ziegler, 2005; Kissler et al., 2005). Immuoblot of Maxi-K channel α-subunit (KCNMA1) in triplicate samples of cells treated either with estrogen (E) or estrogen plus progesterone (E + P). An important action of progesterone during pregnancy is to maintain the uterus in a quiescent state and thereby prevent preterm labor. Circulating serum samples of maternal progesterone during pregnancy were measured by collecting blood from control (n = 3) and P4-treated (n = 3) pregnant mice at E18–E19 in labor (19IL). . 7A). It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. We suggest that restoration of the full-length actins in the pregnant uterus on the day of labor may be indicative of a reorganized, intact contractile architecture, and signals the onset of increased uterine responsiveness. In contrast, there was no band present in cells that were not transfected (–). progesterone action to maintain uterine quiescence may be indirect by inhibiting expression of contractile genes within the uterus and cervix and blocking the production of chemokines that promote chemotaxis of immune cells [11]. Melvyn S. Soloff, Yow Jiun Jeng, Michael G. Izban, Mala Sinha, Bruce A. Luxon, Susan J. Stamnes, Sarah K. England. Smooth muscle α- and γ-actin full length and caspase 3-cleaved levels were normalized to calponin. The effect of exogenous progesterone treatment on the gestational profile of uterine caspase 3 activity is shown. During quiescence, myometrial contractility is inhibited by a variety of biological substances, including progesterone. Western blot analysis was performed on cytoplasmic extracts isolated from uteri of pregnant mice from E13 to E19IL using antibodies that detect both full-length and caspase 3-specific cleavage fragments of smooth muscle α- and γ-actin (n = 3–4 for each gestational time point). B) The effect of exogenous progesterone treatment on the gestational profile of pro-caspase 3 mRNA in the pregnant mouse uterus is shown. As shown in Figure 1, abundant active caspase 3 was observed in the pregnant mouse uterus from E12 to E17, with the highest levels of active caspase 3 detectable on E13, with an observed decline at E15. Figure 6A demonstrates elevated levels of smooth muscle α- and γ-actin caspase 3-specific cleavage fragments in the P4-treated animals as compared with the control animals. Smooth muscle γ-actin is a specific actin found only in smooth muscle. Progesterone and several progesterone metabolites are capable of inhibiting uterine contractility. | With the exception of BDKRB2, E2 + P4 treatment groups were significantly different (, Effect of estradiol (E2) and progesterone, 100 nmol/L (E2 + P4) on intracellular cyclic adenosine monophosphate (cAMP) concentration, with and without forskolin ([FSK], 20 μmol/L) administration. The incision was sutured after pellet implantation, and all mice tolerated the procedure without complication. *Control samples significantly different from progesterone-treated samples (P < 0.05). Epub 2020 Jan 14. Our data suggest that the effects of activated caspase 3 in the pregnant uterus may not be permanent. Epub 2010 Nov 15. The contractile architecture of the cell has been isolated as a target of caspase 3 protease action. In elegant studies by Shynlova et al. 7A). Progesterone acts through the progesterone receptor to direct physiological adaption of the uterus in preparation and completion of pregnancy. Cytoplasmic extracts were isolated from the pregnant uteri of mice from Day 12 of pregnancy (E12) to E19, and uteri from mice in labor (E19IL). As term approached E19, active caspase 3 levels declined to undetectable levels. *Progesterone-treated samples significantly different from control samples (P < 0.05). Progesterone also may act within the myometrial cell to enhance oxytocin receptor degradation and inhibit oxytocin activation of its receptor at the cell surface (Bogacki, 2002). Progesterone serum levels increase from approximately 25 ng/mL during the midluteal phase to 150 ng/mL at the end of pregnancy. To further characterize the mechanisms controlling uterine caspase 3, we analyzed the pro-caspase 3 promoter for PREs. The homogenate was centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant retained as the cytoplasmic fraction. eCollection 2018. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Frozen uterine tissue specimens (n = 3 for each gestational time point) were pulverized and homogenized in Trizol (Invitrogen, Carlsbad, CA). The appropriate timing of the onset of labor is critical to a successful pregnancy, with potentially devastating consequences resulting to both the mother and child with the onset of preterm labor. The appearance of caspase 3-specific cleaved smooth muscle α- and γ-actins intensifies at E18–E19 (Figs. The relative optical density was obtained by normalizing immunoreactive protein band intensity of active caspase 3 and smooth muscle α- and γ-actin to calponin. *ROD for cleaved and full-length smooth muscle α- and γ-actin is significantly different (P < 0.005). The effect of gestational age on the presence of active caspase 3 proteins in the pregnant mouse uterus. P4-treated mice displayed P4 levels at 101.3 ± 20 ng/ml at E18. 2012 Nov;26(11):1857-67. doi: 10.1210/me.2012-1199. We have also highlighted the 70-fold increase in caspase 3-cleaved smooth muscle γ-actin between E13 and E19, and the 5-fold decrease observed between E19 and E19IL (Fig. Rift Valley fever virus targets the maternal-foetal interface in ovine and human placentas. Primer Express software (Applied Biosystems) was used to design gene-specific primers with a preferable amplicon size of 50–150 bp as follows: mouse smooth muscle α-actin (Acta2) primers (forward, 5′-ggctgttttcccatccatcg-3′; reverse, 5′-cccattccaaccattactccctg-3′), mouse smooth muscle γ-actin (Actg2) primers (forward, 5′-tttaccaccactgctgagaggg-3′; reverse, 5′-tcaaaatccagggcaacatagc-3′), mouse pro-caspase 3 primers (forward, 5′-tctgactggaaagccgaaactc-3′; reverse, 5′-tcccactgtctgtctcaatgccac-3′), and mouse acidic ribosomal phosphoprotein primers (Rplp0) (forward, 5′-acctccttcttccaggcttt-3′; reverse, 5′-cccaccttgtctccagtcttt-3′), which was used as a housekeeping reference gene [18]. Aliquots of cytoplasmic proteins, quantified by colorimetric BCA Protein Assay (Promega, Madison, WI) were separated in gradient polyacrylamide gels (Invitrogen) and transferred onto Hybond-P (Amersham Pharmacia, Piscataway, NJ). P4 treatment increased caspase 3 activity levels over 5-fold on E13 and 4-fold on E14. Statistical analysis was performed in Excel 4.0 (Microsoft, Seattle, WA) to identify the mean and SEM. Amplified mRNAs identified by Q-PCR were normalized to a housekeeping gene, acidic ribosomal phosphoprotein (Rplp0) [17]. One report found no differences observed in the contractile potential between nonpregnant and pregnant myometrium [26]. Immunoblot of AKAP12 in triplicate samples of cells treated either with estrogen (E) or estrogen plus progesterone (E + P). for maintaining uterine quiescence are progesterone and/or its related metabolites (such as 5 a-pregnane-3,20-dione [5 -DHP], relaxin and parathyroid hormone related peptide [PTHrP]) (Holtan et al . The removal of progesterone, that is, progesterone withdrawal , directly precedes progression of phase 1 into phase 2 of parturition. Epub 2019 Apr 28. eCollection 2020 Jan. Amazu C, Ma X, Henkes C, Ferreira JJ, Santi CM, England SK. A previous study indicated that progesterone reduces the mean uterine contraction frequency in singleton pregnancy at high risk for preterm birth. All animals delivered prior to E19.5 (1600 h). It belongs to a group of steroid hormones called the progestogens, and is the major progestogen in the body. A single 66.2-kDa band corresponding in size to hER-α was demonstrated in both cell lines transfected with the vector, pQCXIN-hERα (+). PR-B is the principal transcriptional mediator of progesterone action and maintains uterine quiescence, while PR-A represses the transcriptional activity of PR-B and therefore decreases progesterone responsiveness [3, 5, 6, 19]. In most species, peripheral progesterone levels decline before initiation of labor, and treatments that inhibit progesterone synthesis or action cause termination of pregnancy and/or premature deliveries. 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