- Email: [email protected]
Dynamic expression of corticotropin-releasing hormone and urocortin in estrogen induced-cholestasis pregnant rat
Reproductive Toxicology 65 (2016) 179–186
Contents lists available at ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Dynamic expression of corticotropin-releasing hormone and urocortin in estrogen induced-cholestasis pregnant rat Fan Zhou a,b , Bingxin Gao a,b , Chunyan Deng a,b , Guiqiong Huang a,b , Ting Xu a,b , Xiaodong Wang a,b,∗ a b
Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu 610041, China Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China
a r t i c l e
i n f o
Article history: Received 21 October 2015 Received in revised form 25 July 2016 Accepted 29 July 2016 Available online 1 August 2016 Keywords: Placenta Corticotropin-releasing hormone Urocortin Rat Cholestasis Regulation Pathogenesis
a b s t r a c t Intrahepatic cholestasis of pregnancy(ICP) is complicated by acute placental-fetal hypoxia. Corticotropinreleasing hormone(CRH) and urocortin(UCN) are vasodilatory regulators of blood flow in the placenta. An ethinylestradiol(EE)-induced cholestasis rat model was reproduced and serum/placental CRH/UCN were detected during 14–21 days of gestation(DG). Maternal serum or placental CRH/UCN levels in the control rats were relatively consistent during 14–21DG. Serum CRH was reduced in the EE-treated rats compared with the control rats at 21DG. Regarding serum UCN, we observed a decrease at 17DG as well as an increase at 21DG in the EE-treated rats compared with the controls. Moreover, we observed a noticeable reduction of placental CRH/UCN expression at 17 or 19DG in the EE-treated rats compared with the control rats. The serum bile acids levels exhibited an inverse correlation with placental CRH/UCN expression. EEinduced cholestasis rats might serve as a good model to further investigate the pathological mechanism underlying CRH/UCN dysregulation in ICP. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Intrahepatic cholestasis of pregnancy (ICP) is a pregnancyspecific disease in the second or third trimester of pregnancy. The incidence of ICP varies between 0.2% and 2% (in most areas of Europe, America and Asia), is dependent on ethnicity and geographic location and is reported to be 5% in the Yangtze River area of China [1,2]. ICP is characterized by maternal pruritus and abnormally increased maternal serum bile acids and liver transaminases levels, which rapidly return to normal after delivery. The etiology and pathogenesis of ICP has not been fully revealed. Several factors, such as genetics, female sex hormones and immunological and environmental factors, are thought to contribute to the onset of ICP. ICP can affect the fetus and lead to preterm birth (spontaneous or iatrogenic), meconium-stained amniotic fluid and intrauterine fetal death. The prenatal mortality of ICP is as high as 2.25% [3].
Abbreviations: ICP, intrahepatic cholestasis of pregnancy; CRH, cotricotrophin releasing hormone; UCN, urocortin; EE, ethinylestradiol; CRH-R, cotricotrophin releasing hormone receptor; IUFD, intrauterine fetal death; ALT, alanine transaminase; AST, aspartate transaminase. ∗ Corresponding author at: Department of Obstetrics and Gynecology, Sichuan University, Renmin South Road, Chengdu, Sichuan, China. E-mail address: wangxd [email protected] (X. Wang). http://dx.doi.org/10.1016/j.reprotox.2016.07.019 0890-6238/© 2016 Elsevier Inc. All rights reserved.
There is evidence that the sudden intrauterine fetal death in ICP was associated with acute intrauterine fetal hypoxia, which might be related to blood flow dysregulation of the utero-placentalfetal unit [3,4]. In normal conditions, the utero-placental-fetal unit has more than two times of the blood oxygen volume reserve compared with those in hypoxia-stressed conditions. The intervillous space, the lobular vascular volume and their blood update speed in the utero-placental-fetal unit are key compensatory elements for hypoxia. However, ICP patients were found to be complicated with acute utero-placental-fetal unit insufficiency—the placental intervillous space was decreased by 30%, and the lobular vascular volume was decreased by 29% [5–8]. Meanwhile, the mechanism of blood flow regulation in the human placenta is complex and is characterized by high blood flow volume, low vascular resistance and lack of neural regulation [9,10]. The local production of circulation-derived vasoactive substances, including prostaglandins, endothelium-derived relaxing factor, vasoactive peptides and the corticotropin-releasing hormone (CRH) family, especially CRH and urocortin (UCN), is important in blood flow regulation and hypoxia compensatory mechanisms in the uteroplacental-fetal unit. These mechanisms increase the ability to cope with acute fetal hypoxia in late pregnancy and intrapartum fetal asphyxia [9–11].
180
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
In our previous study, the maternal serum and placental CRH/UCN expression were down-regulated in ICP patients [12,13]. However, the placental tissues of ICP patients can only be obtained after delivery, and the patients had always received multiple treatments for ICP. Subcutaneous administration of high doses of ethinylestradiol (EE) in rats induces a predictable and reversible cholestasis [14], and the mechanism of cholestasis is associated with alterations in the fluidity and lipid composition of the liver plasma membrane, driving force for the bile flow, Na+ -K+ -ATPase activity and bile salt transports [15–20]. EE-induced cholestasis pregnant rat serves as an excellent model in various studies relevant to the etiology and pathogenesis of ICP [21,22]. There is also evidence about the expression of CRH in decidualized endometrium and maternal decidua of Sprague-Dawley rats, as well as the involvement of locally produced CRH in mouse embryo implantation and pregnancy maintenance [23–25]. Both of them are in agreement with the hypotheses postulated in humans [11]. Thus, to further evaluate the placental CRH/CRH-R1 and UCN/CRH-R2 expression dynamically and explore their roles in the pathological mechanism of sudden intrauterine fetal death in ICP, we reproduced an EE-induced cholestasis pregnant rat model according to the method reported by Crocenzi [21]. Maternal serum CRH/UCN levels and placental CRH/UCN/CRH-R1/CRH-R2 expression were detected in the EE-induced cholestasis pregnant rats and control rats during 14–21 days of gestation.
2. Materials and methods 2.1. Animals Adult female Sprague-Dawley (SD) rats weighing 250–300 g were mated with adult male SD rats weighing 300–350 g and were checked for vaginal plugs, which indicate pregnancy. The day a vaginal plug was observed was recorded as the first day of gestation. All of the rats were maintained on a standard water and diet ad libitum and were housed in a temperature (15–20 ◦ C) and humidity (40%–70%) controlled room under a constant 12 h light/dark cycle. This study was approved by ethical committees at the West China Second University Hospital of Sichuan University. All rats received humane care according to the criteria prepared by the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Pregnant rats were randomly divided into two experimental groups at day 14 of gestation (n = 24). The EE group was subcutaneously administered EE daily (5 mg/kg body weight, propylene glycol as a solvent, the concentration of EE was 1 mg/ml, Sigma Chemical Co. Shanghai, China) for 5 days starting at day 14 of gestation [21]. The control group was subcutaneously administered propylene glycol (5 ml/kg body weight, Sigma Chemical Co. Shanghai, China) daily for 5 days starting at day 14 of gestation. Surgical procedures were performed at 14, 17 (the day after the third dose of EE or solvent was administered), 19 (the day after the last dose of EE or solvent was administered) and 21 days of gestation (3 days post administration). Animals were anesthetized with a single dose of chloral hydrate (2.5 mg/kg body weight, intraperitoneally). A middle abdominal incision was made rapidly. The placentas (exclude membranes) and blood were sampled within 5 min. At the end of each surgical procedure, the animals were sacrificed by exsanguination. The blood samples were collected using an EDTA-containing tube and were centrifuged for 15 min at 1000 × g at 2–8 ◦ C within 30 min. Alanine transaminase (ALT), aspartate transaminase (AST), total bile acids, total bilirubin and direct bilirubin levels were determined using a fully automatic biochemical analyzer (Siemens Electrical Apparatus Ltd., Germany). Rat maternal serum CRH or
UCN levels were detected using a specific, commercially available ELISA kit (CUSABIO Life Science, China) according to the manufacturer’s instructions. The pre-pregnancy bodyweight (recorded on the day before observation of vaginal plug), maternal bodyweight gain, number of fetal rats/litter, placenta weight (all placentas per litter), fetal rat’s body weight (all foetuses per litter), meconium stained amniotic fluid and intrauterine fetal death were observed and recorded. The significance of meconium stained amniotic fluid in rats has not been fully elucidated. Studies from various types of animals indicated that the increase of utero defecation induced by fetal hypoxia, impaired clearance of meconium and decreased fetal swallow all contribute to meconium stained amniotic fluid [26]. 2.2. Real-time PCR Three placentas were randomly selected from each pregnant rats (except one rat in the control group only had three placentas) using simple random sampling. Total RNA was prepared from placental tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The purity and integrity of the RNA were evaluated by gel electrophoresis. RNA (200 g), quantified by measuring the absorbance at optical density 260 (the ratio of optical density 260/optical density 280 around 1.9-2.0 means high purity of RNA), was reverse transcribed to cDNA using RNase Reverse Transcriptase (Life Technologies, Shanghai, China). All of the primers and probes used for real-time PCR were synthesized by Corelab Biotech (Chengdu, China). The primers were as follows: CRH, F: 5 -CAGCCGTTGAATTTCTTG-3 , R: 5 -GACTTCTGTTGAGGTTCC-3 ; UCN, F: 5 -CAACGACGAGACGACC-3 , R: 5 -ACTTGCCCACCGAATC3 ; CRH-R1, F: 5 -GTGCCTGAGAAACATCAT-3 , R: CRH-R2, F: 5 5 -ACCGAACATCCAGAAGAA-3 ; TGGTGACTTAGTGGACTA-3 , R: 5 -GAAGAGCATGTAGGTGAT-3 ; and -actin, F: 5 -CTGGAGAAGAGCTATGAG-3 , R: 5 -ATGATGGAATTGAATGTAGTT-3 . The probes were as follows: CRH, 5 -CAGCAACCTCAGCCGATTCT UCN, 5 -CCTCACCTTCCACCTGCTG-3 ; CRH-R1, 3; 5 -CACTGGAACCTCATCTCGGC-3 ; CRH-R2, 5 CATCATCCTCGTGCTCCTCATC-3 ; and -actin, 5 ACGGTCAGGTCATCACTATCG-3 . PCR was performed in a reaction mixture consisting of FastStart Universal Probe Master (Roche, Shanghai, China) 10 l, cDNA 5 l, primer 2 l, Taqman probe 1 l and ddH2 O 2 l. PCR program was 95 ◦ C for 10 min; 95 ◦ C for 15 s, 60 ◦ C for 1 min (45 cycles) and 72 ◦ C for 7 min followed by 4 ◦ C (ABI 7900HT, Waltham, MA, USA). All samples were run in triplicate in 96-well optical PCR plates. For analysis, quantitative amounts of mRNA levels were standardized against the internal reference -actin. 2.3. Western blotting Placental tissues (100 mg) and 400 ml lysis buffer (with phenylmethane sulfonyl fluoride) were added to a homogenizing device and were homogenized for 30 min at 4 ◦ C. The homogenate was centrifuged at 12, 000 × g for 5 min at 4 ◦ C, and the supernatant was collected. A bicinchoninic acid assay (Thermo ScientificTM PierceTM , Waltham, MA, USA) was used to determine the protein concentration. Then, samples were separated on an SDS-polyacrylamide gel, and the protein (50 g) was electrophoretically transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, Massachusetts, USA). Next, the membranes were blocked in TBS-T (Tris-buffered saline containing 0.05% Tween 20) containing 5% non-fat dried milk for 2 h at room temperature and incubated with anti-CRH antibodies (Abcam, Shanghai, China) (1:10, 000), anti-UCN antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:800), anti-CRH-R1 antibodies (Proteintech, Rosemont, IL, USA) (1:350) or anti-CRH-R2 antibodies (Acris Antibodies,
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
Rockville, MD, USA) (1:800) overnight at 4 ◦ C. The membranes were washed 3 times with TBS-T and incubated with 1:1, 000 anti-rabbit horseradish-peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 2 h. The ratio of the band intensities to -actin was obtained to quantify the relative protein expression levels and control for sampling errors. 2.4. Statistical analysis Stata 12.1 (Statacorp, College Station, TX, USA) was used for statistical analysis. Pauta criterion (data greater than + 3 or less than − 3 will be rejected) were used to define and reject the outliers. Continuous variables with normal distribution, including the pre-pregnancy weight, maternal body weight gain, number of fetal rats/litter, fetal body weight, placental weight, maternal serum levels of ALT, AST, total bile acids, total bilirubin, direct bilirubin, CRH and UCN, placental CRH/UCN protein expression or placental CRH-R1/CRH-R2 protein expression, were presented as the means ± standard deviation (S.D.) on a litter basis (fetuses were nested within litters). Two way ANOVA was used with the expression in each litter as response, and treatment and gestation day as the main interact effects without considering interaction. Non-normally distributed data was expressed as medians (M) and inter-quartile ranges (IQR) on a litter basis, including mRNA levels of placental CRH, UCN, CRH-R1 or CRH-R2, and the Friedman’s test followed by Dunnett’s Post-hoc test was performed. Fisher’s Exact Test was performed to analyze the differences of meconium stained amniotic fluid or IUFD rates between the EE and control groups on a litter basis (fetuses were nested within litters). A correlation analysis was examined by using Pearson’s correlation (between blood parameters and serum/placental CRH/UCN expression) or Spearman’s correlation (between fetal outcomes and blood parameters or serum/placental CRH/UCN expression). For all statistical testing, a two-tailed P value of <0.05 was considered statistically significant. 3. Results 3.1. Maternal/fetal characteristics and maternal serum biochemical parameters Forty-eight pregnant rats were enrolled in this study, including 24 pregnant rats in the EE group and 24 rats in the control group. The pre-pregnancy weight, maternal bodyweight gain, number of fetal rats/litter and placenta weight exhibited no statistical differences between the two groups (Fig. 1A–C, E). At gestation day 14, 17 and 19, the fetal rats’ body weight demonstrated no significant difference between the EE and control groups. At gestation day 21, the fetal rats’ body weight was lower compared with the control group (Fig. 1D). At day 14 of gestation, the maternal serum ALT, AST, total bile acids, total bilirubin and direct bilirubin levels had no significant differences between the EE and control groups. At 17 and 19 days of gestation, maternal serum ALT and total bile acids levels in the EE group were significantly higher compared with the control group; no significant differences in the AST, total bilirubin or direct bilirubin levels were observed. At 21 days of gestation, the maternal serum AST, total bile acids, total bilirubin and direct bilirubin levels in the EE group were significantly higher than the control group, whereas no significant differences in ALT levels were observed (Fig. 1F–J). 3.2. Pregnancy outcomes In the EE group, no cases of meconium stained amniotic fluid and IUFD were found at day 14 of gestation; at day 17 of gestation,
181
the meconium stained amniotic fluid rate was 16.67% (1/6) and the IUFD rate was 33.33% (2/6); at day 19 of gestation, 16.67% (1/6) had meconium stained amniotic fluid and 33.33% (2/6) had IUFD; meconium stained amniotic fluid and IUFD accounted for 50% (3/6) and 50% (3/6) at day 21 of gestation. In the control group, no cases of meconium stained amniotic fluid and IUFD were found at gestation day 14, 17, 19 or 21. There are no statistical differences in meconium stained amniotic fluid rate and IUFD rate between the EE and control groups. 3.3. Maternal serum CRH and UCN levels Maternal serum CRH levels in the control group were relatively consistent throughout the experiment (Fig. 2A). There was no statistically significant change in maternal serum CRH levels in the EE group throughout the experiment (Fig. 2A). At day 21 of gestation, the difference in maternal serum CRH levels between the EE and control groups achieved statistical significance (Fig. 2A). Maternal serum UCN levels in the control group were relatively consistent throughout the experiment (Fig. 2B). We observed significant differences in maternal serum UCN levels in the EE group throughout the experiment, and the UCN levels at gestation day 17 were lower compared with gestation day 21 (Fig. 2B). At gestation day 17 or 21, the difference in maternal serum UCN levels between the EE and control groups achieved statistical significance (Fig. 2B). 3.4. Placental CRH mRNA and UCN mRNA expression Placental CRH mRNA expression in the control group exhibited no statistically significant change throughout the experiment (Fig. 3A). In the EE group, placental CRH mRNA levels at gestation day 17 and 19 were lower compared with that at gestation day 14 or 21. Meanwhile, at gestation day 17 and 19, placental CRH mRNA levels in the EE group were lower compared with those in the control group (Fig. 3A). Placental UCN mRNA expression in the control group was relatively consistent throughout the experiment (Fig. 3B). In the EE group, placental UCN mRNA expression at gestation day 17 and 19 were lower compared with gestation day 14 or 21. Moreover, at gestation day 17 and 19, placental UCN mRNA levels in the EE group were lower compared with those in the control group (Fig. 3B). 3.5. Placental CRH-R1 mRNA and CRH-R2 mRNA expression Placental CRH-R1 mRNA expression in the EE or control groups was relatively consistent throughout the experiment (Fig. 3C). Placental CRH-R1 mRNA expression demonstrated no significant differences between the EE and control groups at gestation day 14, 17, 19 or 21 (Fig. 3C). There was no statistically significant change in placental CRH-R2 mRNA expression in the EE or control groups throughout the experiment (Fig. 3D). Additionally, at gestation day 17, placental CRH-R2 mRNA levels in the EE group were lower compared with that in the control group (Fig. 3D). 3.6. Placental CRH and UCN expression Placental CRH expression (21 KDa) in the control group exhibited no obvious change throughout the experiment (Fig. 4A). In the EE group, placental CRH levels demonstrated significant differences among gestation day 14, 17, 19 and 21, and those at gestation day 17 or 19 was lower compared with gestation day 14. At gestation day 17 and 19, the differences in placental CRH levels between EE and control groups achieved statistical significance (Fig. 4A). Placental UCN (13 KDa) demonstrated no significant differences in the control group throughout the experiment (P > 0.05) (Fig. 4B).
182
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
Fig. 1. Baseline information and maternal serum biochemical parameters in the EE-treated and control rats. DG, days of gestation; ALT, Alanine transaminase; AST, Aspartate transaminase. Data were expressed as the means ± S.D. after rejected the outliers according to the Pauta criterion (data greater than + 3 or less than − 3 will be rejected) (n = 6). * P < 0.05. The rejected outliers were as follows: two outliers in the ALT levels, two outliers in the AST levels, one outlier in the total bilirubin levels and one outlier in the direct bilirubin levels.
In the EE group, placental UCN levels exhibited significant differences among gestation day 14, 17, 19 and 21, and those at gestation day 17 or 19 was lower compared with gestation day 14 or 21. Moreover, at gestation day 17 and 19, the differences in placental UCN levels between the EE and control groups achieved statistical significance (Fig. 4B). 3.7. Placental CRH-R1 and CRH-R2 expression Our data indicated that placental CRH-R1 expression (48 KDa) in the control group had no obvious change throughout the experiment. In the EE group, placental CRH-R1 levels demonstrated
significant differences among gestation day 14, 17, 19 and 21, and those at gestation day 19 or 21 was lower than gestation day 14 or 17. At gestation day 19 and 21, the differences in placental CRHR1 levels between the EE and control groups achieved statistical significance (Fig. 4C). Our data indicated that placental CRH-R2 expression (21 KDa) in the control group had no significant differences throughout the experiment. There were no statistically significant changes in placental CRH-R2 levels in the EE group throughout the experiment. No significant difference was noticed between the EE and control groups at gestation day 14, 17, 19 or 21 (Fig. 4D).
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
183
Fig. 2. Maternal serum CRH and UCN levels during gestation day 14–21 in the EE-treated and control rats. Data were expressed as the means ± S.D. after rejected the outliers according to the Pauta criterion (data greater than + 3 or less than − 3 will be rejected) (n = 6). * P < 0.05. The rejected outliers were as follows: one outlier in the CRH levels and two outliers in the UCN levels.
3.8. Relationship between maternal biochemical parameters, CRH/UCN expression and fetal outcomes Pearson’s correlation analysis indicated that, in the EE group, maternal serum UCN levels showed a significant correlation with AST levels throughout gestation day 14–21. Placental CRH or UCN expression in the EE group exhibited a significant negative correlation with serum total bile acids levels throughout gestation day 14–21 (Table 1).
4. Discussion Our results revealed the dynamic expression of serum and placental CRH/UCN during gestation day 14–21 in pregnant rats with EE-induced cholestasis. In EE-induced cholestasis pregnant rats, both maternal serum and placental CRH levels were decreased compared with those in the control rats, and the reduction of maternal serum CRH occurred after the decrease of placental CRH. Meanwhile, the maternal serum and the placental UCN expression in the EE-treated rats were reduced simultaneously at gestation day 17 compared with those in the control rats. The maternal serum
UCN levels in the EE-treated rats showed a significant increase and higher than the control rats at gestation day 21. Placental UCN expression in the EE-treated rats remained lower than the control rats at gestation day 19. Our data also revealed a negative correlation between maternal serum total bile acids levels and the expression of placental CRH or UCN in pregnant rats with EE-induced cholestasis. Our study successfully reproduced an EE-induced cholestasis pregnant rat model. The maternal serum liver enzymes, bilirubin and total bile acids levels were elevated in EE administrated pregnant rats. Meanwhile, we noticed decreases of serum total bile acids, total bilirubin or direct bilirubin levels in the control rats at gestation day 21 when compared with gestation day 19. The drop of blood parameters during late pregnancy in rats due to hemodilution was also reported by several studies [27–29]. Meanwhile, no significant differences were found in meconium stained amniotic fluid rate and IUFD rate between the EE and control groups. In 1955, Guillemin [30] first described the hypothalamicderived hormone CRH, and the earliest report of UCN was made by Vaughan [31] in rat brain tissues in 1995. Since then, numerous studies had examined the biochemical properties of CRH and
184
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
Fig. 3. mRNA expression of placental CRH, UCN, CRH-R1 and CRH-R2 during gestation day 14–21 in the EE-treated and control rats. DG, days of gestation. Data were expressed as the medians (M) and inter-quartile ranges (IQR) after rejected the outliers according to the Pauta criterion (data greater than + 3 or less than − 3 will be rejected) (n = 6). * P < 0.05. The rejected outliers were as follows: two outliers in the CRH mRNA expression, two outliers in the UCN mRNA expression, two outliers in the CRH-R1 mRNA expression and one outlier in the CRH-R2 mRNA expression.
Fig. 4. Protein expression of placental CRH, UCN, CRH-R1 and CRH-R2 during gestation day 14–21 in the EE-treated and control rats. Data were expressed as the means ± S.D. after rejected the outliers according to the Pauta criterion (data greater than + 3 or less than − 3 will be rejected) (n = 6). * P < 0.05. The rejected outliers were as follows: two outliers in the CRH expression, three outliers in the UCN levels, one outlier in the CRH-R1 expression and two outliers in the CRH-R2 expression.
Table 1 Correlation analysis between maternal biochemical parameters, CRH expression, UCN expression and fetal outcomes. r Maternal serum CRH (ng/ml)§ Maternal serum UCN (pg/ml)§ Placental CRH§ Placental UCN§ Meconium-stained amniotic fluid rate (%)# IUFD rate (%)#
ALT (U/L)§ −0.04 0.36 −0.10 −0.21 0.80 0.80
AST (U/L)§ 0.09 0.44* −0.18 −0.08 1.00 1.00
Serum bile acids (mmol/L)§ 0.28 −0.07 −0.50* −0.74* 0.40 0.40
Meconium-stained amniotic fluid rate (%)# −0.40 0.80 −0.20 0.80 – –
IUFD rate (%)# −0.40 0.80 −0.20 0.80 – –
Note: Data were analyzed by § Pearson’s correlation or # Spearman’s correlation. CRH, Cotricotrophin releasing hormone; UCN, Urocortin; ALT, Alanine transaminase; AST, Aspartate transaminase; IUFD, Intrauterine fetal death. * P < 0.05.
UCN, especially for their roles in blood flow regulation. Additionally, CRH and UCN from humans and rats are highly homologous,
their amino acids sequences share over 90% homology [9,31]. The blood flow of the utero-placental unit in rats is regulated by vascu-
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
lar resistance, and the process is efficient [32]. Pregnant rats with EE-induced cholestasis might serve as a suitable model for research on dynamical dysregulated expression of placental CRH or UCN in ICP. In this study, the maternal serum CRH levels in pregnant rats were relatively consistent throughout gestation day 14–21. The stabilized maternal serum CRH levels in pregnant rats are inconsistent with those of humans, which are significantly increased in the third trimester of pregnancy and are increased further with the onset of labor [12]. One potential explanation for our findings is that, as lower animals, the maternal serum and placental CRH in rats were kept at a high level during late pregnancy to guarantee enough blood flow supply to multiple placentas. In parallel, the maternal serum UCN levels exhibited no obvious change during gestation day 14–21 in the control rats. The consistent expression of serum UCN levels in rats is concordant with those in humans during late pregnancy [10,13]. In EE-treated pregnant rats, the maternal serum CRH levels were lower than those in the control rats at gestation day 21; the maternal serum UCN levels were lower than those in the control rats at gestation day 17, and were higher than the control rats at gestation day 21. Moreover, the placental CRH and UCN expression in the control rats were also relatively consistent throughout gestation day 14–21. In EE-treated pregnant rats, placental CRH and UCN expression were decreased at gestation day 17 and 19 when compared with those in the control rats. The reduction of placental CRH or UCN expression was not completely consistent with the decrease of maternal serum CRH or UCN levels in pregnant rats with EEinduced cholestasis during gestation day 14–21. The decrease of maternal serum CRH (gestation day 21) was after the placental CRH reduction (gestation day 17) in EE-treated rats. The reduction of maternal serum UCN levels and placental UCN expression in the EE-treated rats (compared with the control rats) both occurred at gestation day 17, but the serum UCN levels returned to normal whereas placental UCN remained lower than the control rats at gestation day 19. At gestation day 21, the placental UCN levels in EE-treated rats exhibited no statistical difference compared with the control rats, whereas maternal serum UCN levels in EE-treated rats were higher than the control rats. Maternal serum CRH or UCN levels could not fully reflect the placental CRH or UCN expression in pregnant rats with EE-induced cholestasis. Our results also indicated that the maternal serum total bile acids levels had an inverse correlation with placental CRH/UCN expression in pregnant rats with EE-induced cholestasis. The elevated serum total bile acids levels in ICP are associated with the risk of complications to the fetus, and serve as an indicator of the severity classification of ICP (maternal serum bile acids levels exceeded 40 micromoles/L would be considered as severe ICP) [1,33,34]. There might be some relationships between the placental CRH/UCN expression and the severity of cholestasis in pregnant rats. Meanwhile, there is evidence that the elevated bile acids in bile duct resection (BDR) rat model could suppress hypothalamic CRH expression [35,36], and this process is mediated by hypothalamic bile acids signaling [37]. However, the hypothalamic CRH might contribute comparatively less to serum CRH levels as there is no evidence that central CRH could be detected in rat serum. There is no literature about the direct role of bile acids on placental CRH/UCN expression. Regarding mRNA expression, placental mRNA expression of CRH or UCN exhibited no obvious change in the control rats, which was consistent with protein expression. Placental CRH mRNA or UCN mRNA expression in pregnant rats with EE-induced cholestasis was significantly lower than those in the control rats at gestation day 17 or 19. We also detected mRNA and protein expression of placental CRH-R1 and CRH-R2 in pregnant rats during gestation day 14–21.
185
There was no significant change in placental CRH-R1 or CRHR2 expression in the control rats throughout the experiment. In EE-treated rats, placental CRH-R1 protein levels were reduced at gestation day 19 and 21 compared with the control rats. Placental CRH-R2 demonstrated no statistical difference between the EE-treated and control rats throughout the experiment. Placental mRNA expression of CRH-R1 and CRH-R2 mRNA in the EE-treated and control rats were relatively consistent throughout the experiment. CRH-R2 mRNA expression in pregnant rats with EE-induced cholestasis was lower than the control rats at gestation day 17. Taken together, these data manifested the dynamic expression of maternal serum CRH/UCN, placental CRH/UCN and placental CRH-1/CRH-R2 in pregnant rats with EE-induced cholestasis, which can not be achieved in ICP. EE-induced cholestasis pregnant rats were accompanied by the reduction of serum and placental CRH or UCN expression, and the maternal serum CRH/UCN levels could not fully reflect the placental CRH/UCN expression. The maternal serum total bile acids levels exhibited an inverse correlation with placental CRH/UCN expression. EE-induced cholestasis pregnant rats might serve as a good model to further investigate the pathological mechanism underlying CRH/UCN dysregulation in ICP. Conflict of interest The authors declare that there is no conflict of interest. Funding This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT0935). Acknowledgments We wish to express our appreciation to the editor and the anonymous reviewers for your insightful comments and suggestions, which have greatly improved the manuscript. References [1] C. Williamson, V. Geenes, Intrahepatic cholestasis of pregnancy, Obstet. Gynecol. 124 (2014) 120–133, http://dx.doi.org/10.1097/AOG. 0000000000000346. [2] X. Liu, J. He, Pay more attention to standardizing the diagnosis and treatment of intrahepatic cholestasis of pregnancy, Zhonghua Fu Chan Ke Za Zhi 46 (2011) 321–323. [3] A.P. Kenyon, J.C. Girling, Obstetric Cholestasis, RCOG Green-top guidelines No 43, 2011, 10.3748/wjg.v19.i43.7639.6. [4] R. Reid, K.J. Ivey, R.H. Rencoret, B. Storey, Fetal complications of obstetric cholestasis, Br. Med. J. 1 (1976) 870–872, http://dx.doi.org/10.1136/bmj.1. 6014.870. [5] D. Joshi, A. James, A. Quaglia, R.H. Westbrook, M.A. Heneghan, Liver disease in pregnancy, Lancet 375 (2010) 594–605, http://dx.doi.org/10.1016/S01406736(09)61495-1. [6] A.L. Costoya, E.A. Leontic, H.G. Rosenberg, M.A. Delgado, Morphological study of placental terminal villi in intrahepatic cholestasis of pregnancy: histochemistry, light and electron microscopy, Placenta 1 (1980) 361–368. [7] K. Kaar, P. Jouppila, J. Kuikka, H. Luotola, J. Toivanen, A. Rekonen, Intervillous blood flow in normal and complicated late pregnancy measured by means of an intravenous 133Xe method, Acta Obstet. Gynecol. Scand. 59 (1980) 7–10. [8] M. He, Z. Liu, X. Wang, Decreased volume of placental lobular villi vessels in patients with intrahepatic cholestasis of pregnancy, Sichuan Da Xue Xue Bao Yi Xue Ban 42 (2011) 797–801 http://europepmc.org/abstract/MED/ 22332545. [9] F. Petraglia, A. Imperatore, J.R.G. Challis, Neuroendocrine mechanisms in pregnancy and parturition, Endocr. Rev. 31 (2010) 783–816, http://dx.doi.org/ 10.1210/er.2009-0019. [10] A.L. Boura, W.A. Walters, M.A. Read, I.M. Leitch, Autacoids and control of human placental blood flow, Clin. Exp. Pharmacol. Physiol. 21 (1994) 737–748. [11] P.P.L.M. Pepels, M.E.A. Spaanderman, J. Bulten, P.B.A.M. Smits, A.R.M.M. Hermus, F.K. Lotgering, C.G.J. Sweep, Placental urocortins and CRF in late gestation, Placenta 30 (2009) 483–490, http://dx.doi.org/10.1016/j.placenta. 2009.03.008.
186
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
[12] F. Zhou, M.M. He, Z.F. Liu, L. Zhang, B.X. Gao, X.D. Wang, Expression of corticotrophin-releasing hormone and its receptor in patients with intrahepatic cholestasis of pregnancy, Placenta 34 (2013) 401–406, http://dx. doi.org/10.1016/j.placenta.2013.02.006. [13] F. Zhou, L. Zhang, Q. Sun, X.D. Wang, Expression of urocortin and corticotrophin-releasing hormone receptor-2 in patients with intrahepatic cholestasis of pregnancy, Placenta 35 (2014) 962–968, http://dx.doi.org/10. 1016/j.placenta.2014.07.019. [14] T.A. Heikel, G.H. Lathe, The effect of oral contraceptive steroids on bile secretion and bilirubin Tm in rats, Br. J. Pharmacol. 38 (1970) 593–601. [15] A.J. Schreiber, F.R. Simon, Estrogen-induced cholestasis: clues to pathogenesis and treatment, Hepatology 3 (1983) 607–613. [16] E.L. Forker, The effect of estrogen on bile formation in the rat, J. Clin. Invest. 48 (1969) 654–663, http://dx.doi.org/10.1172/JCI106023. [17] E.B. Keefee, B.F. Scharschmidt, N.M. Blankenship, R.K. Ockner, Studies of relationship among bile flow, liver plasma membrane NaK-ATPase, and membrane microviscosity in the rat, J. Clin. Invest. 64 (1979) 1590–1598, http://dx.doi.org/10.1172/JCI109620. [18] R.A. Davis, F.J. Kern, R. Showalter, E. Sutherland, M. Sinensky, F.R. Simon, Alterations of hepatic Na+,K+-atpase and bile flow by estrogen: effects on liver surface membrane lipid structure and function, Proc. Natl. Acad. Sci. U. S. A. 75 (1978) 4130–4134. [19] R. Bossard, B. Stieger, B. O’Neill, G. Fricker, P.J. Meier, Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver, J. Clin. Invest. 91 (1993) 2714–2720, http://dx.doi.org/10.1172/ JCI116511. [20] R. Guillemin, B. Rosenberg, Humoral hypothalamic control of anterior pituitary: a study with combined tissue cultures, Endocrinology 57 (1955) 599–607, http://dx.doi.org/10.1210/endo-57-5-599. [21] F.A. Crocenzi, E.J. Sanchez Pozzi, J.M. Pellegrino, C.O. Favre, E.A. Rodriguez Garay, A.D. Mottino, R. Coleman, M.G. Roma, Beneficial effects of silymarin on estrogen-induced cholestasis in the rat: a study in vivo and in isolated hepatocyte couplets, Hepatology 34 (2001) 329–339, http://dx.doi.org/10. 1053/jhep.2001.26520. [22] X. Song, A. Vasilenko, Y. Chen, L. Valanejad, R. Verma, B. Yan, R. Deng, Transcriptional dynamics of bile salt export pump during pregnancy: mechanisms and implications in intrahepatic cholestasis of pregnancy, Hepatology 60 (2014) 1993–2007, http://dx.doi.org/10.1002/hep.27171. [23] I. Athanassakis, V. Farmakiotis, I. Aifantis, A. Gravanis, S. Vassiliadis, Expression of corticotrophin-releasing hormone in the mouse uterus: participation in embryo implantation, J. Endocrinol. 163 (1999) 221–227, JOE03388[pii]. [24] A. Makrigiannakis, E. Zoumakis, S. Kalantaridou, C. Coutifaris, A.N. Margioris, G. Coukos, K.C. Rice, A. Gravanis, G.P. Chrousos, Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance, Nat. Immunol. 2 (2001) 1018–1024, http://dx.doi.org/10.1038/ni719. [25] A. Makrigiannakis, A.N. Margioris, C. Le Goascogne, E. Zoumakis, G. Nikas, C. Stoumaras, A. Psychoyos, A. Gravanis, Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus, Life Sci. 57
(1995) 1869–1875, http://dx.doi.org/10.1016/0024-3205(95)02167-H. [26] S.N. Ahanya, J. Lakshmanan, B.L.G. Morgan, M.G. Ross, Meconium passage in utero: mechanisms, consequences, and management, Obstet. Gynecol. Surv. 60 (2005) 45–56, http://dx.doi.org/10.1097/01.ogx.0000149659.89530.c2, quiz 73–74. [27] E.P.C.T. de Rijk, E. van Esch, G. Flik, Pregnancy dating in the rat: placental morphology and maternal blood parameters, Toxicol. Pathol. 30 (2002) 271–282, http://dx.doi.org/10.1080/019262302753559614. [28] J.C. Kim, H.I. Yun, K.H. Lim, J.E. Suh, M.K. Chung, Haematological values during normal pregnancy in Sprague-Dawley rats, Comp. Haematol. Int. 10 (74) (2000) 74–79, http://dx.doi.org/10.1007/s0058000700112000. [29] J.B. LaBorde, K.S. Wall, B. Bolon, T.S. Kumpe, R. Patton, Q. Zheng, R. Kodell, J.F. Young, Haematology and serum chemistry parameters of the pregnant rat, Lab. Anim. 33 (1999) 275–287, http://dx.doi.org/10.1258/ 002367799780578156. [30] J. Vaughan, C. Donaldson, J. Bittencourt, M.H. Perrin, K. Lewis, S. Sutton, R. Chan, a V Turnbull, D. Lovejoy, C. Rivier, Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor, Nature 378 (1995) 287–292, http://dx.doi.org/10.1038/378287a0. [31] C.J. donaldson, S.W. Sutton, M.H. Perrin, A.Z. Corrigan, K.A. Lewis, J.E. Rivier, J.M. Vaughan, W.W. Vale, Cloning and characterization of human urocortin, Endocrinology 137 (1996) 3896, http://dx.doi.org/10.1210/endo.137.9. 8756563. [32] W. Moll, A. Nienartowicz, H. Hees, K.-H. Wrobel, A. Lenz, Blood flow regulation in the uteroplacental arteries, in: P. Kaufmann, R.K. Miller (Eds.), Placent. Vasc. Blood Flow Basic Res. Clin. Appl., Springer, US, Boston, MA, 1988, pp. 83–96, http://dx.doi.org/10.1007/978-1-4615-8109-3 7. [33] A. Glantz, H.-U. Marschall, L.-A. Mattsson, Intrahepatic cholestasis of pregnancy: relationships between bile acid levels and fetal complication rates, Hepatology 40 (2004) 467–474, http://dx.doi.org/10.1002/hep.20336. [34] E. Wikström Shemer, H. Marschall, J. Ludvigsson, O. Stephansson, Intrahepatic cholestasis of pregnancy and associated adverse pregnancy and fetal outcomes: a 12-year population-based cohort study, BJOG 120 (2013) 717–723, http://dx.doi.org/10.1111/1471-0528.12174. [35] M.G. Swain, M. Maric, Defective corticotropin-releasing hormone mediated neuroendocrine and behavioral responses in cholestatic rats: implications for cholestatic liver disease-related sickness behaviors, Hepatology 22 (1995) 1560–1564, http://dx.doi.org/10.1016/0270-9139(95)90165-5. [36] M. Quinn, Y. Ueno, H.Y. Pae, L. Huang, G. Frampton, C. Galindo, H. Francis, D. Horvat, M. McMillin, S. Demorrow, Suppression of the HPA axis during extrahepatic biliary obstruction induces cholangiocyte proliferation in the rat, Am. J. Physiol. Gastrointest. Liver Physiol. 302 (2012) G182–G193, http://dx. doi.org/10.1152/ajpgi.00205.2011. [37] M. McMillin, G. Frampton, M. Quinn, A. Divan, S. Grant, N. Patel, K. Newell-Rogers, S. DeMorrow, Suppression of the HPA axis during cholestasis can be attributed to hypothalamic bile acid signaling, Mol. Endocrinol. 29 (2015) 1720–1730, http://dx.doi.org/10.1210/me.2015-1087.
Contents lists available at ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Dynamic expression of corticotropin-releasing hormone and urocortin in estrogen induced-cholestasis pregnant rat Fan Zhou a,b , Bingxin Gao a,b , Chunyan Deng a,b , Guiqiong Huang a,b , Ting Xu a,b , Xiaodong Wang a,b,∗ a b
Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu 610041, China Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China
a r t i c l e
i n f o
Article history: Received 21 October 2015 Received in revised form 25 July 2016 Accepted 29 July 2016 Available online 1 August 2016 Keywords: Placenta Corticotropin-releasing hormone Urocortin Rat Cholestasis Regulation Pathogenesis
a b s t r a c t Intrahepatic cholestasis of pregnancy(ICP) is complicated by acute placental-fetal hypoxia. Corticotropinreleasing hormone(CRH) and urocortin(UCN) are vasodilatory regulators of blood flow in the placenta. An ethinylestradiol(EE)-induced cholestasis rat model was reproduced and serum/placental CRH/UCN were detected during 14–21 days of gestation(DG). Maternal serum or placental CRH/UCN levels in the control rats were relatively consistent during 14–21DG. Serum CRH was reduced in the EE-treated rats compared with the control rats at 21DG. Regarding serum UCN, we observed a decrease at 17DG as well as an increase at 21DG in the EE-treated rats compared with the controls. Moreover, we observed a noticeable reduction of placental CRH/UCN expression at 17 or 19DG in the EE-treated rats compared with the control rats. The serum bile acids levels exhibited an inverse correlation with placental CRH/UCN expression. EEinduced cholestasis rats might serve as a good model to further investigate the pathological mechanism underlying CRH/UCN dysregulation in ICP. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Intrahepatic cholestasis of pregnancy (ICP) is a pregnancyspecific disease in the second or third trimester of pregnancy. The incidence of ICP varies between 0.2% and 2% (in most areas of Europe, America and Asia), is dependent on ethnicity and geographic location and is reported to be 5% in the Yangtze River area of China [1,2]. ICP is characterized by maternal pruritus and abnormally increased maternal serum bile acids and liver transaminases levels, which rapidly return to normal after delivery. The etiology and pathogenesis of ICP has not been fully revealed. Several factors, such as genetics, female sex hormones and immunological and environmental factors, are thought to contribute to the onset of ICP. ICP can affect the fetus and lead to preterm birth (spontaneous or iatrogenic), meconium-stained amniotic fluid and intrauterine fetal death. The prenatal mortality of ICP is as high as 2.25% [3].
Abbreviations: ICP, intrahepatic cholestasis of pregnancy; CRH, cotricotrophin releasing hormone; UCN, urocortin; EE, ethinylestradiol; CRH-R, cotricotrophin releasing hormone receptor; IUFD, intrauterine fetal death; ALT, alanine transaminase; AST, aspartate transaminase. ∗ Corresponding author at: Department of Obstetrics and Gynecology, Sichuan University, Renmin South Road, Chengdu, Sichuan, China. E-mail address: wangxd [email protected] (X. Wang). http://dx.doi.org/10.1016/j.reprotox.2016.07.019 0890-6238/© 2016 Elsevier Inc. All rights reserved.
There is evidence that the sudden intrauterine fetal death in ICP was associated with acute intrauterine fetal hypoxia, which might be related to blood flow dysregulation of the utero-placentalfetal unit [3,4]. In normal conditions, the utero-placental-fetal unit has more than two times of the blood oxygen volume reserve compared with those in hypoxia-stressed conditions. The intervillous space, the lobular vascular volume and their blood update speed in the utero-placental-fetal unit are key compensatory elements for hypoxia. However, ICP patients were found to be complicated with acute utero-placental-fetal unit insufficiency—the placental intervillous space was decreased by 30%, and the lobular vascular volume was decreased by 29% [5–8]. Meanwhile, the mechanism of blood flow regulation in the human placenta is complex and is characterized by high blood flow volume, low vascular resistance and lack of neural regulation [9,10]. The local production of circulation-derived vasoactive substances, including prostaglandins, endothelium-derived relaxing factor, vasoactive peptides and the corticotropin-releasing hormone (CRH) family, especially CRH and urocortin (UCN), is important in blood flow regulation and hypoxia compensatory mechanisms in the uteroplacental-fetal unit. These mechanisms increase the ability to cope with acute fetal hypoxia in late pregnancy and intrapartum fetal asphyxia [9–11].
180
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
In our previous study, the maternal serum and placental CRH/UCN expression were down-regulated in ICP patients [12,13]. However, the placental tissues of ICP patients can only be obtained after delivery, and the patients had always received multiple treatments for ICP. Subcutaneous administration of high doses of ethinylestradiol (EE) in rats induces a predictable and reversible cholestasis [14], and the mechanism of cholestasis is associated with alterations in the fluidity and lipid composition of the liver plasma membrane, driving force for the bile flow, Na+ -K+ -ATPase activity and bile salt transports [15–20]. EE-induced cholestasis pregnant rat serves as an excellent model in various studies relevant to the etiology and pathogenesis of ICP [21,22]. There is also evidence about the expression of CRH in decidualized endometrium and maternal decidua of Sprague-Dawley rats, as well as the involvement of locally produced CRH in mouse embryo implantation and pregnancy maintenance [23–25]. Both of them are in agreement with the hypotheses postulated in humans [11]. Thus, to further evaluate the placental CRH/CRH-R1 and UCN/CRH-R2 expression dynamically and explore their roles in the pathological mechanism of sudden intrauterine fetal death in ICP, we reproduced an EE-induced cholestasis pregnant rat model according to the method reported by Crocenzi [21]. Maternal serum CRH/UCN levels and placental CRH/UCN/CRH-R1/CRH-R2 expression were detected in the EE-induced cholestasis pregnant rats and control rats during 14–21 days of gestation.
2. Materials and methods 2.1. Animals Adult female Sprague-Dawley (SD) rats weighing 250–300 g were mated with adult male SD rats weighing 300–350 g and were checked for vaginal plugs, which indicate pregnancy. The day a vaginal plug was observed was recorded as the first day of gestation. All of the rats were maintained on a standard water and diet ad libitum and were housed in a temperature (15–20 ◦ C) and humidity (40%–70%) controlled room under a constant 12 h light/dark cycle. This study was approved by ethical committees at the West China Second University Hospital of Sichuan University. All rats received humane care according to the criteria prepared by the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Pregnant rats were randomly divided into two experimental groups at day 14 of gestation (n = 24). The EE group was subcutaneously administered EE daily (5 mg/kg body weight, propylene glycol as a solvent, the concentration of EE was 1 mg/ml, Sigma Chemical Co. Shanghai, China) for 5 days starting at day 14 of gestation [21]. The control group was subcutaneously administered propylene glycol (5 ml/kg body weight, Sigma Chemical Co. Shanghai, China) daily for 5 days starting at day 14 of gestation. Surgical procedures were performed at 14, 17 (the day after the third dose of EE or solvent was administered), 19 (the day after the last dose of EE or solvent was administered) and 21 days of gestation (3 days post administration). Animals were anesthetized with a single dose of chloral hydrate (2.5 mg/kg body weight, intraperitoneally). A middle abdominal incision was made rapidly. The placentas (exclude membranes) and blood were sampled within 5 min. At the end of each surgical procedure, the animals were sacrificed by exsanguination. The blood samples were collected using an EDTA-containing tube and were centrifuged for 15 min at 1000 × g at 2–8 ◦ C within 30 min. Alanine transaminase (ALT), aspartate transaminase (AST), total bile acids, total bilirubin and direct bilirubin levels were determined using a fully automatic biochemical analyzer (Siemens Electrical Apparatus Ltd., Germany). Rat maternal serum CRH or
UCN levels were detected using a specific, commercially available ELISA kit (CUSABIO Life Science, China) according to the manufacturer’s instructions. The pre-pregnancy bodyweight (recorded on the day before observation of vaginal plug), maternal bodyweight gain, number of fetal rats/litter, placenta weight (all placentas per litter), fetal rat’s body weight (all foetuses per litter), meconium stained amniotic fluid and intrauterine fetal death were observed and recorded. The significance of meconium stained amniotic fluid in rats has not been fully elucidated. Studies from various types of animals indicated that the increase of utero defecation induced by fetal hypoxia, impaired clearance of meconium and decreased fetal swallow all contribute to meconium stained amniotic fluid [26]. 2.2. Real-time PCR Three placentas were randomly selected from each pregnant rats (except one rat in the control group only had three placentas) using simple random sampling. Total RNA was prepared from placental tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The purity and integrity of the RNA were evaluated by gel electrophoresis. RNA (200 g), quantified by measuring the absorbance at optical density 260 (the ratio of optical density 260/optical density 280 around 1.9-2.0 means high purity of RNA), was reverse transcribed to cDNA using RNase Reverse Transcriptase (Life Technologies, Shanghai, China). All of the primers and probes used for real-time PCR were synthesized by Corelab Biotech (Chengdu, China). The primers were as follows: CRH, F: 5 -CAGCCGTTGAATTTCTTG-3 , R: 5 -GACTTCTGTTGAGGTTCC-3 ; UCN, F: 5 -CAACGACGAGACGACC-3 , R: 5 -ACTTGCCCACCGAATC3 ; CRH-R1, F: 5 -GTGCCTGAGAAACATCAT-3 , R: CRH-R2, F: 5 5 -ACCGAACATCCAGAAGAA-3 ; TGGTGACTTAGTGGACTA-3 , R: 5 -GAAGAGCATGTAGGTGAT-3 ; and -actin, F: 5 -CTGGAGAAGAGCTATGAG-3 , R: 5 -ATGATGGAATTGAATGTAGTT-3 . The probes were as follows: CRH, 5 -CAGCAACCTCAGCCGATTCT UCN, 5 -CCTCACCTTCCACCTGCTG-3 ; CRH-R1, 3; 5 -CACTGGAACCTCATCTCGGC-3 ; CRH-R2, 5 CATCATCCTCGTGCTCCTCATC-3 ; and -actin, 5 ACGGTCAGGTCATCACTATCG-3 . PCR was performed in a reaction mixture consisting of FastStart Universal Probe Master (Roche, Shanghai, China) 10 l, cDNA 5 l, primer 2 l, Taqman probe 1 l and ddH2 O 2 l. PCR program was 95 ◦ C for 10 min; 95 ◦ C for 15 s, 60 ◦ C for 1 min (45 cycles) and 72 ◦ C for 7 min followed by 4 ◦ C (ABI 7900HT, Waltham, MA, USA). All samples were run in triplicate in 96-well optical PCR plates. For analysis, quantitative amounts of mRNA levels were standardized against the internal reference -actin. 2.3. Western blotting Placental tissues (100 mg) and 400 ml lysis buffer (with phenylmethane sulfonyl fluoride) were added to a homogenizing device and were homogenized for 30 min at 4 ◦ C. The homogenate was centrifuged at 12, 000 × g for 5 min at 4 ◦ C, and the supernatant was collected. A bicinchoninic acid assay (Thermo ScientificTM PierceTM , Waltham, MA, USA) was used to determine the protein concentration. Then, samples were separated on an SDS-polyacrylamide gel, and the protein (50 g) was electrophoretically transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, Massachusetts, USA). Next, the membranes were blocked in TBS-T (Tris-buffered saline containing 0.05% Tween 20) containing 5% non-fat dried milk for 2 h at room temperature and incubated with anti-CRH antibodies (Abcam, Shanghai, China) (1:10, 000), anti-UCN antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:800), anti-CRH-R1 antibodies (Proteintech, Rosemont, IL, USA) (1:350) or anti-CRH-R2 antibodies (Acris Antibodies,
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
Rockville, MD, USA) (1:800) overnight at 4 ◦ C. The membranes were washed 3 times with TBS-T and incubated with 1:1, 000 anti-rabbit horseradish-peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 2 h. The ratio of the band intensities to -actin was obtained to quantify the relative protein expression levels and control for sampling errors. 2.4. Statistical analysis Stata 12.1 (Statacorp, College Station, TX, USA) was used for statistical analysis. Pauta criterion (data greater than + 3 or less than − 3 will be rejected) were used to define and reject the outliers. Continuous variables with normal distribution, including the pre-pregnancy weight, maternal body weight gain, number of fetal rats/litter, fetal body weight, placental weight, maternal serum levels of ALT, AST, total bile acids, total bilirubin, direct bilirubin, CRH and UCN, placental CRH/UCN protein expression or placental CRH-R1/CRH-R2 protein expression, were presented as the means ± standard deviation (S.D.) on a litter basis (fetuses were nested within litters). Two way ANOVA was used with the expression in each litter as response, and treatment and gestation day as the main interact effects without considering interaction. Non-normally distributed data was expressed as medians (M) and inter-quartile ranges (IQR) on a litter basis, including mRNA levels of placental CRH, UCN, CRH-R1 or CRH-R2, and the Friedman’s test followed by Dunnett’s Post-hoc test was performed. Fisher’s Exact Test was performed to analyze the differences of meconium stained amniotic fluid or IUFD rates between the EE and control groups on a litter basis (fetuses were nested within litters). A correlation analysis was examined by using Pearson’s correlation (between blood parameters and serum/placental CRH/UCN expression) or Spearman’s correlation (between fetal outcomes and blood parameters or serum/placental CRH/UCN expression). For all statistical testing, a two-tailed P value of <0.05 was considered statistically significant. 3. Results 3.1. Maternal/fetal characteristics and maternal serum biochemical parameters Forty-eight pregnant rats were enrolled in this study, including 24 pregnant rats in the EE group and 24 rats in the control group. The pre-pregnancy weight, maternal bodyweight gain, number of fetal rats/litter and placenta weight exhibited no statistical differences between the two groups (Fig. 1A–C, E). At gestation day 14, 17 and 19, the fetal rats’ body weight demonstrated no significant difference between the EE and control groups. At gestation day 21, the fetal rats’ body weight was lower compared with the control group (Fig. 1D). At day 14 of gestation, the maternal serum ALT, AST, total bile acids, total bilirubin and direct bilirubin levels had no significant differences between the EE and control groups. At 17 and 19 days of gestation, maternal serum ALT and total bile acids levels in the EE group were significantly higher compared with the control group; no significant differences in the AST, total bilirubin or direct bilirubin levels were observed. At 21 days of gestation, the maternal serum AST, total bile acids, total bilirubin and direct bilirubin levels in the EE group were significantly higher than the control group, whereas no significant differences in ALT levels were observed (Fig. 1F–J). 3.2. Pregnancy outcomes In the EE group, no cases of meconium stained amniotic fluid and IUFD were found at day 14 of gestation; at day 17 of gestation,
181
the meconium stained amniotic fluid rate was 16.67% (1/6) and the IUFD rate was 33.33% (2/6); at day 19 of gestation, 16.67% (1/6) had meconium stained amniotic fluid and 33.33% (2/6) had IUFD; meconium stained amniotic fluid and IUFD accounted for 50% (3/6) and 50% (3/6) at day 21 of gestation. In the control group, no cases of meconium stained amniotic fluid and IUFD were found at gestation day 14, 17, 19 or 21. There are no statistical differences in meconium stained amniotic fluid rate and IUFD rate between the EE and control groups. 3.3. Maternal serum CRH and UCN levels Maternal serum CRH levels in the control group were relatively consistent throughout the experiment (Fig. 2A). There was no statistically significant change in maternal serum CRH levels in the EE group throughout the experiment (Fig. 2A). At day 21 of gestation, the difference in maternal serum CRH levels between the EE and control groups achieved statistical significance (Fig. 2A). Maternal serum UCN levels in the control group were relatively consistent throughout the experiment (Fig. 2B). We observed significant differences in maternal serum UCN levels in the EE group throughout the experiment, and the UCN levels at gestation day 17 were lower compared with gestation day 21 (Fig. 2B). At gestation day 17 or 21, the difference in maternal serum UCN levels between the EE and control groups achieved statistical significance (Fig. 2B). 3.4. Placental CRH mRNA and UCN mRNA expression Placental CRH mRNA expression in the control group exhibited no statistically significant change throughout the experiment (Fig. 3A). In the EE group, placental CRH mRNA levels at gestation day 17 and 19 were lower compared with that at gestation day 14 or 21. Meanwhile, at gestation day 17 and 19, placental CRH mRNA levels in the EE group were lower compared with those in the control group (Fig. 3A). Placental UCN mRNA expression in the control group was relatively consistent throughout the experiment (Fig. 3B). In the EE group, placental UCN mRNA expression at gestation day 17 and 19 were lower compared with gestation day 14 or 21. Moreover, at gestation day 17 and 19, placental UCN mRNA levels in the EE group were lower compared with those in the control group (Fig. 3B). 3.5. Placental CRH-R1 mRNA and CRH-R2 mRNA expression Placental CRH-R1 mRNA expression in the EE or control groups was relatively consistent throughout the experiment (Fig. 3C). Placental CRH-R1 mRNA expression demonstrated no significant differences between the EE and control groups at gestation day 14, 17, 19 or 21 (Fig. 3C). There was no statistically significant change in placental CRH-R2 mRNA expression in the EE or control groups throughout the experiment (Fig. 3D). Additionally, at gestation day 17, placental CRH-R2 mRNA levels in the EE group were lower compared with that in the control group (Fig. 3D). 3.6. Placental CRH and UCN expression Placental CRH expression (21 KDa) in the control group exhibited no obvious change throughout the experiment (Fig. 4A). In the EE group, placental CRH levels demonstrated significant differences among gestation day 14, 17, 19 and 21, and those at gestation day 17 or 19 was lower compared with gestation day 14. At gestation day 17 and 19, the differences in placental CRH levels between EE and control groups achieved statistical significance (Fig. 4A). Placental UCN (13 KDa) demonstrated no significant differences in the control group throughout the experiment (P > 0.05) (Fig. 4B).
182
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
Fig. 1. Baseline information and maternal serum biochemical parameters in the EE-treated and control rats. DG, days of gestation; ALT, Alanine transaminase; AST, Aspartate transaminase. Data were expressed as the means ± S.D. after rejected the outliers according to the Pauta criterion (data greater than + 3 or less than − 3 will be rejected) (n = 6). * P < 0.05. The rejected outliers were as follows: two outliers in the ALT levels, two outliers in the AST levels, one outlier in the total bilirubin levels and one outlier in the direct bilirubin levels.
In the EE group, placental UCN levels exhibited significant differences among gestation day 14, 17, 19 and 21, and those at gestation day 17 or 19 was lower compared with gestation day 14 or 21. Moreover, at gestation day 17 and 19, the differences in placental UCN levels between the EE and control groups achieved statistical significance (Fig. 4B). 3.7. Placental CRH-R1 and CRH-R2 expression Our data indicated that placental CRH-R1 expression (48 KDa) in the control group had no obvious change throughout the experiment. In the EE group, placental CRH-R1 levels demonstrated
significant differences among gestation day 14, 17, 19 and 21, and those at gestation day 19 or 21 was lower than gestation day 14 or 17. At gestation day 19 and 21, the differences in placental CRHR1 levels between the EE and control groups achieved statistical significance (Fig. 4C). Our data indicated that placental CRH-R2 expression (21 KDa) in the control group had no significant differences throughout the experiment. There were no statistically significant changes in placental CRH-R2 levels in the EE group throughout the experiment. No significant difference was noticed between the EE and control groups at gestation day 14, 17, 19 or 21 (Fig. 4D).
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
183
Fig. 2. Maternal serum CRH and UCN levels during gestation day 14–21 in the EE-treated and control rats. Data were expressed as the means ± S.D. after rejected the outliers according to the Pauta criterion (data greater than + 3 or less than − 3 will be rejected) (n = 6). * P < 0.05. The rejected outliers were as follows: one outlier in the CRH levels and two outliers in the UCN levels.
3.8. Relationship between maternal biochemical parameters, CRH/UCN expression and fetal outcomes Pearson’s correlation analysis indicated that, in the EE group, maternal serum UCN levels showed a significant correlation with AST levels throughout gestation day 14–21. Placental CRH or UCN expression in the EE group exhibited a significant negative correlation with serum total bile acids levels throughout gestation day 14–21 (Table 1).
4. Discussion Our results revealed the dynamic expression of serum and placental CRH/UCN during gestation day 14–21 in pregnant rats with EE-induced cholestasis. In EE-induced cholestasis pregnant rats, both maternal serum and placental CRH levels were decreased compared with those in the control rats, and the reduction of maternal serum CRH occurred after the decrease of placental CRH. Meanwhile, the maternal serum and the placental UCN expression in the EE-treated rats were reduced simultaneously at gestation day 17 compared with those in the control rats. The maternal serum
UCN levels in the EE-treated rats showed a significant increase and higher than the control rats at gestation day 21. Placental UCN expression in the EE-treated rats remained lower than the control rats at gestation day 19. Our data also revealed a negative correlation between maternal serum total bile acids levels and the expression of placental CRH or UCN in pregnant rats with EE-induced cholestasis. Our study successfully reproduced an EE-induced cholestasis pregnant rat model. The maternal serum liver enzymes, bilirubin and total bile acids levels were elevated in EE administrated pregnant rats. Meanwhile, we noticed decreases of serum total bile acids, total bilirubin or direct bilirubin levels in the control rats at gestation day 21 when compared with gestation day 19. The drop of blood parameters during late pregnancy in rats due to hemodilution was also reported by several studies [27–29]. Meanwhile, no significant differences were found in meconium stained amniotic fluid rate and IUFD rate between the EE and control groups. In 1955, Guillemin [30] first described the hypothalamicderived hormone CRH, and the earliest report of UCN was made by Vaughan [31] in rat brain tissues in 1995. Since then, numerous studies had examined the biochemical properties of CRH and
184
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
Fig. 3. mRNA expression of placental CRH, UCN, CRH-R1 and CRH-R2 during gestation day 14–21 in the EE-treated and control rats. DG, days of gestation. Data were expressed as the medians (M) and inter-quartile ranges (IQR) after rejected the outliers according to the Pauta criterion (data greater than + 3 or less than − 3 will be rejected) (n = 6). * P < 0.05. The rejected outliers were as follows: two outliers in the CRH mRNA expression, two outliers in the UCN mRNA expression, two outliers in the CRH-R1 mRNA expression and one outlier in the CRH-R2 mRNA expression.
Fig. 4. Protein expression of placental CRH, UCN, CRH-R1 and CRH-R2 during gestation day 14–21 in the EE-treated and control rats. Data were expressed as the means ± S.D. after rejected the outliers according to the Pauta criterion (data greater than + 3 or less than − 3 will be rejected) (n = 6). * P < 0.05. The rejected outliers were as follows: two outliers in the CRH expression, three outliers in the UCN levels, one outlier in the CRH-R1 expression and two outliers in the CRH-R2 expression.
Table 1 Correlation analysis between maternal biochemical parameters, CRH expression, UCN expression and fetal outcomes. r Maternal serum CRH (ng/ml)§ Maternal serum UCN (pg/ml)§ Placental CRH§ Placental UCN§ Meconium-stained amniotic fluid rate (%)# IUFD rate (%)#
ALT (U/L)§ −0.04 0.36 −0.10 −0.21 0.80 0.80
AST (U/L)§ 0.09 0.44* −0.18 −0.08 1.00 1.00
Serum bile acids (mmol/L)§ 0.28 −0.07 −0.50* −0.74* 0.40 0.40
Meconium-stained amniotic fluid rate (%)# −0.40 0.80 −0.20 0.80 – –
IUFD rate (%)# −0.40 0.80 −0.20 0.80 – –
Note: Data were analyzed by § Pearson’s correlation or # Spearman’s correlation. CRH, Cotricotrophin releasing hormone; UCN, Urocortin; ALT, Alanine transaminase; AST, Aspartate transaminase; IUFD, Intrauterine fetal death. * P < 0.05.
UCN, especially for their roles in blood flow regulation. Additionally, CRH and UCN from humans and rats are highly homologous,
their amino acids sequences share over 90% homology [9,31]. The blood flow of the utero-placental unit in rats is regulated by vascu-
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
lar resistance, and the process is efficient [32]. Pregnant rats with EE-induced cholestasis might serve as a suitable model for research on dynamical dysregulated expression of placental CRH or UCN in ICP. In this study, the maternal serum CRH levels in pregnant rats were relatively consistent throughout gestation day 14–21. The stabilized maternal serum CRH levels in pregnant rats are inconsistent with those of humans, which are significantly increased in the third trimester of pregnancy and are increased further with the onset of labor [12]. One potential explanation for our findings is that, as lower animals, the maternal serum and placental CRH in rats were kept at a high level during late pregnancy to guarantee enough blood flow supply to multiple placentas. In parallel, the maternal serum UCN levels exhibited no obvious change during gestation day 14–21 in the control rats. The consistent expression of serum UCN levels in rats is concordant with those in humans during late pregnancy [10,13]. In EE-treated pregnant rats, the maternal serum CRH levels were lower than those in the control rats at gestation day 21; the maternal serum UCN levels were lower than those in the control rats at gestation day 17, and were higher than the control rats at gestation day 21. Moreover, the placental CRH and UCN expression in the control rats were also relatively consistent throughout gestation day 14–21. In EE-treated pregnant rats, placental CRH and UCN expression were decreased at gestation day 17 and 19 when compared with those in the control rats. The reduction of placental CRH or UCN expression was not completely consistent with the decrease of maternal serum CRH or UCN levels in pregnant rats with EEinduced cholestasis during gestation day 14–21. The decrease of maternal serum CRH (gestation day 21) was after the placental CRH reduction (gestation day 17) in EE-treated rats. The reduction of maternal serum UCN levels and placental UCN expression in the EE-treated rats (compared with the control rats) both occurred at gestation day 17, but the serum UCN levels returned to normal whereas placental UCN remained lower than the control rats at gestation day 19. At gestation day 21, the placental UCN levels in EE-treated rats exhibited no statistical difference compared with the control rats, whereas maternal serum UCN levels in EE-treated rats were higher than the control rats. Maternal serum CRH or UCN levels could not fully reflect the placental CRH or UCN expression in pregnant rats with EE-induced cholestasis. Our results also indicated that the maternal serum total bile acids levels had an inverse correlation with placental CRH/UCN expression in pregnant rats with EE-induced cholestasis. The elevated serum total bile acids levels in ICP are associated with the risk of complications to the fetus, and serve as an indicator of the severity classification of ICP (maternal serum bile acids levels exceeded 40 micromoles/L would be considered as severe ICP) [1,33,34]. There might be some relationships between the placental CRH/UCN expression and the severity of cholestasis in pregnant rats. Meanwhile, there is evidence that the elevated bile acids in bile duct resection (BDR) rat model could suppress hypothalamic CRH expression [35,36], and this process is mediated by hypothalamic bile acids signaling [37]. However, the hypothalamic CRH might contribute comparatively less to serum CRH levels as there is no evidence that central CRH could be detected in rat serum. There is no literature about the direct role of bile acids on placental CRH/UCN expression. Regarding mRNA expression, placental mRNA expression of CRH or UCN exhibited no obvious change in the control rats, which was consistent with protein expression. Placental CRH mRNA or UCN mRNA expression in pregnant rats with EE-induced cholestasis was significantly lower than those in the control rats at gestation day 17 or 19. We also detected mRNA and protein expression of placental CRH-R1 and CRH-R2 in pregnant rats during gestation day 14–21.
185
There was no significant change in placental CRH-R1 or CRHR2 expression in the control rats throughout the experiment. In EE-treated rats, placental CRH-R1 protein levels were reduced at gestation day 19 and 21 compared with the control rats. Placental CRH-R2 demonstrated no statistical difference between the EE-treated and control rats throughout the experiment. Placental mRNA expression of CRH-R1 and CRH-R2 mRNA in the EE-treated and control rats were relatively consistent throughout the experiment. CRH-R2 mRNA expression in pregnant rats with EE-induced cholestasis was lower than the control rats at gestation day 17. Taken together, these data manifested the dynamic expression of maternal serum CRH/UCN, placental CRH/UCN and placental CRH-1/CRH-R2 in pregnant rats with EE-induced cholestasis, which can not be achieved in ICP. EE-induced cholestasis pregnant rats were accompanied by the reduction of serum and placental CRH or UCN expression, and the maternal serum CRH/UCN levels could not fully reflect the placental CRH/UCN expression. The maternal serum total bile acids levels exhibited an inverse correlation with placental CRH/UCN expression. EE-induced cholestasis pregnant rats might serve as a good model to further investigate the pathological mechanism underlying CRH/UCN dysregulation in ICP. Conflict of interest The authors declare that there is no conflict of interest. Funding This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT0935). Acknowledgments We wish to express our appreciation to the editor and the anonymous reviewers for your insightful comments and suggestions, which have greatly improved the manuscript. References [1] C. Williamson, V. Geenes, Intrahepatic cholestasis of pregnancy, Obstet. Gynecol. 124 (2014) 120–133, http://dx.doi.org/10.1097/AOG. 0000000000000346. [2] X. Liu, J. He, Pay more attention to standardizing the diagnosis and treatment of intrahepatic cholestasis of pregnancy, Zhonghua Fu Chan Ke Za Zhi 46 (2011) 321–323. [3] A.P. Kenyon, J.C. Girling, Obstetric Cholestasis, RCOG Green-top guidelines No 43, 2011, 10.3748/wjg.v19.i43.7639.6. [4] R. Reid, K.J. Ivey, R.H. Rencoret, B. Storey, Fetal complications of obstetric cholestasis, Br. Med. J. 1 (1976) 870–872, http://dx.doi.org/10.1136/bmj.1. 6014.870. [5] D. Joshi, A. James, A. Quaglia, R.H. Westbrook, M.A. Heneghan, Liver disease in pregnancy, Lancet 375 (2010) 594–605, http://dx.doi.org/10.1016/S01406736(09)61495-1. [6] A.L. Costoya, E.A. Leontic, H.G. Rosenberg, M.A. Delgado, Morphological study of placental terminal villi in intrahepatic cholestasis of pregnancy: histochemistry, light and electron microscopy, Placenta 1 (1980) 361–368. [7] K. Kaar, P. Jouppila, J. Kuikka, H. Luotola, J. Toivanen, A. Rekonen, Intervillous blood flow in normal and complicated late pregnancy measured by means of an intravenous 133Xe method, Acta Obstet. Gynecol. Scand. 59 (1980) 7–10. [8] M. He, Z. Liu, X. Wang, Decreased volume of placental lobular villi vessels in patients with intrahepatic cholestasis of pregnancy, Sichuan Da Xue Xue Bao Yi Xue Ban 42 (2011) 797–801 http://europepmc.org/abstract/MED/ 22332545. [9] F. Petraglia, A. Imperatore, J.R.G. Challis, Neuroendocrine mechanisms in pregnancy and parturition, Endocr. Rev. 31 (2010) 783–816, http://dx.doi.org/ 10.1210/er.2009-0019. [10] A.L. Boura, W.A. Walters, M.A. Read, I.M. Leitch, Autacoids and control of human placental blood flow, Clin. Exp. Pharmacol. Physiol. 21 (1994) 737–748. [11] P.P.L.M. Pepels, M.E.A. Spaanderman, J. Bulten, P.B.A.M. Smits, A.R.M.M. Hermus, F.K. Lotgering, C.G.J. Sweep, Placental urocortins and CRF in late gestation, Placenta 30 (2009) 483–490, http://dx.doi.org/10.1016/j.placenta. 2009.03.008.
186
F. Zhou et al. / Reproductive Toxicology 65 (2016) 179–186
[12] F. Zhou, M.M. He, Z.F. Liu, L. Zhang, B.X. Gao, X.D. Wang, Expression of corticotrophin-releasing hormone and its receptor in patients with intrahepatic cholestasis of pregnancy, Placenta 34 (2013) 401–406, http://dx. doi.org/10.1016/j.placenta.2013.02.006. [13] F. Zhou, L. Zhang, Q. Sun, X.D. Wang, Expression of urocortin and corticotrophin-releasing hormone receptor-2 in patients with intrahepatic cholestasis of pregnancy, Placenta 35 (2014) 962–968, http://dx.doi.org/10. 1016/j.placenta.2014.07.019. [14] T.A. Heikel, G.H. Lathe, The effect of oral contraceptive steroids on bile secretion and bilirubin Tm in rats, Br. J. Pharmacol. 38 (1970) 593–601. [15] A.J. Schreiber, F.R. Simon, Estrogen-induced cholestasis: clues to pathogenesis and treatment, Hepatology 3 (1983) 607–613. [16] E.L. Forker, The effect of estrogen on bile formation in the rat, J. Clin. Invest. 48 (1969) 654–663, http://dx.doi.org/10.1172/JCI106023. [17] E.B. Keefee, B.F. Scharschmidt, N.M. Blankenship, R.K. Ockner, Studies of relationship among bile flow, liver plasma membrane NaK-ATPase, and membrane microviscosity in the rat, J. Clin. Invest. 64 (1979) 1590–1598, http://dx.doi.org/10.1172/JCI109620. [18] R.A. Davis, F.J. Kern, R. Showalter, E. Sutherland, M. Sinensky, F.R. Simon, Alterations of hepatic Na+,K+-atpase and bile flow by estrogen: effects on liver surface membrane lipid structure and function, Proc. Natl. Acad. Sci. U. S. A. 75 (1978) 4130–4134. [19] R. Bossard, B. Stieger, B. O’Neill, G. Fricker, P.J. Meier, Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver, J. Clin. Invest. 91 (1993) 2714–2720, http://dx.doi.org/10.1172/ JCI116511. [20] R. Guillemin, B. Rosenberg, Humoral hypothalamic control of anterior pituitary: a study with combined tissue cultures, Endocrinology 57 (1955) 599–607, http://dx.doi.org/10.1210/endo-57-5-599. [21] F.A. Crocenzi, E.J. Sanchez Pozzi, J.M. Pellegrino, C.O. Favre, E.A. Rodriguez Garay, A.D. Mottino, R. Coleman, M.G. Roma, Beneficial effects of silymarin on estrogen-induced cholestasis in the rat: a study in vivo and in isolated hepatocyte couplets, Hepatology 34 (2001) 329–339, http://dx.doi.org/10. 1053/jhep.2001.26520. [22] X. Song, A. Vasilenko, Y. Chen, L. Valanejad, R. Verma, B. Yan, R. Deng, Transcriptional dynamics of bile salt export pump during pregnancy: mechanisms and implications in intrahepatic cholestasis of pregnancy, Hepatology 60 (2014) 1993–2007, http://dx.doi.org/10.1002/hep.27171. [23] I. Athanassakis, V. Farmakiotis, I. Aifantis, A. Gravanis, S. Vassiliadis, Expression of corticotrophin-releasing hormone in the mouse uterus: participation in embryo implantation, J. Endocrinol. 163 (1999) 221–227, JOE03388[pii]. [24] A. Makrigiannakis, E. Zoumakis, S. Kalantaridou, C. Coutifaris, A.N. Margioris, G. Coukos, K.C. Rice, A. Gravanis, G.P. Chrousos, Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance, Nat. Immunol. 2 (2001) 1018–1024, http://dx.doi.org/10.1038/ni719. [25] A. Makrigiannakis, A.N. Margioris, C. Le Goascogne, E. Zoumakis, G. Nikas, C. Stoumaras, A. Psychoyos, A. Gravanis, Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus, Life Sci. 57
(1995) 1869–1875, http://dx.doi.org/10.1016/0024-3205(95)02167-H. [26] S.N. Ahanya, J. Lakshmanan, B.L.G. Morgan, M.G. Ross, Meconium passage in utero: mechanisms, consequences, and management, Obstet. Gynecol. Surv. 60 (2005) 45–56, http://dx.doi.org/10.1097/01.ogx.0000149659.89530.c2, quiz 73–74. [27] E.P.C.T. de Rijk, E. van Esch, G. Flik, Pregnancy dating in the rat: placental morphology and maternal blood parameters, Toxicol. Pathol. 30 (2002) 271–282, http://dx.doi.org/10.1080/019262302753559614. [28] J.C. Kim, H.I. Yun, K.H. Lim, J.E. Suh, M.K. Chung, Haematological values during normal pregnancy in Sprague-Dawley rats, Comp. Haematol. Int. 10 (74) (2000) 74–79, http://dx.doi.org/10.1007/s0058000700112000. [29] J.B. LaBorde, K.S. Wall, B. Bolon, T.S. Kumpe, R. Patton, Q. Zheng, R. Kodell, J.F. Young, Haematology and serum chemistry parameters of the pregnant rat, Lab. Anim. 33 (1999) 275–287, http://dx.doi.org/10.1258/ 002367799780578156. [30] J. Vaughan, C. Donaldson, J. Bittencourt, M.H. Perrin, K. Lewis, S. Sutton, R. Chan, a V Turnbull, D. Lovejoy, C. Rivier, Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor, Nature 378 (1995) 287–292, http://dx.doi.org/10.1038/378287a0. [31] C.J. donaldson, S.W. Sutton, M.H. Perrin, A.Z. Corrigan, K.A. Lewis, J.E. Rivier, J.M. Vaughan, W.W. Vale, Cloning and characterization of human urocortin, Endocrinology 137 (1996) 3896, http://dx.doi.org/10.1210/endo.137.9. 8756563. [32] W. Moll, A. Nienartowicz, H. Hees, K.-H. Wrobel, A. Lenz, Blood flow regulation in the uteroplacental arteries, in: P. Kaufmann, R.K. Miller (Eds.), Placent. Vasc. Blood Flow Basic Res. Clin. Appl., Springer, US, Boston, MA, 1988, pp. 83–96, http://dx.doi.org/10.1007/978-1-4615-8109-3 7. [33] A. Glantz, H.-U. Marschall, L.-A. Mattsson, Intrahepatic cholestasis of pregnancy: relationships between bile acid levels and fetal complication rates, Hepatology 40 (2004) 467–474, http://dx.doi.org/10.1002/hep.20336. [34] E. Wikström Shemer, H. Marschall, J. Ludvigsson, O. Stephansson, Intrahepatic cholestasis of pregnancy and associated adverse pregnancy and fetal outcomes: a 12-year population-based cohort study, BJOG 120 (2013) 717–723, http://dx.doi.org/10.1111/1471-0528.12174. [35] M.G. Swain, M. Maric, Defective corticotropin-releasing hormone mediated neuroendocrine and behavioral responses in cholestatic rats: implications for cholestatic liver disease-related sickness behaviors, Hepatology 22 (1995) 1560–1564, http://dx.doi.org/10.1016/0270-9139(95)90165-5. [36] M. Quinn, Y. Ueno, H.Y. Pae, L. Huang, G. Frampton, C. Galindo, H. Francis, D. Horvat, M. McMillin, S. Demorrow, Suppression of the HPA axis during extrahepatic biliary obstruction induces cholangiocyte proliferation in the rat, Am. J. Physiol. Gastrointest. Liver Physiol. 302 (2012) G182–G193, http://dx. doi.org/10.1152/ajpgi.00205.2011. [37] M. McMillin, G. Frampton, M. Quinn, A. Divan, S. Grant, N. Patel, K. Newell-Rogers, S. DeMorrow, Suppression of the HPA axis during cholestasis can be attributed to hypothalamic bile acid signaling, Mol. Endocrinol. 29 (2015) 1720–1730, http://dx.doi.org/10.1210/me.2015-1087.