Effects of estrogen and estrogenic compounds, 4-tert-octylphenol, and bisphenol A on the uterine contraction and contraction-associated proteins in rats

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Highlights

  • We examined the effects of EDCs on uterine contractions.

  • E2 and EDCs all increased oxytocin-related pathway.

  • The prostaglandin-related signaling was reduced by E2 and EDCs.

  • E2 enhanced the contractility, while EDCs significantly decreased it.

  • Uterine cell contraction was differently regulated by E2 and EDCs.

Abstract

We examined the effects of estradiol (E2), 4-tert-octylphenol (OP), and bisphenol A (BPA) on uterine contractions in immature rats. The expression and localization of contraction-associated proteins (CAPs), and contractility of rat uterus with a collagen gel contraction assay were analyzed. E2, OP, and BPA all increased oxytocin (OT)-related pathway, while the prostaglandin-related signaling was reduced. Interestingly, E2 and estrogenic compounds showed distinct effects on the contractile activity of uterine cells. E2 enhanced the contractility, while OP and BPA significantly decreased it. Immunohistochemical analysis of CAPs showed distinct regulation of prostaglandin F receptor localization by E2 and estrogenic compounds, which may explain the different contractile activities of those reagents. In summary, we demonstrate that E2, OP, and BPA regulate CAP expression in a similar manner in the immature rat uterus, however, the effects on contractile activity were modulated differently. These findings suggest that OP and BPA interfere with uterine contractility.

Introduction

The uterus is a major female reproductive organ composed of a well-differentiated endometrium, a thick muscular myometrium, and an outer serosal layer. There has been marked progress toward understanding the physiology and pathophysiology of the endometrium, which has resulted in many important interventions for menstrual function, conception, and contraception. In contrast, relatively few studies regarding the role of the myometrium in reproductive organs are available despite growing concerns of the importance of the uterine muscle layer. Myometrial function may be of vital importance in physiological processes such as the menstrual cycle, sperm and embryo transport, implantation, and parturition. The uterus contracts frequently throughout the menstrual cycle. These contractions have been called endometrial or contractile waves and appear to involve only the sub-endometrial layer of the myometrium (Aguilar and Mitchell, 2010). During the early follicular phase, the uterus contracts once or twice per min. Contraction frequency increases to 3–4 times per min as ovulation approaches. During the luteal phase, frequency of the contractions declines, possibly in preparation for implantation and pregnancy (Aguilar and Mitchell, 2010). If implantation fails, the contractile activity increases during menstruation and sometimes induces menstrual pain due to labor-like contractions of the uterus. Abnormalities in uterine contractility in terms of contractile amplitude and frequency are thought to contribute to various clinical problems including infertility and abortion (Dallot et al., 2003, Richards et al., 1998).

The physiological functions of two main uterine tissues, the endometrium and myometrium, are regulated differently by steroid hormones, eicosanoids, and peptides (Riemer and Heymann, 1998). Progesterone (P4) promotes myometrial relaxation and sustains pregnancy while estrogen (E2) increases uterine contractility during parturition (Dawe et al., 1982, Wu et al., 2004, Csapo et al., 1971). The principal mechanism through which P4 and E2 regulate myometrial contractility is by modulating the expression of genes encoding contraction-associated proteins (CAPs) (Mesiano and Welsh, 2007). Some important CAPs include the OT receptor (OTR) and prostaglandin (PG)-F (PGF) receptor (FP), the PG-metabolizing enzyme 15-hydroxy-PG-dehydrogenase (PGDH), and the gap-junction protein connexin-43 (CNX43) (Mesiano, 2007). Although regulation of uterine contraction by steroid hormones has been done during pregnancy and parturition, only a few studies have focused on the non-pregnant stage.

Recently, there have been considerable concerns that xenobiotics such as bisphenol A (BPA) and 4-tert-octylphenol (OP) may have adverse effects on humans, wildlife, and aquatic organisms by disrupting endocrine function. These compounds have therefore been referred to as endocrine disrupting chemicals (EDCs) (Nilsson, 2000). BPA is used extensively in the production of polycarbonate and epoxy resins, and serves as a non-polymer additive in plastics such as polyvinyl chloride (PVC) and water pipes (Welshons et al., 2006). The global demand for BPA is expected to grow from 3.9 million tons in 2006 to about 5 million tons in 2010 (Tsai, 2006). Alkylphenol compounds such as OP are also classified as EDCs (Nimrod and Benson, 1996). OP is an important industrial chemical used as a surfactant in detergents, paints, and herbicides (Nimrod and Benson, 1996). This hydrophobic compound is highly adsorbed in soil, sludge, and sediment (Othman et al., 2012). It has been reported that approximately 50,000 tons of OP are produced annually worldwide, and the levels of OP in sediments and aquatic environments are up to 670 μg/kg and 200 μg/L, respectively (Othman et al., 2012, Isobe et al., 2001). Previously, we suggested that BPA and OP exert estrogenic effects on the uterus by regulating E2-target genes and uterotrophic activity (An et al., 2002). Since E2 has been shown to regulate uterine contractile activity (Dawe et al., 1982, Wu et al., 2004), it is highly possible that EDCs could also modulate this activity.

In the present study, we examined the effects of E2, BPA, and OP on rat uterine contractions in the immature state. To explore the mechanism underlying E2 and EDC activities, the expression of CAPs was analyzed. Contractility of the rat uterus was further evaluated using a three dimensional (3D) collagen gel contraction assay.

Section snippets

Reagents and chemicals

BPA, E2, and corn oil were purchased from Sigma Chemical Co. (St. Louis, MO, USA). OP was obtained from Fluka Chemie (Seoul, Republic of Korea). Goat anti-oxytocin receptor (OTR), rabbit anti-actin, and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-goat IgG antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit antibody specific for FP was obtained from Cayman Chemical (Ann Arbor, MI, USA).

Animals and treatments

Immature Sprague–Dawley (SD; 40 females) rats were

Transcriptional regulation of CAPs by E2, OP, and BPA

The regulation of CAPs including OT, OTR, PDGH, FP, and CNX43 was examined. In our initial experiment, the rats were administrated with E2 (40 μg/kg/BW), OP (10, 100, and 500 mg/kg/BW), and BPA (10, 100, and 500 mg/kg/BW) for 3 d. Uterine weight was then measured (please see Supplemental Fig. S1A) to estimate the estrogenic activity of OP and BPA under our experimental conditions. As we expected, E2 as well as OP and BPA increased uterine weight in a dose-dependent manner. To further confirm the

Discussion

It has been known that EDCs have adverse effects on female and male reproductive organs. These compounds can cause reproductive problems by decreasing sperm count and quality, increasing the number of testicular germ cells and causing male breast cancer, cryptorchidism, hypospadias, miscarriages, irregularities of the menstrual cycle, and infertility (Balabanic et al., 2011). In the infertile female patients, serum levels of EDCs are significantly higher, which interfere with endocrine

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012R1A1A2041975).

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