Involvement of the orphan nuclear receptor SF-1 in the effect of PCBs, DDT and DDE on the secretion of steroid hormones and oxytocin from bovine granulosa cells

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Abstract

Polychlorinated biphenyls (PCBs), DDT and its metabolite (DDE) belong to estrogen-like endocrine disruptors. However, though their activity is approximately 1000-fold lower than the activity of estradiol (E2), this steroid's high concentration in follicular fluid and incubation media does not inhibit the influence of these xenobiotics. It was hypothesized that these xenobiotics might affect Steroidogenic Factor-1 (SF-1) and impair ovary function. To test this hypothesis, granulosa cells were obtained from ovarian follicles >1 or <1 cm in diameter, which were treated with PCB-77, PCB-153, DDT or DDE (each at 10 ng/ml), alone or jointly with an SF-1 antagonist (F0160). Treatment with the SF-1 antagonist inhibited (P < 0.05) the secretion of P4 from cells of both sizes of follicles, as induced (P < 0.05) by an SF-1 activator (HxP), DDE or PCB-153. All xenobiotics and HxP stimulated (P < 0.05) the synthesis and secretion of oxytocin (OT). However, the effect on mRNA expression for NP-I/OT, which is OT precursor, was inhibited (P < 0.05) by F0160 in all cultures treated with PCB-77, except for granulosa cells derived from follicles <1 cm. Moreover, F0160 inhibited the effect on OT secretion of HxP, as well as all xenobiotics except for PCB-77 and DDE, in granulosa cells derived from follicles <1 cm. Xenobiotic treatment did not affect (P > 0.05) the expression for SF-1 mRNA. It is suggested that the SF-1 receptor may be involved in the adverse effects of xenobiotics on P4 secretion as well as the synthesis and secretion of OT.

Introduction

Polychlorinated biphenyls (PCBs), 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and its environmental metabolite 1,1-bis(p-chlorophenyl)-2,2-bichloroethane (DDE) are permanently present in the environment as a side effect of industrialization and the chemicals that are still used in agriculture, and these PCBs substantially disrupt the reproduction of animals and humans (Corona-Cruz et al., 1999, Roselli et al., 2000). They belong to the large group of endocrine disruptors which, after entering the body, disrupt regulatory pathways by binding to the estradiol receptor – ER (Ho et al., 2008), stimulating the aryl hydrocarbon receptor – AhR (Wojtowicz et al., 2005), acting as anti-androgens (Wilson et al., 2008) or inhibiting the interactions of steroid hormone transporting proteins with their ligands (Hodgert Jury et al., 2000). Even though, DDT, DDE, PCB 77 and PCB 153 are recognized as estrogen-like xenobiotics (Nesaretnam et al., 1996, Cogliano, 1998, Li et al., 2008), their biological activity is over 1000-fold lower than that of estradiol (E2) (Matthews et al., 2000, Safe et al., 2001). These xenobiotics at a concentration of 1–10 ng/ml, can interfere with the steroidogenesis, synthesis and secretion of oxytocin (OT) from luteal and granulosa cells in vitro (Mlynarczuk et al., 2005, Mlynarczuk et al., 2009). It should be emphasized that the E2 concentrations in the follicular fluid of preovulatory follicles were determined to be in the range of 691–1023 ng/ml (Landau et al., 2000). In addition, while the amount of E2 secreted into the culture medium by granulosa cells after 72 h of incubation was 200–300 pg/ml, this concentration did not inhibit the negative impact of PCBs, DDT or DDE (Mlynarczuk et al., 2005, Mlynarczuk et al., 2009). Oxytocin, which is produced in follicles, functions as an autocrine factor and is involved in the growth and maturation of the follicle (Okuda et al., 1997). Moreover, it also plays a role in ovulation, enhances the luteinization of granulosa and theca interna cells (Tallam et al., 2000), and shifts follicular steroidogenesis toward progesterone (P4) production during cellular luteinization after the preovulatory LH surge (Brendtson et al., 1996, Jo and Fortune, 2002). However, the gene encoding the OT precursor (neurophysin I/oxytocin, NP-I/OT) is not a direct transcriptional target of the E2 receptor transcription factor, so it is unlikely that these xenobiotics affect this gene via the estradiol receptor. It is known that the “orphan” nuclear receptor Steroidogenic Factor 1 (SF-1, NR5A1), which is found in the steroidogenic cells of fetuses and adult animals (Parker et al., 2002, Walther et al., 2006), is responsible for the increase of NP-I/OT gene transcription in cattle (Wehrenberg et al., 1994), although the physiological ligand for SF-1 has not yet been identified (Benoit et al., 2006). SF-1 is also responsible for regulating the expression of genes that affect sex determination and the development of reproductive organs (WT1, DAX1) (Ozisik et al., 2002), the cyclic regulation of the reproductive processes (GnRHR, LHB, FSHR, PRLR) (Barnhart and Mellon, 1994, Holvorson et al., 1996) and metabolism (HDLR, SHP, SRB1) (Marchal et al., 1998, Zhao et al., 2001) by binding to response elements in the promoter regions of these genes, either as a monomer or a dimer (Benoit et al., 2006) in conjunction with a number of cofactors (SOX-9, WT-1, SHP) (Mlynarczuk and Rekawiecki, 2010). Moreover, SF-1 increases the expression of StAR protein (Christenson and Devoto, 2003, Manna et al., 2002), as well as several enzymes involved in ovarian (Honda et al., 1993, Leers-Sucheta et al., 1997, Michael et al., 1995) and adrenal (Morohashi et al., 1992, Zhang and Mellon, 1996) steroidogenesis. It has been shown that expression of SF-1 in rodents can be autoregulated (Falender et al., 2003). In addition, SF-1 is a transcription factor for the gene Amh1, which encodes the protein MIS and evokes the regression of the Müllerian duct during the prenatal period (Shen et al., 1994), and functions in postnatal life as one of the factors that controls follicle growth (Durlinger et al., 1999). Thus, SF-1 is considered to be an important factor for the proper course of reproduction, which has been confirmed by experiments in which the SF-1 gene was knocked out in the gonads (Jeyasuria et al., 2004). One of its potential xenoligands is the herbicide atrazine, which increases the expression of genes regulated by SF-1 (Fan et al., 2007, Pogrmic-Majkic et al., 2010). These data, and especially the ability of DDT, DDE and estrogen-like PCBs to induce increased of synthesis and secretion of OT from bovine ovarian steroidogenic cells, suggest that these compounds may adversely affect reproductive processes by acting on the SF-1 receptor.

The aim of this study was to evaluate the effect of DDT, DDE, PCB 77 and PCB 153 on (a) E2, P4 and OT secretion and (b) mRNA expression for NP-I/OT and SF1, in granulosa cells.

Section snippets

Cell preparation

Ovaries from healthy cows or sexually mature heifers were collected from a slaughterhouse within 15–20 min of slaughter, immersed in ice-cold saline (0.9% NaCl supplemented with 10 IU/ml penicillin, 100 μg/ml streptomycin, 2 μg/ml amphotericin and 100 μg/ml l-glutamine) and transported to the laboratory within 1 h. Granulosa cells were obtained by a vigorous rinse, followed by the aspiration of follicular fluid from two groups of ovarian follicles: <1 cm (small) and >1 cm (large) in diameter. Harvested

Results

The combination of studied xenobiotics with F0160 at the dose 1 × 10−5 M, decreased cells viability (P < 0.05) obtained from follicles >1 cm. However, when F0160 was used at a dose 1 × 10−6 M, only Actinomycin D significantly decreased (P < 0.001) cellular viability (Fig. 1). The same effect was observed in cells cultures from follicles <1 cm (data not shown). Therefore we assume the observed changes in granulosa cells response were not induced by cytotoxic effect of the xenobiotics when the lower dose of

Discussion

The increase in P4 and OT secretion evoked by the xenobiotics used in this study confirms the data obtained in our earlier investigations (Mlynarczuk et al., 2005, Mlynarczuk et al., 2009). However, the main aim of this work was to verify the hypothesis that SF-1 acts as an intermediate in mediating the effect xenobiotics on the synthesis and secretion of these hormones. Considering the low affinity of the xenobiotics used for the estrogen receptor (ER) (Matthews et al., 2000) on the one hand

Acknowledgments

We thank Dr. G.L. Williams (Texas A&M University, Beeville, USA) for estradiol antiserum, Dr. G. Kotwica and Dr. S. Okrasa (University of Warmia and Mazury, Olsztyn, Poland) for oxytocin and progesterone antisera, respectively. FSH (AFP-4261-A) was purchased from Dr. A.F. Parlow (Pituitary Hormones & Antisera Center, Torrance, CA, USA). This study was supported by a grant (N/N308 006136) from the National Science Centre and by the Polish Academy of Sciences.

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