Prenatal and neonatal exposure to the antiandrogen flutamide alters connexin 43 gene expression in adult porcine ovary

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Prenatal and neonatal exposure to the antiandrogen flutamide alters connexin 43 gene expression in adult porcine ovary M. Durlej*, K. Knapczyk-Stwora, M. Duda, I. Kopera-Sobota, A. Hejmej, B. Bilinska, M. Slomczynska Department of Endocrinology and Tissue Culture, Institute of Zoology, Jagiellonian University, Krakow, Poland Received 27 May 2010; received in revised form 7 August 2010; accepted 9 August 2010-08-30

Abstract Connexin 43 (Cx43) is the predominant gap junction protein within porcine ovary and is required for proper follicle and corpus luteum (CL) development. Recent research suggests maternally or neonatally mediated effects of antiandrogens on reproductive function during adulthood, notably those dependent on gap junctional communication. The current study was conducted to determine whether late gestational or neonatal exposure to the antiandrogen flutamide influences Cx43 gene expression in the adult porcine ovary. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 posnatally. After animals reached sexual maturity, the ovaries were collected from treated and nontreated (control) pigs. Expression of Cx43 mRNA and protein was determined for preantral and antral follicles and for CLs. In addition, 3␤-hydroxysteroid dehydrogenase (3␤-HSD) expression and progesterone concentration were determined for luteal tissues. In preantral follicles, Cx43 mRNA was down-regulated (P ⬍ 0.01) following maternal and neonatal flutamide exposure. In large antral follicles, Cx43 mRNA was up-regulated (P ⬍ 0.01) after neonatal flutamide administration. Immunofluorescence showed that Cx43 expression decreased (P ⬍ 0.001) in preantral follicles and increased (P ⬍ 0.001) in large antral follicles following flutamide exposure. In luteal tissues, Cx43 and 3␤-HSD expression and progesterone concentration decreased (P ⬍ 0.01) after postnatal flutamide treatment. Overall, these results suggest the involvement of androgens in the regulation of Cx43 expression in pig ovary. Moreover, alteration of Cx43 expression by the administration of flutamide during particular prenatal and neonatal time periods may affect porcine follicle development, as well as CL formation and function. © 2011 Elsevier Inc. All rights reserved. Keywords: Connexin 43; Flutamide; Androgens; Ovary; Pig

1. Introduction Recent reports have documented adverse maternally or neonatally mediated effects of antiandrogens on male reproductive function during adulthood, notably those dependent on gap junctional communication

* Corresponding author. Department of Endocrinology and Tissue Culture, Institute of Zoology, Jagiellonian University, Ingardena 6, 30060 Karkow, Poland. Tel.: ⫹48 12 663 24 66; fax: ⫹48 12 634 07 85. E-mail address: [email protected] (M. Durlej).

[1–3]. Little is known about the corresponding effects in females, but these are worthy of elucidation. The formation of porcine ovarian follicles starts on day 56 post coitum, but follicles surrounded by a single layer of squamous pregranulosa cells are present on day 106 post coitum. Primary follicles with cuboidal granulosa cells do not occur prenatally [4]. Thus, in pigs, the assembly of primordial follicles and their subsequent transition to the primary stage occur in the late gestational and neonatal period. These critical processes in ovarian biology may determine female fertil-

0739-7240/11/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2010.08.003

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ity in adulthood [5,6]. Although androgens are proposed to have a small role in these events, studies on primates [7,8] have increased understanding of their function in the promotion of early follicular development. Moreover, we have demonstrated the presence of androgen receptors (ARs) in porcine fetal ovaries at different stages of gestation [9], which suggests a capacity to respond to androgens and/or antiandrogenic factors. Gap junctions represent plasma membrane channels that connect adjacent cells in most mammalian tissues and mediate electrical and metabolic cell– cell coupling [10]. It has been documented that gap junctions built of connexin 43 (Cx43) are necessary for ovarian follicle development and oocyte growth [11–13], as well as for the formation, function, and regression of corpus luteum (CL) [14,15]. The expression of Cx43 within porcine ovarian follicles was detected between granulosa cells from the primary to the antral stage, increasing with follicular growth, and also between theca cells of antral follicles [16 –19]. In addition, based on results from mice lacking Cx43, this connexin is also required for primordial germ cell survival and migration and for early stages of folliculogenesis, up to primary follicles [20]. Thus, Cx43 is required during early stages of folliculogenesis and might be controlled by androgens, as indicated above [7–9]. Based on our recent evidence that flutamide leads to changes of Cx43 expression in the gonads of prepubertal pigs [21], we hypothesize that androgen deficiency during prenatal and neonatal windows may alter Cx43 expression, leading to abnormal folliculogenesis in adult pig ovaries and disturbed CL function. Alterations in ovarian function are often not discovered until puberty, but are manifested later in adult life [22]. Flutamide (2-methyl-N-[4-nitro-30-(trifluoromethyl)phenyl] propamide) is a nonsteroidal, pure antiandrogenic compound shown to bind the ARs and block androgen action [23]. Flutamide promotes AR translocation to the nucleus and DNA binding, but nevertheless fails to initiate transcription, inhibiting the AR signaling pathway [24]. Hence, the aim of this study was to quantify Cx43 mRNA expression using realtime PCR and localize Cx43 protein immunologically in preantral and antral follicles and CL of adult pigs following prenatal or neonatal flutamide exposure. 2. Materials and methods 2.1. Animals and experiment design Sexually mature crossbred gilts (n ⫽ 6; Large White ⫻ Polish Landrace) that exhibited 2 estrous cycles of normal duration were used for this study. Gilts were

observed for estrous behavior twice daily and mated to a fertile boar at the onset of estrus and again 12 and 24 h later. The first day of estrus was designated as day 0. Pregnant pigs were randomly divided into three groups. Animals in the first group served as a control (treated with vehicle). Gilts of the second group were injected with flutamide (Sigma–Aldrich, St. Louis, Missouri, USA) starting at day 80 post coitum (GD80). Gilts of the third group were not injected during pregnancy, but their newborn female offspring were treated with flutamide starting at day 2 post partum (PD2). Flutamide was suspended in corn oil and administered subcutaneously at a dose of 50 mg/kg body weight [according to [25,26]] five times every second day. The flutamide exposure was based on our previous studies demonstrating changes in Cx43 expression in the neonatal and immature porcine gonads [19,21]. Female offspring from control pigs (n ⫽ 3) and from pigs treated with flutamide during pregnancy (GD80; n ⫽ 3), as well as piglets injected with flutamide at day 2 post partum (PD2; n ⫽ 5) were maintained until sexual maturity. The use of the animals was approved by the National Commission of Bioethics at Jagiellonian University, Krakow, Poland (No. 4/2008). 2.2. Tissue collection Ovaries were obtained from gilts exhibiting 2 estrous cycles of normal duration. Prior to slaughter at a local abattoir, the body weight and anogenital distance (AGD) were measured. Preantral follicles (mechanically dissected from ovarian cortex), large antral follicles (7–10 mm in diameter), and midluteal CL (obtained between days 8 and 11 of estrous cycle) were excised. The ovaries were obtained from follicular phase for follicles and from luteal phase for CL collection. The collection times were consistent between experimental groups. Collected tissues were fixed in 10% buffered formalin for immunostaining or frozen in liquid nitrogen for RNA isolation. 2.3. RNA preparation and reverse transcription polymerase chain reaction (PCR) Isolation of total cellular RNA, including a 15-min DNAse I treatment, was carried out using the NucleoSpin RNA II kit (Macherey-Nagel GmbH & Co., Düren, Germany) according to the manufacturer’s specifications, eluting the RNA with 40 ␮L RNAse-free water prior to storage at ⫺80 °C. The RNA concentration was determined by an A260 measurement in a spectrophotometer Smart Plus (Bio-Rad Laboratories, GmbH,

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München, Germany) and a volume equivalent to 1 ␮g of RNA was taken for reverse transcription. Reverse transcription was performed using a highcapacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was prepared in a 20-␮L volume using the random primers, dNTP mix, RNAse inhibitor, and MultiScribe reverse transcriptase. As a negative control RNase-free water was substituted for the mRNA template in the reverse transcription reaction to exclude any possible genomic DNA contamination. Reverse transcription was performed in a Veriti thermal cycler (Applied Biosystems) with a temperature cycling program of 10 min at 25 °C, 2 h at 37 °C, and 5 min at 85 °C, with subsequent cooling to 4 °C. Samples were kept at ⫺20 °C until further analysis. PCR reactions were performed with a reaction mixture containing 1 ␮L of cDNA, 10 ␮M forward and reverse primers obtained from Institute of Biochemistry and Biophysics PAS (Warsaw, Poland), 10 mM of dinucleotide triphosphate, 10⫻ PCR buffer, and 2 units of DyNAzyme II polymerase (Finnzymes, Espoo, Finland) in a Veriti thermal cycler. The primer sequences used for PCR amplifications were as follows: for AR, forward (5=-CACATTGAAGGCTATGAGTG-3=) and reverse (5=-CCCATCCAGGAGTACTGAAT-3=) [27]; for Cx43, forward (5=-GGT GGA CTG TTT CCT CTC TCG-3=) and reverse (5=-GGA GCA GCC ATT GAA ATA AGC-3=); and for glyceraldehyde-3-phosphate dehydrogenase (GADPH) forward (5=-GGA CTC ATG ACC ACG GTC CAT-3=) and reverse (5=-TCA GAT CCA CAA CCG ACA CGT-3=) [28]. GAPDH was used as an internal control. After a denaturation cycle at 95 °C for 4 min, the temperature cycling conditions for Cx43 were 35 cycles consisting of denaturation at 95 °C for 30 sec, annealing at 65 °C for 30 sec, and extension at 72 °C for 45 sec, with acquisition at 72 °C. For GAPDH, after a denaturation cycle at 94 °C for 4 min, the conditions were 35 cycles consisting of denaturation at 94 °C for 45 sec, annealing at 57 °C for 45 sec, and extension at 72 °C for 90 sec, followed by final extension at 72 °C for 7 min. PCR products (10 ␮L) were run on 2% agarose gels containing ethidium bromide together with a readyload 100-bp DNA ladder marker (Promega, Southampton, UK) and bands were digitally imaged under UV illumination. AR, Cx43, and GAPDH mRNA were detected in the sample by presence of 242-, 232-, or 220-bp amplification products, respectively.

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2.4. TaqMan real-time PCR and data analysis Real-time PCR analyses were performed using the StepOne Real-Time PCR system (Applied Biosystems). The mRNA expression levels of the AR and Cx43 were quantified in each sample using TaqMan Gene Expression Assays (Applied Biosystems) as follows: for AR assay ID, Ss03822350_s1; for Cx43 assay ID, Ss03374839_u1. GAPDH levels were determined as an endogenous control assay (Applied Biosystems, assay ID, Ss03375629_u1). Quantitative PCR was performed with 200 ng of cDNA, 1 ␮L gene expression assay, and 10 ␮L TaqMan PCR master mix (Applied Biosystems) in a final volume of 20 ␮L. After 2 min of incubation at 50 °C, the thermal cycling conditions were 10 min at 95 °C followed by 40 repeats of 15 sec at 95 °C and 1 min at 60 °C to determine the cycle threshold number (Ct) for quantitative measurement. Relative quantification (RQ) was obtained using the 2⫺⌬⌬Ct method, adjusting the AR and Cx43 mRNAs expression to the expression of GAPDH mRNA and taking the adjusted expression in the control group as reference (RQ ⫽ 1) [29]. 2.5. Cx43 immunofluorescence in preantral and antral follicles and quantitative analysis Tissue sections (5 ␮m) were immersed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven (3 ⫻ 4 min, 750 W) to retrieve antigenicity. Endogenous peroxidase activity was prevented by incubation with 0.3% H2O2 in Tris-buffered saline (TBS, pH 7.4) and nonspecific binding sites were blocked by using 5% normal goat serum (Sigma–Aldrich) for 40 min at room temperature (RT). For visualizing the Cx43, sections were first incubated overnight at 4 °C in a humidified chamber with rabbit polyclonal anti-Cx43 antibody (1: 2000; Sigma–Aldrich). After being rinsed in TBS with 0.1% Tween 20, the sections were incubated with farred fluorescent Alexa Fluor 633 goat anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA) at 1:100 dilution for 1.5 h in the dark. Finally, the slides were mounted in Vectashield medium for fluorescence with 4=6-diamidino-2-phenylindole (DAPI; Vector Labs, Burlingame, CA, USA) and viewed under a Zeiss confocal laser scanning microscope LSM510 (GmbH, Jena, Germany). Quantitative analysis was performed on at least 10 different sections of each examined animal (three follicles per slide) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Image acquisition was performed with constant values for contrast, brightness, and pinhole and only the red

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channel was used for quantitative analysis. The results of staining detected in color images were converted into grayscale. A constant arbitrary value of threshold was then used to create binary images. Pixels corresponding to fluorescent labeling were counted and expressed as the percentage of positive pixels per unit of granulosa or theca cell area. 2.6. Cx43 and 3␤-HSD immunohistochemistry in CL and quantitative analysis For Cx43 and 3␤-HSD immunohistochemistry, CL sections were treated as described above for Cx43 immunofluorescence until primary antibody application [for details see [19,30]]. In this procedure, the primary antibody for Cx43 and its dilution were the same as for immunofluorescence. Immunostaining for 3␤-HSD was performed using a rabbit polyclonal antirecombinant mouse 3␤-HSD antibody (1:1000; a generous gift from Professor A. H. Payne, Stanford University School of Medicine, Stanford, CA, USA). Next, for both antigens, biotinylated secondary antibody, goat antirabbit IgG (1:300, 1.5 h at RT; Vector Labs) was applied. Finally, the detection of the antigens was accomplished using avidin– biotin–peroxidase complex (1:100, 40 min at RT in the dark; StreptABComplex-HRP, Dako/AS, Glostrup, Denmark). The color of the reaction was developed in TBS containing 0.01% H2O2, 0.05% diaminobenzidine, and 0.07% imidazole. Slides were dehydrated and mounted in DPX (Fluka Chemie GmbH, Buchs, Switzerland). Negative controls were performed by substituting the primary antibody with nonimmune rabbit immunoglobulin G (IgG). Slides with Cx43 were counterstained with Mayer’s hematoxylin. The sections were photographed using a Nikon Eclipse E200 microscope attached to a Coolpix 5400 digital camera (Nikon, Tokyo, Japan) with corresponding software. To quantitatively evaluate the intensity of immunoreaction, the digital images from at least 10 different sections (1 CL per slide) of each examined animal were analyzed using ImageJ software. The intensity of Cx43 and 3␤-HSD staining was expressed as a relative optical density (ROD) and calculated using the formula ROD ⫽ ODspecimen/ODbackground ⫽ log(GLblank/ GLspecimen)/log(GLblank/GLbackground), where GL is the gray level for the stained area (specimen) and unstained area (background) and blank is the gray level measured after the slide was removed from the light path [31]. 2.7. Progesterone (P4) concentration P4 concentration evaluation in homogenates of CL excised from adult porcine ovaries was determined us-

ing a radioimmunoassay as described previously by Szoltys et al. [32]. Appropriate aliquots were extracted with 2.5 mL n-hexane (Sigma–Aldrich). All samples were assayed in duplicate. Progesterone was measured using [1,2,6,7-3H]progesterone (specific activity 96 Ci/ mmol; Amersham International plc, Buckinghamshire, UK) and an antibody induced in sheep against 11␣hydroxyprogesterone succinyl: BSA (a generous gift from Professor B. Cooke, University of Glasgow, Glasgow, Scotland). Series of other steroids were tested for cross-reactivity with the following results: with pregnenolone 1.8%, with corticosterone 1.5%, with 17␣hydroxyprogesterone 0.8%, and with testosterone 0.1%. Binding of other steroids was below 0.1%. The limit of sensitivity of the assay was 20 pg/mL. Coefficients of variation within and between assays were below 5.0% and 9.8%, respectively. The concentration of P4 is expressed picograms per milligram as means ⫾ SD from at least five CL per control and experimental groups. 2.8. Statistical analysis Statistical analysis was performed using the Statistica 5.1 program (StatSoft, Inc., Tulsa, OK, USA). Differences between control and flutamide-treated groups were assessed using Student’s t test. The data were statistically evaluated with significance at P⬍ 0.05. The values are expressed as means ⫾ standard deviation (SD). 3. Results 3.1. AGD and body weight AGD (a marker of androgenicity) was greater (P ⬍ 0.05) in control and GD80 groups than in PD2 group (25 ⫾ 0.01, 24 ⫾ 0.02, and 18.4 ⫾ 0.32 mm, respectively). Body weight was not affected by flutamide treatment (the average for all groups was 92 ⫾ 2.4 kg). 3.2. Expression of mRNA for AR and Cx43 in ovarian follicles and CL The expression of mRNA for AR and Cx43 in preantral follicles (Fig. 1A), large antral follicles (Fig. 1B), and CL (Fig. 1C) was revealed using the reverse transcriptase PCR technique. Electrophoresis displayed PCR amplicons of the predicted sizes: 242 bp for AR, 232 bp for Cx43, and 220 bp for GAPDH in both control ovaries and those from flutamide-treated animals. In preantral follicles, AR and Cx43 mRNAs were down-regulated (P ⬍ 0.01) following GD80 and PD2

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Fig. 1. AR and Cx43 mRNAs expression in preantral follicles (A, A’), large antral follicles (B, B’), and corpora lutea (C, C’) of adult pig ovary from control, GD80, and PD2 groups. As an intrinsic control, the GAPDH mRNA level was measured in the same samples. (A, B, C) Representative gels electrophoresis of qualitative expression of AR, Cx43, and GAPDH mRNAs in pig follicles and CL from control, GD80, and PD2 groups. (A’, B’, C’) Relative expression of mRNA for AR and Cx43 in control and flutamide-treated adult porcine ovaries determined using quantitative real-time PCR analysis. Relative quantification (RQ) is expressed as means ⫾ SD. Asterisks indicate statistically significant differences in the expression of Cx43 gene between control and experimental groups (Student’s t test; P ⬍ 0.01). Control (n ⫽ 3), GD80 (n ⫽ 3), PD2 (n ⫽ 5).

flutamide exposure (Fig. 1A’). In large antral follicles, AR mRNA expression was up-regulated (P ⬍ 0.01) in GD80 and PD2 groups, but Cx43 mRNA expression was up-regulated (P ⬍ 0.01) only in the PD2 group (Fig. 1B’). In the CL, AR but not Cx43 mRNA expression was up-regulated (P ⬍ 0.01) in the PD2 group (Fig. 1C’). 3.3. Localization of Cx43 in porcine ovarian follicles Immunofluorescence revealed punctate Cx43 localization on the borders of granulosa cells of preantral follicles in control (Fig. 2A), GD80 (Fig. 2B), and PD2

(Fig. 2C) groups. In large antral follicles, Cx43 was observed between granulosa and theca cells of both control (Fig. 2D) and flutamide-exposed pigs (Fig. 2E and F). Cx43 displayed punctate (Fig. 2D and E) to linear (Fig. 2F) expression on the borders of granulosa cells and punctate expression within the theca compartment (Fig. 2D–F) of antral follicles. In preantral follicles, Cx43 expression decreased (P ⬍ 0.001) in GD80 and PD2 groups (Fig. 2G). In large antral follicles, Cx43 expression was greatest (P ⬍ 0.001) in the PD2 group, less in the GD80 group, and least in the control group (Fig. 2H; shaded bars). Within the theca layer,

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Fig. 2. Representative micrographs of Cx43 staining in preantral (A–C) and antral (D–F) follicles from the ovary of control and flutamide-treated adult pigs. Immunoreactive proteins were visualized using an Alexa Fluor 633 detection system (red). Nuclei were counterstained with DAPI (blue). Preantral follicles displayed punctate Cx43 immunoreactivity on the borders of granulosa cells (arrows) in control (A), GD80 (B), and PD2 (C) groups. Large antral follicles displayed punctate to linear Cx43 immunoreactivity on the borders of granulosa cells (arrows) and theca cells (open arrowheads) in control (D), GD80 (E), and PD2 (F) groups. Charts represent quantitative analysis of Cx43 in preantral follicles (G) and large antral follicles (H) of control, GD80, and PD2 groups. Bars express means ⫾ SD (shaded bars, granulosa cells; open bars, theca cells). Asterisks denote significant differences (Student’s t test, P ⬍ 0.001). Control (n ⫽ 3), GD80 (n ⫽ 3), PD2 (n ⫽ 5). G, granulosa cells; T, theca cells; scale bar ⫽ 25 ␮m.

Cx43 expression was unaffected by flutamide treatment (Fig. 2H, open bars).

luteal cell hypertrophy and cytoplasmic vacuolization (Fig. 3E).

3.4. Localization of Cx43 and 3␤-HSD in porcine CL

3.5. Luteal tissue P4 concentration

Immunohistochemistry demonstrated that Cx43 was present as a punctate staining between luteal cells in control (Fig. 3A), GD80 (Fig. 3B), and PD2 (Fig. 3C) groups. The intensity of Cx43 immunostaining decreased significantly (P ⬍ 0.05) following flutamide administration at PD2 (Fig. 3G). 3␤-HSD was localized in the cytoplasm of luteal cells from control pigs (Fig. 3D) and those from the GD80 (Fig. 3E) and PD2 (Fig. 3F) groups. The intensity of 3␤-HSD staining was lower (P ⬍ 0.01) in the PD2 group compared with the GD80 and control groups (Fig. 3H). It was notable that exposure to flutamide at GD80 was associated with

The concentration of P4 in luteal tissues from control and GD80 groups was higher (P ⬍ 0.01) when compared with the PD2 group (3501 ⫾ 294, 3384 ⫾ 204, and 1966 ⫾ 151 pg/mg, respectively). 3.6. CL cysts Histological examination by routine hematoxylin and eosin staining revealed the presence of corpus luteum cysts in the PD2 group (Fig. 4). The cavity of the cysts was surrounded by a thick, luteinized wall with an internal layer of nonluteal cells.

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Fig. 3. Representative micrographs of Cx43 (A–C) and 3␤-HSD (D–F) staining in midluteal CL from the ovaries of control and flutamide-treated adult pigs. Cx43 was observed as punctate staining on the borders of luteal cells (arrows) in control (A), GD80 (B), and PD2 (C) groups. 3␤-HSD revealed cytoplasmic expression in luteal cells (arrows) from control pigs (D) and those from the GD80 (E) and PD2 (F) groups. Control sections, in which the primary antibodies were replaced by rabbit IgG, did not exhibit any positive staining (C and F, insets). Charts represent the intensity of Cx43 (G) and 3␤-HSD (H) immunostaining expressed as a relative optical density (ROD) in control and flutamide-exposed (GD80, PD2) pigs. Bars represent means ⫾ SD. Asterisks denote significant differences at P ⬍ 0.05 for Cx43 and P ⬍ 0.01 for 3␤-HSD (Student’s t test). Control (n ⫽ 3), GD80 (n ⫽ 3), PD2 (n ⫽ 5). Scale bar ⫽ 50 ␮m (A–C) or 25 ␮m (D–F).

4. Discussion

4.1. Mechanism of Cx43 gene regulation by androgens

Genomic effects of androgens are mediated via nuclear ARs and androgens have the ability to up- or down-regulate their own receptors [33,34]. Changes in AR mRNA or protein expression result in altered androgen signaling, which may induce disturbances in androgen-dependent processes during adult life, including Cx43 regulation. Our previous research revealed no influence of flutamide exposure on Cx43 expression in the gonads of neonatal piglets [19] and changes in the expression of Cx43 in the gonads of prepubertal boars and gilts [21]. However, we expected overt effects in the adult porcine ovary after reprogramming via altered AR signaling following flutamide administration during critical developmental windows (GD80 or PD2).

As shown here, exposure to flutamide during GD80 and PD2 led to alterations in AR mRNA levels in preantral and large antral follicles and the CL of adult pigs. These findings are associated with changes in Cx43 mRNA in preantral and antral porcine follicles, but did not correspond with data from CL. Several studies have demonstrated that steroid hormones, including estrogens, progesterone, and thyroid hormones, regulate Cx43 expression [35–38]. In the rat myometrium, estrogens upregulate Cx43 mRNA, whereas P4 antagonizes this increase [36]. It is suggested that the mechanism by which the aforementioned steroids modulate the synthesis of Cx43 in myometrium occurs at the transcriptional level. This regulation is mediated through Fos/Jun proteins (AP-1 complex), which bind

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of maternal or neonatal exposure to estrogens/androgens [49]. On the other hand, Tang et al. [50] proposed that the manifestation of early life epigenetic reprogrammed gene expression is dependent on adult ovarian steroids and changes over the course of natural aging of the animal. Nonetheless, the present data reinforce the notion that androgens may be involved in the regulation of Cx43 gene expression. 4.2. Effect of flutamide on Cx43 expression in preantral follicles

Fig. 4. Representative micrograph (hematoxylin and eosin staining) depicting CL cyst from the ovary of pig following flutamide administration at PD2. Asterisk indicates the cavity of the cyst surrounded by an internal layer of nonluteal cells (arrows). LC, luteal cells; scale bar ⫽100 ␮m.

to the promoter of the gene encoding Cx43. Specifically, P4 has been shown to reduce c-fos and c-jun expression, whereas estradiol induced its increase [39,40]. The reported Cx43 regulation by estrogens is not surprising in light of the fact that the Cx43 promoter contains a series of half-palindromic estrogen-responsive elements [41,42]. Unfortunately, the mechanism by which androgens regulate Cx43 is still unknown. As reported by Cyr et al. [43], epididymis from orchidectomized rats had increased Cx43 expression, suggesting that androgens regulate Cx43 expression in this organ. Additionally, Huynh et al. [44] revealed a castration-induced increase of Cx43 mRNA and protein, indicating the regulation of Cx43 gene by androgens in rat prostate gland. The AR is a transcriptional factor that functions primarily via interaction with androgen-responsive elements in the promoter regions of target genes [45]. Although no androgen-responsive element has been identified in the Cx43 gene, the Cx43 promoter includes several activator protein (AP), cyclic adenosine monophosphate (cAMP), and specific protein (SP) sites [46 – 48]. We hypothesize that this is the mechanism of Cx43 regulation by androgens because in other gene promoters these sites have been shown to interact with hormone receptors, including AR, and activate transcription. Another explanation of how exposure to flutamide during GD80 or PD2 contributes significantly to adult Cx43 expression outcomes may involve epigenetic modifications. Recent studies have found that sex steroids have the ability to influence the methylation and acetylation state of DNA, resulting in long-term effects

As reported by Melton et al. [18], Cx43 plays a pivotal role in the first stages of porcine folliculogenesis, including the transition of primordial to primary follicles. The expression of Cx43 in granulosa cells of primordial follicles was undetectable, but observed in primary follicles. Therefore, it is suggested that enhanced transcription of Cx43 is associated with activation of follicular growth and development and can be used as a marker of these events [18]. In the present study, exposure to flutamide during periods (GD80 or PD2) critical for Cx43 expression caused down-regulation of Cx43 mRNA and protein in the granulosa cells of preantral follicles. We hypothesize that decreased Cx43 expression following flutamide administration may lead to disturbances in normal follicle development. Gap junction communication is required for granulosa cell proliferation during follicle maturation [13,14]. In developing chick retina, diminished Cx43 expression reduced cell proliferation [51]. Furthermore, decreased Cx43 abundance was observed during follicular atresia in pigs [28] and cows [52]. If reduced gap junction signaling after flutamide exposure is involved in the induction of atresia, the number of follicles ripening to ovulation might be reduced, thus decreasing fertility in pigs. Furthermore, Li and Mather [53] showed that the addition of the gap junction inhibitor, lindane, to granulosa– oocyte cultures prevents follicle formation. Thus, gap junction communication may be also important for cellular reorganization, such as in antrum development [53]. In summary, a deficiency of Cx43 in the granulosa cells of porcine preantral follicles following GD80 or PD2 flutamide exposure would be expected to hamper the morphogenesis of ovarian follicles by preventing granulosa cell proliferation or inducing atresia. 4.3. Effect of flutamide on Cx43 expression in large antral follicles In pig antral follicles, Cx43 is expressed within granulosa and theca cells layers [16,17]. It has been

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proposed that gap junctions play a more important role in granulosa cells than in theca cells, which can be explained by the fact that the granulosa layer is avascular [54]. In contrast, the theca layer contains a fine network of capillaries, which can supply nutrients, hormones, and signaling molecules by passive diffusion [54]. Herein, we demonstrated the greatest Cx43 expression between granulosa cells after GD80 and PD2 flutamide administration and unaffected Cx43 expression between theca cells. Thus, signaling through gap junctions seem to be less important in the theca than in granulosa cells. These results are opposite to those from preantral follicles. It is possible that the difference in Cx43 expression between preantral and antral follicles after flutamide exposure is that after antrum formation expression is influenced by follicle-stimulating hormone (FSH) and luteinizing hormone (LH) [12,17]. We hypothesized that the high Cx43 expression in large antral follicles might affect ovulation or the subsequent luteinization. So far, the LH surge downregulates Cx43 in preovulatory follicles [11,17], but the level of LH might be not enough to break down gap junctions and facilitate ovulation. We observed properly formed CLs in each examined ovary, but found CL cysts following PD2 flutamide administration. The cysts included a large cavity surrounded by luteal cells, which indicates incomplete or rupture luteinization. This kind of cysts is rare [55] and is characterized by intense endocrine activity [56]. On the other hand, their presence causes morphological changes in the endothelium of oviduct and uterus mucosa, which are proposed as a cause of persistent infertility in culled pigs [55].

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tion between Cx43 expression and the secretion of progesterone, which is the major function of CL. Our results are in agreement with those from sheep [61], which demonstrated inhibition of Cx43 mRNA expression by Cx43 siRNA and decreased progesterone production by luteal cells. This suggests that Cx43 is involved in the regulation of luteal steroidogenesis in sheep. Moreover, as shown previously, Cx43 is involved in the regulation of endocrine function of adrenal cells [62], and channels formed by Cx43 are crucial to steroidogenesis in bovine adrenal tissue [63]. Recently, a positive correlation between increased Cx43 protein expression and enhanced insulin secretion by the pancreas was demonstrated [64]. This evidence indicates that Cx43 contributes to the regulation of hormone production in several endocrine glands, including the CL. Nevertheless, Cx43 seems to be only a part of the complex mechanism of normal CL function. 5. Conclusions In conclusion, the present data demonstrate that exposure to flutamide during crucial developmental windows (GD80 or PD2) influences Cx43 mRNA and protein expression in adult porcine ovaries. Moreover, altered Cx43 expression may affect the development of ovarian follicles or the formation and function of porcine CL. Although we showed an association between AR and Cx43 mRNA expression, these results did not fully support the hypothesis of Cx43 regulation by androgens. In future studies, it will be important to determine the molecular mechanism of this regulation.

4.4. Effect of flutamide on Cx43 expression in CL Acknowledgments Cx43 is probably a major protein that forms gap junctional channels in the CL [14]. Previous reports from cows [57,58], sheep [59], and baboons [60] demonstrated the comparable pattern of Cx43 expression in CL during growth, differentiation, and regression. The expression of Cx43 in luteal tissue was greatest during the early and midluteal phases and decreased during the late luteal phase of the estrous cycle [57– 60]. Recently, Cx43 was proposed as being involved in the regulation of progesterone secretion by luteal tissue [61]. The present study revealed that exposure to flutamide during the neonatal window resulted in diminished Cx43 protein expression in CL, although Cx43 mRNA was at the same level as in the control. The concentration of P4 in the homogenates of luteal tissues sharply decreased, and this parallels the low intensity of 3␤-HSD staining. As shown here, there is an associa-

The authors are grateful to Professor Marek Koziorowski and Dr Anna Tabecka-Lonczynska (Department of Physiology and Reproduction of Animals, University of Rzeszow, Poland) for their care animals, assistance with flutamide treatment, and collection of ovaries. This work was financially supported by the Ministry of Science and Higher Education, Grant N N303339835, and in part by Grant N N303017737. References [1] Kelce WR, Gray LE. Environmental antiandrogens as endocrine disruptors. In: Naz RK, ed. Endocrine disruptors. Effects on male and female reproductive systems. New York: CRC Press; 1999:247–278. [2] Anway MD, Rekow SS, Skinner MK. Comparative anti-androgenic actions of vinclozolin and flutamide on transgenerational

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