Peroxisome proliferator-activated receptor (PPAR) isoforms are differentially expressed in peri-implantation porcine conceptuses

Theriogenology 101 (2017) 53e61

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Peroxisome proliferator-activated receptor (PPAR) isoforms are differentially expressed in peri-implantation porcine conceptuses Agnieszka Blitek*, Magdalena Szymanska Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 February 2017 Received in revised form 18 May 2017 Accepted 10 June 2017 Available online 13 June 2017

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors. PPARs are critical regulators of glucose homeostasis and lipid metabolism, and affect cell proliferation and differentiation. In the current study, we examined (1) the profiles of PPARA, PPARD, and PPARG mRNA expression and DNA binding activity in porcine conceptuses collected on Days 10e11 (spherical and tubular conceptuses), 11e12 (filamentous conceptuses), 13e14, and 15e16 (elongated conceptuses) of pregnancy, (2) the presence of PPARA, PPARD, and PPARG proteins in Days 10, 12, and 15 conceptuses. Moreover, we analyzed the abundance of retinoid X receptor (RXR; PPARs heterodimer partner) transcripts as well as the correlation between PPARs mRNA expression and the expression of genes important for and/or associated with elongation of porcine conceptuses: aromatase (CYP19A1), prostaglandin endoperoxide synthase 2 (PTGS2), glucose transporter 1 (SLC2A1), and interleukin 1B (IL1B). PPARA mRNA expression in conceptuses did not change during Days 10e14 of gestation, but was greater on Days 15e16 compared to Days 10e11 (P < 0.05). A considerable increase in PPARD and PPARG mRNA expression was observed in filamentous conceptuses from Days 11e12 compared to spherical and tubular conceptuses from Days 10e11 (P < 0.01), followed by a decrease on Days 13e14 and 15e16 (P < 0.05). PPARA, PPARD, and PPARG proteins were present in conceptus tissue demonstrating nuclear localization clearly visible on Days 12 and 15 of pregnancy. DNA binding activity of the PPARD isoform was greater in filamentous conceptuses from Days 11e12 than in spherical and tubular conceptuses from Days 10e11 (P < 0.01). Moreover, concentrations of active PPARD and PPARG proteins in nuclear fractions of conceptus tissue were greater on Days 11e12 compared to Days 13e14 and 15e16 of pregnancy (P < 0.05). RXRA, RXRD, and RXRG mRNA expression in conceptuses increased on Days 11e12 compared to Days 10e11 (P < 0.05). PPARD and PPARG mRNA expression showed strong positive correlations with PTGS2 mRNA expression (P < 0.0001). Additionally, PPARD gene expression correlated with SLC2A1 and IL1B mRNA expression (P < 0.01). Collectively, these results indicate that among all three PPARs expressed in peri-implantation porcine conceptuses, PPARD and PPARG may be involved in conceptus elongation before implantation. © 2017 Elsevier Inc. All rights reserved.

Keywords: Pig Pregnancy Conceptus development Peroxisome proliferator-activated receptors Implantation

1. Introduction Peroxisome proliferator-activated receptors (PPARs) are members of a nuclear receptor superfamily of ligand-dependent transcription factors. Until now, three isoforms of PPAR named PPARA, PPARD and PPARG have been described, each encoded by a separate gene. For complete activation, PPARs must heterodimerize with retinoid X receptors (RXR) to form a PPAR/RXR complex that binds to a specific DNA sequence called a PPAR-response element in a

* Corresponding author. E-mail address: [email protected] (A. Blitek). http://dx.doi.org/10.1016/j.theriogenology.2017.06.013 0093-691X/© 2017 Elsevier Inc. All rights reserved.

target gene [1,2]. Activity of PPARs can be regulated by a broad range of both natural and synthetic factors. Arachidonic acid and its metabolites are among the endogenous compounds, while synthetic ligands are represented mainly by fibrates, thiazolidinediones, and non-steroidal anti-inflammatory drugs [2e4]. PPARs are expressed in a broad range of tissues and control the expression of a wide array of genes mainly involved in the regulation of energy and glucose homeostasis, lipid metabolism, and cell proliferation and differentiation [1,5,6]. Increasing evidence suggests that PPARs are critical players in immune and inflammatory responses [1,7,8]. A role of PPAR isoforms has also been reported for female reproductive functions [9e12]. Activation of PPARs was observed during steroidogenesis, tissue remodeling, and

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angiogenesis in the ovary [9,13]. Spatiotemporal expression of PPARD and/or PPARG was detected in the endometrium of mice, rats, pigs, sheep and cattle [14e19], indicating their involvement in the process of embryo implantation. In the porcine endometrium, PPARG mRNA expression was low until Day 15 of pregnancy; then, significantly increased and maintained high until Day 30. Endometrial PPARA and PPARD genes showed similar profiles of expression on Days 5e30 of pregnancy, with a decrease detected on Days 11e12 (the period of the maternal recognition of pregnancy) and 22 to 30 (the end of implantation) [19]. In mice, Ppard expression was markedly up-regulated in the stroma surrounding implanting blastocysts and was abundantly expressed in decidua during the post-implantation period [14]. Moreover, disruption of the Ppard or Pparg genes in this species was lethal to embryos due to placental dysfunction [20e22]. In the human placenta, PPARG was involved in hCG synthesis and trophoblast cell differentiation [23,24], trophoblast invasion [25] and fatty acid uptake [26]. Pre-implantation mouse and bovine embryos express Ppard and PPARG, respectively [27,28]. Moreover, the expression of PPARA, PPARD, and PPARG in ovine conceptuses changes along with the stage of conceptus development during the peri-implantation period [15]. Recent data clearly showed that PPARG is an essential regulator of ovine conceptus elongation, because inhibition of PPARG protein synthesis resulted in growth retardation and malformation of conceptuses on Day 14 of gestation [29]. In ruminants and pigs, elongation of conceptuses during the peri-implantation period is the process, which allows extensive contact between trophectoderm and maternal uterine epithelium [30,31]. In contrast to sheep, there are no data available showing the presence of PPARs and their possible role in peri-implantation porcine conceptuses. Therefore, the current study was conducted to examine (1) the profiles of mRNA expression and DNA binding activity of PPARA, PPARD, and PPARG in porcine conceptuses collected on Days 10e16 of pregnancy, and (2) PPARA, PPARD, and PPARG protein localization in conceptuses of different morphology collected on Days 10 (spherical), 12 (filamentous), and 15 (elongated) of gestation. Moreover, we analyzed the abundance of RXR transcripts in conceptus samples as well as the correlation between expression levels of all three PPARs mRNA and the expression of genes important for and/or associated with the elongation of porcine conceptuses: aromatase (CYP19A1; cytochrome P450 family 19 subfamily A member 1), prostaglandin endoperoxide synthase 2 (PTGS2), glucose transporter 1 (SLC2A1) and interleukin 1B (IL1B). 2. Materials and methods 2.1. Animals and sample collection All procedures involving the use of animals were conducted in accordance with the national guidelines for agricultural animal care and were approved by the Animal Ethics Committee, University of Warmia and Mazury in Olsztyn, Poland. Thirty-four crossbred gilts (Polish Landrace x Duroc) of similar genetic background from one commercial herd were used. Gilts that exhibited two estrous cycles of normal length were checked daily for the next estrus behavior with intact males. Then, gilts were bred 12 and 24 h after detection of estrus. The day of the second breeding was considered the first day of pregnancy. Gilts were slaughtered on Days 10 (n ¼ 7), 11 (n ¼ 5), 12 (n ¼ 5), 13 (n ¼ 5), 14 (n ¼ 5), 15 (n ¼ 3), and 16 (n ¼ 4) of pregnancy. Conceptuses were recovered by gently flushing uterine horns with 20 mL of PBS (pH 7.4). The day of pregnancy was confirmed by the size and morphology of conceptuses. On Day 11, spherical, tubular and filamentous forms of conceptuses were obtained. Conceptuses from all gilts were assigned to the following groups/days: Days 10e11

(spherical conceptuses with a diameter of 3e8 mm and tubular conceptuses with a length of 10e25 mm), Days 11e12 (filamentous conceptuses), Days 13e14 and Days 15e16 (elongated conceptuses). Spherical and tubular conceptuses were classified into one group based on the lack of significant gene expression changes as demonstrated previously [32]. Conceptuses were snap-frozen in liquid nitrogen and stored at 80  C until total RNA isolation and nuclear fraction preparation. For immunohistochemistry, Days 10, 12, and 15 conceptuses were fixed in 4% paraformaldehyde solution, dehydrated in a graded series of ethanol, and embedded in paraffin. Additionally, adipose tissue and liver were collected and used as positive control tissues for the presence of PPAR proteins during immunostaining procedure [1,2]. 2.2. Total RNA isolation and real-time PCR Total RNA was extracted from frozen conceptuses using a Total RNA Prep Plus kit (A&A Biotechnology, Gdansk, Poland) and treated with DNase I (Invitrogen Life Technologies Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. Samples were reverse transcribed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Briefly, the RT reaction mix contained 1 x RT buffer, 1 mM dNTP mix, 2.5 mM RT random primers, 1 U/mL RNase inhibitor, and 2.5 U/mL MultiScribe™ Reverse Transcriptase. First, the RNA was denatured at 70  C for 10 min, then the RT reaction was carried out at 25  C for 10 min, 37  C for 120 min and then 85  C for 5 min. Diluted cDNA from RT-PCR was used to analyze quantitative gene expression with an ABI Viia7 Sequence Detection System (Life Technologies). To evaluate PPARA, PPARD, PPARG, RXRA, RXRD, RXRG, CYP19A1, PTGS2, SLC2A1, IL1B, ACTB (b-actin), and GAPDH (glyceraldehyde-3phosphate dehydrogenase) gene expression, 15 ng of complementary cDNA was amplified using PPARA (Ss03380164_u1), PPARD (Ss03394198_g1), PPARG (Ss03394829_m1), RXRA (Ss033753 49_u1), RXRD (Ss04247366_m1), RXRG (Ss03373714_m1), CYP19A1 (Ss03384876_u1), PTGS2 (Ss03394692_m1), SLC2A1 (Ss033747 47_s1), IL1B (Ss04321151_m1), ACTB (Ss03376081_u1) and GAPDH (Ss03375435_u1) TaqMan Gene Expression assays (Life Technologies). Each PCR reaction (10 ml) was performed in duplicates in a 384-well plate using the following conditions: initial denaturation for 10 min at 95  C, followed by 40 cycles of 15 s of denaturation at 95  C and then 60 s of annealing at 60  C. Data obtained by Real-time PCR were analyzed using the Miner method [33] as previously described [34]. NormFinder software [35] was used to select the most stable reference genes. Analysis indicated overall the same stability value in ACTB and GAPDH gene expression (0.349). Therefore, all expression data for each target gene were divided by geometric averaging of ACTB and GAPDH (the stability value was 0.123). 2.3. ELISA of DNA binding activity Nuclear fractions of frozen conceptus samples were prepared using the Nuclear Extraction Kit (10009277; Cayman Chemicals, Ann Arbor, MI, USA) and used to analyze the DNA binding activity of PPAR isoforms. For this, the PPARA Transcription Factor Assay Kit (10006915; Cayman Chemicals), PPARD Transcription Factor Assay Kit (10006914; Cayman Chemicals) and PPARG Transcription Factor Assay Kit (10006855; Cayman Chemicals) were used. Each kit included a 96-well plate coated with a specific double stranded DNA sequence containing the peroxisome proliferator response element (PPRE). PPARs contained in nuclear extracts bound specifically to the PPRE, then each PPAR isoform was detected by the addition of specific primary antibody. A secondary antibody

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2.5. Statistical analysis All statistical analyses were performed using GraphPad PRISM v. 6.0 (GraphPad Software, Inc., San Diego, CA). To test PPARA, PPARD, PPARG, RXRA, RXRD, RXRG, CYP19A1, PTGS2, SLC2A1, and IL1B mRNA expression as well as PPARA, PPARD, and PPARG DNA binding activity in conceptus tissue, one-way ANOVA followed by Bonferroni's post-hoc test was used. All numerical data are expressed as mean ± SEM, and means were considered to be statistically different at P < 0.05. Correlations between PPARA, PPARD, or PPARG transcript abundance and mRNA expression of CYP19A1, PTGS2, SLC2A1, and IL1B were assessed using the Pearson test. 3. Results 3.1. PPARA, PPARD, and PPARG mRNA expression The mRNA expression of all three PPAR isoforms varied in developing conceptuses collected during the peri-implantation period (Fig. 1). PPARA mRNA expression in the porcine conceptuses did not change significantly on Days 10e14, but was greater on Days 15e16 compared to Days 10e11 (P < 0.05). A considerable increase in PPARD gene expression was observed in filamentous conceptuses from Days 11e12 of pregnancy compared to spherical and tubular conceptuses from Days 10e11 (P < 0.01), followed by a decrease in conceptuses from Days 13e14 and 15e16 (P < 0.01). An almost 10-fold increase in PPARG mRNA expression was observed in filamentous conceptuses collected on Days 11e12 compared to spherical and tubular conceptuses on Days 10e11 (0.041 ± 0.005 vs. 0.0049 ± 0.002; P < 0.0001). After Day 12, the abundance of PPARG transcripts decreased (P < 0.05, when compared to Days 11e12), but was still greater than on Days 10e11 (P < 0.01).

PPARA mRNA

(relative abundance)

0.010

b ab

0.005

ab

a 0.000

10-11

11-12

13-14

15-16

a

a

13-14

15-16

c

c

13-14

15-16

PPARD mRNA

0.08

(relative abundance)

For immunostaining, serial conceptus, liver and adipose tissue sections at 4 mm were mounted on SuperFrost Plus microscope €ser; Braunschweig, Germany), and paraffin was slides (Menzel-Gla removed by heating slides to 60  C and washing in xylene. After samples were rehydrated in a graded series of ethanol (100%e70% [v/v]), 30% (v/v) hydrogen peroxide was added for 30 min to block endogenous peroxidase activity. Antigenic sites were unmasked in citrate buffer (10 mM sodium citrate, 0.05% Tween 20; pH 6.0) at 99  C for 15 min. After that, SEA BLOCK Blocking Buffer (Cat. No. 37727; Thermo Scientific, Rockford, IL, USA) was added for 1 h. The primary antibodies used were: rabbit polyclonal anti-human PPARA (dilution 1:20; sc-9000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-human PPARD (dilution 1:20; sc-7197, Santa Cruz Biotechnology, Inc.), or mouse monoclonal anti-human PPARG (dilution 1:20; sc-7273, Santa Cruz Biotechnology, Inc.). Incubation with primary antibodies was performed overnight at 4  C. Subsequently, sections were treated with biotinylated goat anti-rabbit IgG (1:500; Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) or biotinylated horse antimouse IgG (1:400; Vectastain ABC kit, Vector Laboratories). Negative control staining was accomplished by replacing the primary antibody with rabbit IgG negative control (I-1000, Vector Laboratories) or mouse IgG1 isotype control (ab170190, abcam, Cambridge, UK). Slides were examined under a light microscope and photographed using an Olympus digital camera.

0.06

b 0.04

0.02

a

0.00 10-11

11-12

0.08

PPARG mRNA

2.4. Immunohistochemistry

0.015

(relative abundance)

conjugated to HRP was added to provide colorimetric readout. All assays were performed according to the manufacturer's protocol. Absorbance was measured at 450 nm and calculated per total protein content determined [36] in nuclear extracts.

55

0.06

b

0.04

0.02

a 0.00 10-11

11-12

days of pregnancy Fig. 1. Expression of PPARA, PPARD, and PPARG mRNA in porcine conceptuses collected on Days 10e16 of pregnancy. Values from Real-time PCR for PPARs were normalized to geometric averaging of GAPDH and ACTB gene expression. Column labeled 10e11 contains data for spherical and tubular conceptuses collected on Days 10e11; column labeled 11e12 is for filamentous conceptuses collected on Days 11e12; remaining columns are for elongated conceptuses collected on Days 13e14 and 15e16 of pregnancy. Data are expressed as the mean ± SEM (n ¼ 6e10). Bars with various letters are significantly different.

3.2. PPARA, PPARD, and PPARG DNA binding activity The use of PPARA-, PPARD-, and PPARG-specific transcription factor assay kits allowed detection of each PPAR isoform protein in nuclear extracts of conceptus tissue. In this way, we analyzed the presence of active, DNA-bound PPAR proteins. The profiles of PPARA, PPARD, and PPARG proteins activity changed during the studied period of conceptus development (Fig. 2). Higher concentrations of PPARA were observed in nuclear extracts of conceptus tissue from Days 10e11 and 11e12 of pregnancy when compared to Days 15e16 (P < 0.05). The greatest content of PPARD transcription factor was detected in filamentous conceptuses collected on Days

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(relative absorbance)

DNA binding activity

0.05

0.03

(relative absorbance)

DNA binding activity

a

a

0.02

ab

0.01

1.2

10-11

11-12

13-14

b 15-16

PPARD

1.0

b

0.8 0.6 0.4

a

a

a

0.03

10-11

11-12

13-14

15 - 16

PPARG a

0.02

0.01

ab

b b

0.00

10-11

11-12

13-14

3.4. RXRA, RXRD, and RXRG mRNA expression RXRA mRNA expression in conceptuses increased in filamentous forms collected on Days 11e12 compared to spherical and tubular forms from Days 10e11 (P < 0.05), and remained stable until Days 15e16 (Fig. 4). The greatest RXRD mRNA expression was detected in filamentous conceptuses from Days 11e12 compared both to spherical and tubular conceptuses from Days 10e11 (P < 0.01) and to elongated conceptuses from Days 13e16 (P < 0.05). A considerable increase of RXRG gene expression was observed in filamentous conceptuses from Days 11e12 compared to spherical and tubular conceptuses from Days 10e11 (P < 0.05). After Day 12, RXRG mRNA expression showed a transient decrease detected on Days 13e14 (P < 0.05; when compared to Days 11e12). 3.5. CYP19A1, PTGS2, SLC2A1, and IL1B mRNA expression and their correlation with the abundance of PPAR transcripts

0.2 0.0

(relative absorbance)

observed in conceptuses from Days 12 and 15. Moreover, weak immunoreaction was detected in the cytoplasm of trophoblast cells. Only spherical Day 10 conceptuses showed uniform cellular staining for all PPARs. Adipose tissue was used as a positive control tissue for PPARD and PPARG protein expression, while liver was used as a positive tissue for PPARA protein presence. PPAR isoforms showed strong nuclear expression in a respective positive control tissue.

0.04

0.00

DNA binding activity

PPARA

15 - 16

days of pregnancy Fig. 2. DNA binding activity of PPARA, PPARD, and PPARG in porcine conceptuses collected on Days 10e16 of pregnancy. Column labeled 10e11 contains data for spherical and tubular conceptuses collected on Days 10e11; column labeled 11e12 is for filamentous conceptuses collected on Days 11e12; remaining columns are for elongated conceptuses collected on Days 13e14 and 15e16 of pregnancy. Values are expressed as the mean ± SEM (n ¼ 5e7). Bars with various letters are significantly different.

11e12 when compared to spherical and tubular (Days 10e11; P < 0.01) or elongated (Days 13e14 and 15e16; P < 0.01) conceptuses. Similar to PPARD, the concentration of active PPARG protein was greater in conceptuses from Days 11e12 compared to conceptuses from Days 13e14 and 15e16 (P < 0.05) of pregnancy. 3.3. PPARA, PPARD, and PPARG protein localization As demonstrated in Fig. 3, immunoreactive PPAR proteins were present in spherical, filamentous and elongated forms of conceptuses collected on Days 10, 12, and 15 of pregnancy; respectively. A strong nuclear staining of PPARA, PPARD and PPARG isoforms was

Profiles of CYP19A1, PTGS2, SLC2A1, and IL1B mRNA expression in peri-implantation porcine conceptuses are depicted in Fig. 5. Elevated expression of CYP19A1 mRNA in conceptuses collected on Days 10e12 was followed by a decrease observed in conceptuses from Days 13e14 (P < 0.05) and maintained low until Days 15e16 (P < 0.01, when compared to Days 10e11). The greatest expression of PTGS2 mRNA was detected in filamentous conceptuses from Days 11e12 compared to conceptuses from all other days studied (P < 0.01). SLC2A1 and IL1B genes showed similar profiles of expression with an increase observed between Days 10e11 and 11e12 (P < 0.01), followed by a dramatic decrease detected in conceptuses collected on Days 13e14 (P < 0.001, when compared to Days 11e12). Low level of SLC2A1 and IL1B transcripts was maintained until Days 15e16 (P < 0.05, when compared to Days 10e11 and P < 0.001, when compared to Days 11e12). Table 1 shows coefficients of correlation between PPARA, PPARD or PPARG transcript abundance and the expression of CYP19A1, PTGS2, SLC2A1 or IL1B genes in conceptuses on Days 10e16 of pregnancy. PPARA mRNA expression displayed no correlation with the expression of CYP19A1, PTGS2, SLC2A1, or IL1B. In contrast to PPARA, PPARD and PPARG genes showed a strong positive correlation with PTGS2 mRNA expression (P < 0.0001). Moreover, PPARD expression correlated with SLC2A1 (P < 0.0001) and IL1B (P < 0.01) gene expression. 4. Discussion In the pig, blastocysts undergo a rapid transition from spherical to filamentous forms between Days 10 and 12 of pregnancy, achieving their final length until Day 16 [30,31]. Conceptus transformation involves cellular hypertrophy and remodeling, migration of trophectoderm and extra-embryonic endoderm cells, reduction in blastocyst diameter, and an increased conceptus length and weight [31,37]. Because activation of PPARs plays an important role in transport, cellular uptake, and metabolism of lipids and glucose, PPARs appear to be essential regulators of conceptus elongation. Our tested hypothesis was that the expression of PPAR isoforms varies during the process of porcine conceptus development within

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Fig. 3. PPARA, PPARD, and PPARG protein localization in porcine conceptuses of different morphology collected on Days 10 (spherical conceptuses), 12 (filamentous conceptuses), and 15 (elongated conceptuses) of pregnancy. Liver was used as a positive control tissue for PPARA protein presence, while adipose tissue was used as a positive control tissue for PPARD and PPARG proteins. Arrows indicate nuclear staining. NC e negative controls accomplished by replacing the primary antibody with rabbit IgG negative control (liver and Day 15 conceptus) or mouse IgG1 isotype control (adipose tissue); scale bars, 20 mm.

the peri-implantation period. Indeed, the results of this study showed that both mRNA expression and DNA binding activity of PPARD and PPARG in porcine conceptuses changed substantially along with conceptus transformation. Moreover, mRNA expression of PPARD highly correlated with mRNA expression of PTGS2, SLC2A1 and IL1B, which are all known to participate in conceptus development. These results indicate that PPARs are involved in the

process of porcine conceptus elongation preceding implantation. Very little data are available concerning PPARA expression and role in peri-implantation embryos. The absence of reproductive defects in Ppara-deficient mice suggests that this transcription factor is not essential for female fertility in this species [38]. However, expression of PPARA was demonstrated in human and rat term placentas [39]. During early pregnancy, abundant

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RXRA mRNA

(relative abundance)

0.20

0.15

b 0.10

b

b

13-14

15-16

a

0.05

0.00

10-11

11-12

RXRD mRNA

(relative abundance)

0.005 0.004

b 0.003 0.002

a

a

a

13-14

15-16

0.001 0.000

10-11

11-12

RXRG mRNA

(relative abundance)

0.00015

b

0.00010

0.00005

ab a

a

0.00000 10-11

11-12

13-14

15-16

days of pregnancy Fig. 4. Expression of RXRA, RXRD, and RXRG mRNA in porcine conceptuses collected on Days 10e16 of pregnancy. Values from Real-time PCR for RXRs were normalized to geometric averaging of GAPDH and ACTB gene expression. Column labeled 10e11 contains data for spherical and tubular conceptuses collected on Days 10e11; column labeled 11e12 is for filamentous conceptuses collected on Days 11e12; remaining columns are for elongated conceptuses collected on Days 13e14 and 15e16 of pregnancy. Data are expressed as the mean ± SEM (n ¼ 6e10). Bars with various letters are significantly different.

concentrations of PPARA transcripts were detected in ovine trophoblasts on Day 7 followed by very low expression on Days 9e17 [15], which is the period of conceptus elongation and initial implantation in this species [40]. In the current study, PPARA mRNA expression was greater in elongated porcine conceptuses collected on Days 15e16 of gestation than in spherical and tubular conceptuses from Days 10e11. In the pig, initial attachment of trophectoderm to the uterine epithelium starts at approximately Day 13 of gestation, with a more stable adhesion observed on Day 16 [41]. Therefore, up-regulation of PPARA in elongated conceptuses indicates the importance of this isoform in the process of implantation rather than in rapid conceptus elongation. However, the activity of PPARA protein binding to PPRE showed the opposite

profile to its mRNA, with lower values found in conceptus tissue on Days 15e16 compared to Days 10e12. Nevertheless, the lack of differences in DNA binding activity between spherical and filamentous forms of conceptuses led us to conclude that PPARA may not be the primary PPAR isoform involved in the rapid elongation of conceptus trophoblast occurring on Days 10e12 of gestation in the pig. Until now, there has been only one available report showing the presence of PPARD and PPARG proteins in pig conceptuses: both isoforms were detected in trophoblast cells of Day 25 pregnant sows [16]. In the current study, we examined expression and activation of both PPAR isoforms in conceptuses collected during the peri-implantation period. Interestingly, the expression of PPARD and PPARG mRNA was substantially increased in filamentous conceptuses collected on Days 11e12 compared to spherical and tubular conceptuses from Days 10e11. Moreover, the highest content of active DNA-bound PPARD protein was found in conceptus tissue on Days 11e12. The expression profile of PPARG protein was similar to that of PPARD. Therefore, the present results clearly indicate a possible role of PPARD and PPARG in the process of conceptus elongation in the pig. Similarly, PPARD and PPARG transcript abundance increased gradually between Days 7 and 14 of ovine pregnancy, as conceptus development progressed [15]. Further in utero loss-of-function studies showed that PPARG is an essential regulator of conceptus elongation in the sheep, because blocking PPARG mRNA translation, but not that of PPARD, resulted in severely growth-retarded conceptuses found in the uterine lumen on Day 14 of gestation [29]. The expression of PPARG isoform was also intensively studied in bovine peri-implantation Day 15 conceptuses. The abundance of PPARG transcripts increased substantially in tubular compared to ovoid conceptuses, with a further increase of transcripts in filamentous conceptuses compared to both ovoid and tubular forms. Moreover, functional analysis pointed to PPARG as an important upstream regulator of transcriptome changes observed during conceptus elongation in cattle [42]. In contrast to ruminants, a role of Ppard among all PPAR isoforms seems to be crucial for mouse blastocyst development. While Ppard gene expression increased from the 8-cell to the blastocyst stage, both Ppara and Pparg were not expressed [43]. Moreover, activation of Ppard stimulates cell proliferation, protects cells from apoptosis, and promotes blastocyst hatching in mice [43,44]. In vitro development of porcine embryos was also enhanced in the presence of iloprost [45], which is a synthetic ligand of PPARD receptor. However, detailed mechanisms of intracellular iloprostdependent signaling in embryos remain to be elucidated. Nevertheless, results of the current study indicate that similar to other species, PPARD and/or PPARG activation is important for conceptus development in the pig. A previous study showed positive immunostaining for PPARD and PPARG proteins in porcine trophoblast tissue on Day 25 of pregnancy [16]. However, no data concerning PPARA protein localization in pig embryos/trophoblasts were available until now. The current study is the first demonstrating the presence of all three PPAR isoforms in pig conceptuses collected on Days 10, 12, and 15 of pregnancy. Immunoreactive PPAR proteins were present in spherical, filamentous and elongated forms of conceptuses. A strong nuclear staining of PPARA, PPARD, and PPARG isoforms was observed in Days 12 and 15 conceptuses, what confirms that PPARs are active in trophoblast tissue. Only Day 10 spherical conceptuses showed uniform cellular staining for PPARs. In the present study, we detected the transcripts for all PPARs' heterodimerization partners in the porcine conceptus tissue. Interestingly, greater RXRA, RXRD, and RXRG gene expression was observed in filamentous conceptuses from Days 11e12 compared to spherical and tubular conceptuses from Days 10e11. It indicates

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Fig. 5. Expression of CYP19A1, PTGS2, SLC2A1, and IL1B mRNA in porcine conceptuses collected on Days 10e16 of pregnancy. Values from Real-time PCR for studied genes were normalized to geometric averaging of GAPDH and ACTB gene expression. Column labeled 10e11 contains data for spherical and tubular conceptuses collected on Days 10e11; column labeled 11e12 is for filamentous conceptuses collected on Days 11e12; remaining columns are for elongated conceptuses collected on Days 13e14 and 15e16 of pregnancy. Data are expressed as the mean ± SEM (n ¼ 6e10). Bars with various letters are significantly different.

important role of RXRs during conceptus elongation. Simultaneous up-regulation of PPARD, PPARG, RXRA, and RXRG gene expression was also reported for ovine conceptuses during their elongation between Days 12 and 14 of pregnancy [15]. During the period of rapid elongation from the spherical to filamentous form on Days 10e12 of pregnancy, porcine conceptuses secrete high amounts of estrogens, mainly E2, which is a pregnancy recognition signal in this species [31,46]. Accordingly, aromatase (a member of the estrogen biosynthetic pathway) expression increases dramatically in tubular conceptuses compared to spherical ones, with a further increase occurring in filamentous conceptuses [47,48]. As we demonstrated here, no correlation was detected between each PPAR and CYP19A1 mRNA expression. Although activation of PPARs can affect synthesis and/or metabolism of steroids in the ovary [4,9], current results do not confirm a relationship between PPARs and E2 synthesis in porcine conceptus tissue. Besides E2, developing porcine conceptuses synthesize and secrete prostaglandins (PG) and IL1B, which may have auto-, para-, and intra-crine effects on both endometrial functions and conceptus development [49,50]. Both PTGS2 (key enzyme in PG synthesis) and

Table 1 Correlations between PPARA, PPARD, and PPARG mRNA abundance and expression of genes important for and/or associated with conceptus elongation. Genea

CYP19A1

SLC2A1

PTGS2

IL1B

PPARA PPARD

0.27 0.35

PPARG

0.11

0.05 0.77 P < 0.0001 0.31

0.12 0.97 P < 0.0001 0.62 P < 0.0001

0.17 0.48 P < 0.01 0.30

a PPAR ¼ peroxisome proliferator-activated receptor; CYP19A1 ¼ cytochrome P450 family 19 subfamily A member 1; SLC2A1 ¼ solute carrier family 2 member 1; PTGS2 ¼ prostaglandin endoperoxide synthase 2; IL1B ¼ interleukin 1 beta.

IL1B mRNA expression are highly up-regulated in filamentous forms of conceptuses [51e53]. Additionally, we analyzed expression of the SLC2A1 gene, which encodes the membrane transporter responsible for glucose uptake, and glucose is an important energy source for developing embryos [54,55]. We observed an increase in PTGS2, SLC2A1, and IL1B mRNA expression in filamentous conceptuses compared to spherical/tubular conceptuses. Our current study showed that PPARA mRNA expression did not correlate with genes selected as important for conceptus elongation in the pig. Interestingly, PPARD mRNA expression correlated with SLC2A1 and IL1B transcript abundance. Moreover, the level of both PPARD and PPARG mRNA expression highly correlated with the expression of PTGS2 mRNA. IL1B, which is an inducer of phospholipase A2, may regulate arachidonic acid release from cell membranes, thus ensuring membrane fluidity necessary for trophoblast remodeling and contributing to PG synthesis [56]. Because PG are endogenous ligands of PPAR, it is possible that PTGS2-derived PGs, including PGE2, PGI2, or PGD2 may act via PPARD or PPARG activation to stimulate elongation-related genes in porcine conceptuses. Among 482 transcripts differentially expressed in conceptuses during their transition from spherical to filamentous forms were genes involved in lipid biosynthesis and metabolism, amino acid transport, ATPase activity, and cell motility and growth [32]. Moreover, our present results are in agreement with those obtained for bovine conceptuses, in which the level of PPARG mRNA correlated with the levels of PTGS2, SLCO2A1 (PG transporter) and SLC27A6 (fatty acid transporter) gene expression [42]. Therefore, we suggest that, similar to ruminants, activation of PPARs is necessary for pig conceptus elongation prior to implantation. 5. Conclusions In summary, this is the first demonstration of the presence of all

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three PPAR isoforms in porcine peri-implantation conceptuses. The profiles of PPARD and PPARG mRNA expression and activity of protein to bind to specific DNA sequence as well as up-regulation of RXRs mRNA expression in peri-implantation conceptuses indicate important roles of PPAR isoforms in porcine conceptus elongation. This conclusion is supported by existence of the correlation between PPARD and PPARG mRNA expression and the abundant expression of genes involved in conceptus development. However, the intracellular signaling pathways involved in PPAR-dependent survival and proliferation of porcine conceptuses remain to be determined. Competing interests The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

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