Domestic Animal Endocrinology 53 (2015) 17–25

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Ovarian stimulation with human chorionic gonadotropin and equine chorionic gonadotropin affects prostacyclin and its receptor expression in the porcine oviduct I. Ma1ysz-Cymborska, A. Andronowska* Department of Hormonal Action Mechanisms, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 February 2015 Received in revised form 14 March 2015 Accepted 3 April 2015

Prostaglandins are well-known mediators of crucial events in the female reproductive tract, eg, early embryo development and implantation. Prostacyclin (PGI2) is the most synthesized prostaglandin in the human oviduct during the postovulatory period, indicating its important role in supporting and regulating the oviductal environment. The present study was undertaken to determine the influence of insemination and ovarian stimulation with human chorionic gonadotropin (hCG)/equine chorionic gonadotropin (eCG) on PGI2 synthesis in the porcine oviduct on day 3 post coitus. Mature gilts (n ¼ 25) were assigned into 2 experiments. In experiment I, gilts were divided into cyclic (control; n ¼ 5) and inseminated (control; n ¼ 5) groups. In experiment II, there were 3 groups of animals: inseminated (n ¼ 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n ¼ 5), and superovulated/inseminated (1,500 IU eCG,1,000 IU hCG; n ¼ 5) gilts. Parts of oviducts (isthmus and ampulla) were collected 3 days after phosphate-buffered saline treatment (cyclic gilts of experiment I) or insemination (all other groups). Expression of messenger RNA for PGI2 synthase (PGIS) and its receptor (IP) was measured by real-time reverse transcription polymerase chain reaction (real-time RT PCR) and protein levels using Western blots. Concentrations of the PGI2 metabolite 6-keto PGF1a were evaluated by enzyme immunoassay and localization of PGIS and IP in the oviductal tissues using immunohistochemical staining. Insemination by itself increased PGIS protein levels in the oviductal isthmus (P < 0.05) and IP protein expression in the ampulla (P < 0.05). The concentration of 6-keto PGF1a increased significantly in the oviductal ampulla after insemination (P < 0.05). Induction of ovulation decreased IP protein levels in the oviductal ampulla (P < 0.05), whereas superovulation reduced IP levels in both parts of the oviduct (P < 0.01). Synthesis of 6-keto PGF1a was reduced by induction of ovulation and by superovulation in the oviductal ampulla (P < 0.05). Immunohistochemical staining confirmed the presence of PGIS in the muscular layer of the isthmus and both mucosa and muscular layers of the ampulla. IPpositive cells were observed in both mucosal and muscular layers of the isthmus and ampulla. This study showed for the first time that PGI2 synthesis and IP expression are insemination dependent. Moreover, ovarian stimulation with hCG/eCG decreases IP expression and 6-keto PGF1a concentrations in porcine oviducts. Therefore, disturbances in PGI2/IP expression and synthesis may lead to disruption of the oviductal environment and, in turn, perturbed development of embryos and their transport to the uterus. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Pig Oviduct Prostacyclin hCG eCG

* Corresponding author. Tel.: þ48 89 5393120; fax: þ48 89 524 01 24. E-mail address: [email protected] (A. Andronowska). 0739-7240/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2015.04.002

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1. Introduction

2. Materials and methods

Prostaglandins (PGs) are well-known proinflammatory mediators, which exert many actions in the body. The biosynthesis of PGs requires conversion of arachidonic acid to PGG2 and then the unstable endoperoxide PGH2, catalyzed by PGH synthase, also called cyclooxygenase (COX-1 and COX-2). Subsequently, PGH2 is catalyzed by individual PGs synthases to: prostacyclin (PGI2) by PGI synthase (PGIS), PGE by PGE synthase, and PGF2a by PGF synthase. Different cells of all parts of the body synthesize PGs on a variety of stimuli [1,2]. It is well documented that, in the female reproductive tract, PGs are crucial factors in key processes such as fertilization, embryo development, and implantation [3,4]. A multiplicity of PGI2 actions result from binding with the IP receptor, which belongs to a group of receptors coupled with G proteins [5]. Predominantly, PGI2 binding to IP induces the Gs-dependent signaling pathway, leading to an increase in cAMP [6]. Nevertheless, various cells seem to activate different G-type signaling pathways via binding of PGI2 with IP. For example, in cultured preadipocytes and human erythroleukemia cell line cells, incubation with PGI2 stimulates the Gg-dependent PGI2 signaling pathway leading to increased Ca2þ concentrations in these cells [7,8]. Although it was revealed that 40% to 50% of the PGs synthesized in human oviducts was PGI2 [9], PGE2 and PGF2a are still the most studied PGs in this part of the female reproductive tract [4]. The influence of oviduct-derived PGI2 on mouse embryos in vitro is well established [10,11]. Supplementing culture media with PGI2 increased the frequency of mouse embryo hatching as well as implantation and live births [12,13]. However, in the porcine oviduct there is still very little information about PGI2 synthesis. Since Kim et al [14] documented that iloprost, a PGI2 analogue, stimulates oocyte meiotic maturation and early development in pigs, it could be assumed that PGI2 synthesis in the oviduct is crucial for ensuring an environment beneficial for early embryo development. Moreover, PGI2 is also considered as a relaxant factor, which may regulate contractions of oviductal smooth muscle and in this way indirectly participate in gamete and embryo transport [15]. Therefore, PGI2 seems to be a crucial factor determining the function of the oviduct. Although there is evidence that PGI2 synthesis and release in the porcine uterus during the estrous cycle is influenced by many factors, such as steroid hormones [16], knowledge about the regulation of PGI2 synthesis in the porcine oviduct is still inadequate. Our previous study revealed that ovarian stimulation with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) to induce ovulation and evoke superovulation completely changed the synthesis of PGE2 and PGF2a [17]. Therefore, we propose that disturbances of proper embryo development and viability, which were documented after administering hCG/eCG [18] may be connected with alterations in PGI2 production and expression of its receptor in porcine oviducts. The present study was undertaken to examine, for the first time, whether insemination by itself and with hCG/eCG affect PGI2 synthesis in the porcine oviduct on day 3 post coitus (p.c.).

2.1. Experimental scheme All experimental procedures involving use of animals were approved by the Animal Ethics Committee, University of Warmia and Mazury in Olsztyn (No. 69/2008/N). Twentyfive crossbreed gilts of similar age (5–5.5 mo), weight (100– 110 kg), and genetic background were observed daily for the onset of estrus. After exhibiting one natural cycle, gilts were divided into 2 experiments. 2.1.1. Experiment 1: effect of insemination on PGIS and IP expression and 6-keto PGF1a concentration in the porcine oviduct Ten mature gilts were assigned to the cyclic (control; n ¼ 5) or inseminated (n ¼ 5) groups. The onset of estrus (day 0) was determined as the day of occurrence of a standing reflex in the presence of a boar. Gilts in the cyclic (control) group received a 100 mL intrauterine infusion of phosphate-buffered saline (PBS; pH 7.4), whereas the other group was inseminated twice via a transcervical catheter with 100 mL of semen (containing 2.5  109 spermatozoa), diluted in Safe Cell Plus commercial extender (IMV Technologies, Szczecinek, Poland), 12 and 24h after detection of their third estrus. The ratio of neat semen-tosemen extender was determined according to the concentration and motility of the spermatozoa. 2.1.2. Experiment 2: influence of hCG and eCG treatment on PGIS and IP expression and 6-keto PGF1a concentration in porcine oviducts Gilts (n ¼ 15) were divided into 3 groups: inseminated (control group; n ¼ 5), induced ovulation/inseminated (n ¼ 5), and superovulated/inseminated (n ¼ 5). Day 0 was determined as the day of occurrence of a standing reflex in the presence of a boar. Gilts that were only inseminated (control group) received 100 mL of diluted semen, via a transcervical catheter, 12 and again 24 h after detection of their third estrus. Between days 12 and 16 of their second estrous cycle, gilts of the induced ovulation group were injected with a single dose of 750 IU eCG (Folligon; Intervet, Boxmeer, The Netherlands), followed by 500 IU hCG (Chorulon; Intervet) 72 h later. For superovulation, gilts received 1,500 IU eCG followed by 1,000 IU hCG 72 h later between days 12 and 16 of their second estrus cycle. Gilts of both hormonally treated groups received 100-mL intrauterine infusions of diluted semen via transcervical catheter 24 and again 48 h after hCG administration (as described by Ma1yszCymborska et al [2013, 2014]). 2.2. Sample collection Oviducts (isthmus and ampullary parts) were collected immediately after slaughter, 3 days after the second insemination, and frozen in liquid nitrogen for total RNA and protein extraction or fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin for immunohistochemical staining. Each oviduct was flushed with 2 mL of PBS to obtain oocytes/ embryos for verifying the effectiveness of insemination. Corpora lutea were counted to evaluate the superovulation yield (as described by Ma1ysz-Cymborska et al [2014]).

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2.3. Total RNA extraction, reverse transcription, and real-time quantitative polymerase chain reaction (PCR) Total RNA was extracted from porcine oviducts using a commercial kit (Total RNA Prep Plus kit; A&A Biotechnology, Gdansk, Poland). The RNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc, DE, USA), and RNA quality was assessed using a Bioanalyzer Agilent 2100 (Agilent Technologies, Waldbronn, Germany). The integrity number of the isolated RNA ranged from 7.0 to 9.0. Next, RNA was incubated with DNase I (Invitrogen Life Technologies, Inc, Carlsbad CA, USA) according to the manufacturer’s instructions. Subsequently, the reverse transcription mix was added (1X 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; High Capacity cDNA Reverse Transcription kit; Applied Biosystems, Foster City, CA, USA). Briefly, RNA was denatured according to the manufacturer’s protocol. In addition, 2 types of RT controls were made, one without complementary DNA (cDNA) and a second one without the reverse transcriptase. The cDNA obtained was diluted in nuclease-free water (Promega) and stored at 80 C until real-time PCR analysis. Diluted cDNA (5 ng/mL) were used for RT-PCR analysis with the 7900HT Fast Real-Time PCR System (Applied Biosystems). Each sample contained 3-mL (15 ng) cDNA, 1.5-mL RNAse-free water (Promega), 5-mL TaqMan Universal MasterMix II, with UNG (Life Technologies) and 0.5-mL TaqMan assays (Life Technologies): PTGIS (Ss03374149_m1), PTGIR–IP (designed and sequenced based on XM_003355952.2 National Center for Biotechnology Information reference sequence). The following PCR conditions were performed: an initial denaturation step (15 min at 95 C), followed by 40 cycles of denaturation (15 s at 95 C) and annealing (60 s at 60 C). All samples for each gene were assayed in duplicate and run on the same plate (384 wells). Each PCR run included a nontemplate control with water added instead of cDNA and an RT negative control for each gene. Stability of the reference genes b-actin (Ss03376081_u1), cyclophilin (Ss03394780_g1), and GAPDH (Ss03375435_u1) was assessed using the statistical algorithm Normfinder 2.0. Based on the results of this analysis, the most stably expressed gene was b-actin. Expression of bactin in the oviduct was constant in both experiments; therefore, data obtained from the real-time PCR for PGIS and IP were normalized using the ratio of examined messenger RNA (mRNA) to the b-actin mRNA. Quantification of gene expression was performed using the comparative Ct method [19]. 2.4. Western blot analysis Total fractions (50 mg per sample) of homogenized oviducts were dissolved in a sodium dodecyl sulfate (SDS) gelloading buffer (50-mM Tris-HCl, pH 6.8; 4% SDS, 20% glycerol, and 2% b-mercaptoethanol), heated to 95 C for 5 min, and separated on 10 or 12% (for PGIS and IP, respectively) SDS-polyacrylamide gel electrophoresis. Next, proteins were electroblotted onto 0.45-mm pore size polyvinylidene difluoride membranes using wet transfer in a transfer buffer (20-mM Tris-HCl buffer, pH 8.2; 150-mM glycine, 20% methanol) for 1.5 h. Subsequently, nonspecific binding sites were blocked with 5% nonfat

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instant milk dissolved in TBS-T buffer (Tris-buffered saline, 0.1% Tween-20) for 1.5 h at 24 C. Then, blots were incubated overnight at 4 C with primary antibodies specific for each protein: polyclonal rabbit anti-PGIS antibody (1:150; Cayman Chemicals, Ann Arbor, MI, USA), or polyclonal rabbit anti-IP antibody (1:200, Cayman Chemicals). The next day, membranes were washed in TBS-T, 3 times for 10 min each, and incubated with alkaline phosphatase– conjugated secondary anti-rabbit IgG antibodies (1:10,000; Sigma–Aldrich Co) at 24.2 C for 1.5 h and washed another 3 times in TBS-T. Next, immunodetection was done using a standard alkaline phosphatase visualization method. Each sample was standardized against b-actin (1:3000; Abcam, Cambridge, UK) expression on each blot. Negative controls for the primary antibody were done using specific blocking peptides (Cayman Chemicals). Blots were photographed and counted using the VersaDoc MP 4000 System (Bio-Rad Laboratories, Inc, CA, USA). 2.5. Immunohistochemistry Immunohistochemistry was done using paraffin blocks, cut to 6-mm sections using a Reichert-Leica microtome (Reichert Inc, NY, USA) and transferred onto Superfrost silane-coated slides (Menzel Gläser, Braunschweig, Germany) in a water bath. Next, the sections were incubated at 56 C for 40 min and then deparaffinized in xylene 2 times for 5 min each. Subsequently, sections were rehydrated in a graded series of ethanol (100%, 96%, 70%, H2O) for 5 min per step at 24.5 C, incubated in citrate buffer (sodium citrate, 0.05% Tween-20; pH 6.00) for 5 min, and denatured in citrate buffer at 90 C for 40 min. Then, the sections were cooled to 24.5 C for 30 min and washed 3 times in TBS containing 0.2% Triton X-100 (Sigma–Aldrich Co) for 10 min, followed by incubation in 10% hydrogene peroxide for 20 min at 24.6 C. Subsequently, the sections were washed 3 times in TBS for 10 min each and incubated in 0.75% glycine for 30 min at 24.5 C. Next, the sections were washed 3 times in TBS for 10 min each at 24.6 C and blocked in a blocking buffer: PBS, 0.1% bovine serum albumin, and 5.5% thimerosal, containing 10% NGS (Normal Goat Serum; Sigma–Aldrich Co) for 1 h at 24.8 C. After that, slides were incubated overnight at 24.5 C with primary rabbit polyclonal antibodies diluted in blocking buffer/10% NGS (PGIS 1:100; IP 1:50; Cayman Chemicals, Inc). The next day, sections were washed 3 times in TBS for 10 min each, incubated with biotinylated secondary antibody (goat antirabbit IgG, 1:400; Vector Laboratories, Inc, Burlingame, CA, USA) for 1 h at 25.3 C, and washed 3 times in TBS for 10 min each. Subsequently, the sections were incubated with A and B solutions of the Vectastain ABC kit (Vector Laboratories), as described by the manufacturer, then washed 3 times in TBS, incubated in DAB solution (Sigma–Aldrich Co), counterstained in Mayer’s hematoxylin, and dehydrated. A negative control staining of primary antibody was accomplished by using blocking peptides (Cayman Chemicals, Inc) suitable for each protein, whereas a secondary antibody control was performed by replacing the primary antibodies with 10% NGS (Sigma–Aldrich Co). Sections were mounted and analyzed using a Zeiss Axio Imager Z1 microscope (Zeiss, Germany).

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2.6. Enzyme immunoassay of 6-keto PGF1a Concentrations of 6-keto PGF1a in oviductal homogenates were determined using a commercial Enzyme immunoassay kit (Cayman Chemicals) according to the manufacturer’s protocol. The sensitivity of the assay was 1.6 pg/mL, and intra-assay and interassay coefficients of variance were 3.9% and 7.8%, respectively.

2.7. Statistical analysis Statistical analyses were performed using GraphPad Prism 6.0 (Graphpad Software, Inc, San Diego, CA, USA). The remaining analyses were conducted using 2-way analysis of variance, followed by Bonferroni’s post hoc test to determine the effects of treatments (insemination, induction of ovulation, and superovulation) and anatomic location (isthmus vs ampulla). All numerical data are presented as the mean  standard error of the mean, and differences were considered statistically significant at the 95% confidence level (P < 0.05). 3. Results 3.1. Influence of insemination on PGIS and IP expression and 6-keto PGF1a concentration in porcine oviducts 3.1.1. Messenger RNA expression of PGIS and IP after insemination Insemination did not affect PGIS and IP mRNA expression in the isthmus and in the ampulla (Fig. 1A, B). However, expression of mRNA for these factors was different in the 2 oviduct regions. Expression of PGIS was much lower in the ampulla than in the isthmus (P < 0.01; Fig. 1A). Similarly, mRNA of IP was lower in the ampulla than the isthmus of both groups of animals (P < 0.05; Fig. 1B). 3.1.2. Protein expression of PGIS and IP after insemination Western blot analysis revealed that insemination by itself increased the PGIS protein level in the isthmus of the oviduct (P < 0.05, Fig. 1C), without any changes in the ampulla. The IP protein level was higher in the ampulla, but not in the isthmus, of inseminated gilts compared with cyclic gilts (P < 0.05, Fig. 1D). There were significant differences in IP protein expression between the oviductal isthmus and ampulla. Levels of IP were much lower in the ampulla than the isthmus of cyclic and inseminated gilts (P < 0.001 and P < 0.05, respectively; Fig. 1D). 3.1.3. The concentration of 6-keto PGF1a in porcine oviducts after insemination The concentration of 6-keto PGF1a was significantly increased in the oviductal ampulla of inseminated gilts compared with the cyclic group (P < 0.05; Fig. 2). There were no changes in 6-keto PGF1a concentration after insemination in the isthmus of the oviduct, nor any differences between the 2 parts of the oviduct of cyclic and inseminated gilts (Fig. 2).

3.2. Effect of hCG and eCG on mRNA expression of PGIS and IP in oviductal tissues 3.2.1. Messenger RNA expression of PGIS and IP after hCG and eCG treatment No significant differences in mRNA expression of PGIS and IP in the oviductal isthmus and ampulla after stimulation with eCG/hCG (Fig. 3A, B) were observed. Only in the inseminated gilts without hormonal manipulation (control group), there was a significant decrease of IP mRNA expression in the oviductal ampulla vs the isthmus (P < 0.05; Fig. 3B). 3.2.2. Protein expression of PGIS and IP after hCG and eCG treatment There was no significant difference in the protein level profile of PGIS in the ampulla of both hormone-stimulated groups (Fig. 3C). Induction of ovulation reduced the IP protein level in the ampulla (P < 0.01; Fig. 3D), whereas superovulation decreased IP levels in both parts of the oviduct (P < 0.001; Fig. 3D). Examination of differences in PGIS and IP proteins expression between the 2 regions of the oviduct revealed reduced PGIS protein levels in the ampulla compared with the isthmus in inseminated only (P < 0.01; Fig. 3C), induced ovulation (P < 0.05; Fig. 3C), and superovulated/ inseminated gilts (P < 0.01; Fig. 3C). Similarly, IP protein levels were lower in the ampulla compared with the isthmus of inseminated only and induced ovulation/inseminated groups of gilts (P < 0.05 and P < 0.0001, respectively, Fig. 3D). 3.2.3. The concentration of 6-keto PGF1a in porcine oviducts after hCG and eCG treatment Induction of ovulation and superovulation both significantly decreased 6-keto PGF1a concentrations in the oviductal ampulla in comparison with the inseminated only gilts (P < 0.05; Fig. 4). There were no significant differences in 6-keto PGF1a concentration after hCG and eCG treatments in the isthmus of the oviduct nor any differences between the 2 parts of the oviducts of all 3 groups (Fig. 4). 3.3. Cellular localization of PGIS and IP in the porcine isthmus and ampulla In the oviductal isthmus, PGIS-positive cells were observed only in the smooth muscle cells (Fig. 5A–C). In the ampulla of the oviduct, strong PGIS staining was found in the muscular layer (Fig. 5I, K). However, there was also a weak PGIS-positive staining in epithelial and lamina propria cells of the mucosa (Fig. 5J). IP-positive cells were observed in smooth muscle cells and epithelial cells of the mucosa of the isthmus (Fig. 5E–G). In the ampulla, strong IP staining was found in the smooth muscle cells and apical parts of the mucosal epithelium of the ampulla (Fig. 5M–O). A weak IPpositive staining was also observed in lamina propria cells of the mucosa of the ampulla (Fig. 5N). 4. Discussion Among all parts of the reproductive tract, the most important just after ovulation is the oviduct. Because of its anatomic and physiological properties, the oviduct provides the proper environment for sequential occurrence of

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Fig. 1. Influence of insemination on mRNA (A,B) and protein (C,D) expression of PGIS and IP in oviductal isthmus and ampulla, expressed as the mean  SEM of ratios relative to b-actin. (E) Representative western blot images of PGIS and IP. Mean values with different letters denote a significant difference (P < 0.05; small letters for isthmus and capital for ampulla). Asterisks represent differences between isthmus and ampulla in cyclic and inseminated groups of gilts (*P < 0.05, **P < 0.01, ***P < 0.001).

gamete maturation, fertilization, and early embryo development. Although PGI2 is the most synthesized PG in the human oviduct [9], the most established PGs in the oviduct, are still PGE2 and PGF2a. Therefore, the present study was performed to determine for the first time the possible role of PGI2 and its receptor in the oviduct on day 3 p.c. and the effects of hCG and eCG on oviductal PGI2 synthesis. 4.1. Insemination The oviductal environment may be modulated by spermatozoa and oocytes in different ways including gene

expression and protein/cytokine synthesis [20,21]. Our present study revealed that the porcine oviduct synthesizes high levels of PGI2 on day 3 p.c. Similar to PGE2, PGI2 is considered to be a relaxant factor [22]. Therefore, a 2-fold increase of the IP receptor in the ampulla may be crucial for proper oviductal contractions and, in turn, the velocity of embryo transport to the uterus. This idea is strongly supported by our immunohistochemical staining results for PGI2 and its receptor. A large amount of PGIS in oviductal smooth muscle and its absence in oviductal mucosa of the isthmus suggests a role for PGI2 in muscle contractions. However, tubal transport of

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The influence of PGI2 on sperm motility is still in question [12,26]. However, we suggest that an abundant level of PGI2 in the oviductal ampulla, the region where fertilization takes place, may be crucial for the functions and/or performance of spermatozoa. It is well known that in the oviductal ampulla, more than 50% of all epithelial cells are secretory cells [27]. Knowing that PGI2 together with NO stimulates vascular permeability [28,29], we propose that increased PGI2 levels in the oviductal ampulla may be connected with intensified oviductal fluid formation on day 3 p.c. Altogether, increased synthesis of PGI2 correlated with higher expression of IP receptor in the oviductal ampulla after insemination, strongly suggesting a role for PGI2 in porcine early embryo development in the postovulatory period. Fig. 2. Concentration of 6-keto PGF1a in the isthmus and ampulla of the oviduct after insemination. Data are expressed as the mean  SEM. Mean values with different letters denote a significant difference (P < 0.05; small letters for isthmus and capital for ampulla).

gametes and embryos could not be effective without the cilia on the epithelial cells of the oviduct. The ciliary beat frequency (CBF) is dependent on hormonal changes during the estrous cycle [23]. PGE2 and PGF2a stimulated the CBF of rabbit oviducts in vitro [24]. Therefore, we presume that increased CBF on days 2 to 3 after insemination, which was reported previously [23,25], may be an effect of increased PGI2 and IP levels in the oviductal ampulla, leading to elevated levels of cAMP and calcium ions.

4.2. hCG and eCG stimulation Induction of ovulation and superovulation using hCG and eCG are well-known tools for animal breeding improvement [30,31]. Nevertheless, there is growing concern about their possible adverse effects on the environment of the female reproductive tract and on embryos. Our previous research revealed that use of hCG/eCG affected PGE2 and PGF2a and the vascular endothelial growth factor (VEGF) system [32]. Similarly, our present study indicated for the first time that ovarian stimulation also influences PGI2 and IP expression in porcine oviducts on day 3 p.c.

Fig. 3. Effects of induction of ovulation and superovulation on mRNA (A,B) and protein (C,D) expression of PGIS and IP in the oviductal isthmus and ampulla. Data are expressed as the mean  SEM of ratios relative to b-actin. Mean values with different letters denote a significant difference (P < 0.05; small letters for isthmus and capital for ampulla). Asterisks represent differences between isthmus and ampulla in inseminated, induced ovulation and superovulated groups of gilts (*P < 0.05, **P < 0.01, ****P < 0.0001).

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Fig. 4. Concentrations of 6-keto PGF1a in the isthmus and ampulla of the oviduct after induction of ovulation and superovulation. Data are expressed as the mean  SEM. Mean values with different letters denote a significant difference (P < 0.05; small letters for isthmus and capital for ampulla).

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Stimulation with hCG and eCG did not influence mRNA and protein expression of PGIS, either in the isthmus or in the ampulla. Surprisingly, there was a significant decrease of the PGI2 metabolite 6-keto PGF1a in the oviductal ampulla. Because it is COX-2, but not COX-1, that is predominantly responsible for PGI2 synthesis [33,34], reduced levels of 6-keto PGF1a could be explained by disturbances in COX-2 expression. However, our previous study revealed that in the ampulla hCG/eCG did not affect COX-2 protein expression [17]. Therefore, the decrease in PGI2 synthesis may be a direct effect of hCG and eCG on PGIS activity. On the other hand, because VEGF stimulates PGI2 production via VEGFR2 (KDR/Flk-1; [35]), reduced levels of KDR protein in the oviductal ampulla, as revealed in our previous research [32], may be the reason for disturbances in PGI2 synthesis in this part of the oviduct. The ampulla of the oviduct is the longest and the most important region of the tube, which produces two-thirds of the total daily oviductal fluid secretion [36]. Many factors determine fluid secretion, like steroid hormones, VEGF, or PGs [37,38]. The increased capacity to synthesize PGI2,

Fig. 5. The cellular localization of PGIS in isthmus (A – magnification x100; B,C – magnification x400) and ampulla of the oviduct (I – magnification x100; J,K – magnification x400). Localization of IP positive cells in oviductal isthmus (E – magnification x100; F,G – magnification x400) and ampulla (M – magnification x100; N,O – magnification x400). Negative controls of isthmus (D – magnification x100, H – magnification x400) and ampulla (L – magnification x100, P – magnification x400).

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occurring on days 2 to 3 in mice [13] and porcine oviducts (present study), may indicate an important role of PGI2 in tubal fluid production and release. Moreover, it is established that oviduct-derived PGI2 is crucial for porcine oocyte maturation, as manifested by enhanced blastocyst structure and survival [14]. Thus, disturbances in PGI2 synthesis, which were caused by hCG/eCG stimulation as revealed in the present study, may be the reason for disturbances in early embryo development. The main physiological functions of PGI2 are made possible via binding with the membrane IP receptor. Binding of PGI2 to IP induces cell-specific signaling through activation of Gs, Gg, and Gi pathways [39]. Gg-dependent PGI2 signaling leads to increased Ca2þ concentrations in HEL cells [7]. For that reason, we propose that the IP receptor may play an important role in CBF, which is calciumdependent [23]. As revealed recently, CBF increased on days 1 to 3 p.c. to support proper gamete fertilization and embryo transport to the uterus [23]. Therefore, disorders in IP signaling caused by hCG and/or eCG, as revealed in the present study, may affect proper gamete interactions and embryo transport. Moreover, the higher concentrations of calcium in the isthmus of the bovine oviduct seemed to stimulate the sperm acrosome reaction [40]. Consequently, reduced levels of IP in the oviductal isthmus and ampulla of hCG/eCG–stimulated pigs, found in the present study, may lead to disturbances in sperm functions. On the other hand, IP through activation of GS increases cAMP levels [6] and IP3 3-kinase activity, resulting in decreased muscle tone [41]. As a result, lowered levels of IP may affect oviductal contractions and in turn accelerate transport of gametes and embryos through the oviduct. In consequence, it would lead to premature arrival of embryos in the uterus, thus desynchronizing embryo stage and uterine receptivity. Collectively, in the present study, we revealed for the first time that PGI2 synthesis increases in the porcine oviduct after insemination. This may be beneficial for sperm activation, fertilization, and early embryo development. Moreover, we documented that hormones routinely used for induction of ovulation and superovulation strongly affect PGI2 synthesis and IP expression. Therefore, failures in achieving full efficiency of pig breeding may be a consequence of disturbances in PGI2/IP expression in the porcine oviduct during the postovulatory period.

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Acknowledgments

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This study was supported by the National Science Centre (grant no, 2012/05/N/NZ9/02444) and Polish Ministry for Science and Higher Education (grant no, N N311 334436) and will be part of the primary author’s PhD thesis. Author of the article was supported by the European Union within the European Social Fund.

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Ovarian stimulation with human chorionic gonadotropin and equine chorionic gonadotropin affects prostacyclin and its receptor expression in the porcine oviduct.

Prostaglandins are well-known mediators of crucial events in the female reproductive tract, eg, early embryo development and implantation. Prostacycli...
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