Experimental design
Animals and general management have been described earlier in a study on cytokine expression in the gilt oviduct [6]. Crossbred Landrace/Yorkshire gilts, about 8-9 months old, were brought from the University experimental herd (Swedish University of Agricultural Sciences) and euthanized (stunned by captive bolt followed by exsanguination). In brief, the gilts were inseminated with 100 mL of seminal plasma (SP, n = 4), spermatozoa in extender (Beltsville thawing solution, BTS [17], SPZ, n = 4) or extender (BTS) alone (n = 4). In four control gilts the disposable insemination catheter (Goldenpig™, IMV, L’Aigle, France) was inserted without any fluid being inseminated (control, n = 4). The gilts were euthanized 35-40 h after insemination when tissue samples were collected, frozen in liquid nitrogen and stored at -80°C. Uterine samples were collected from the mesometrial side, 20-30 cm from the tip of the uterine horn. Oviductal samples were collected from the isthmus as well as from the infundibulum. The experimental study was approved by the Ethical Committee for Experimentation with Animals, Uppsala, Sweden (SLU.kv.Fe.2006.5.4.-15).
Semen preparation and insemination
The semen collection and preparation is detailed in [6]. In brief, semen was collected from four boars with proven fertility, then pooled and centrifuged. At every sampling the motility of the spermatozoa was examined. The SP was thereafter separated from the sperm layer, centrifuged twice to remove any remaining spermatozoa and stored at -20°C until insemination.
Spermatozoa were isolated from semen using the single layer centrifugation technique (SLC, [14]). Briefly, a layer of semen extended with BTS was carefully layered on top of a colloid solution (Androcoll™-P; SLU) and centrifuged. The sperm pellet generated was transferred to a new tube containing BTS and washed by centrifugation. BTS was added to generate a dose of 100 mL containing 5 × 109 spermatozoa for insemination.
Gilts were inseminated once in their second or third oestrus at 15-20 h after the first signs of standing reflex (about 15-20 h before expected ovulation).
Quantification of endometrial PMNs
Quantification of PMNs in the endometrium was performed previously [7]. In brief, paraffin-embedded uterine samples were sectioned and stained with hematoxylin-eosin. Blind evaluation of slides was performed using a light microscope with a ×40 objective. PMNs were counted in the surface epithelium and in the sub-epithelial connective tissue using an ocular micrometer, where the ocular field length and area obtained corresponded to 0.25 mm and 6.25 × 10-2 mm2 of tissue, respectively. Ten random areas were chosen for evaluation in two different sections from each animal. Results are presented as the number of PMNs per ocular micrometer length (number of cells/OML) in the surface epithelium as well as per ocular micrometer area (number of cells/OMA) in the sub-epithelial connective tissue [7].
RNA extraction
Frozen oviductal tissues (isthmus and infundibulum) were cut in cross sections, resulting in a longitudinal length of 2-3 mm per piece. Endometrial samples were obtained by separating the endometrium from the myometrium, and cutting it into pieces of about 15-20 mm3. Tissues were homogenized using a micropestle; total RNA was extracted using 1 mL of TRIzol® reagent (Invitrogen Ltd., Paisley, UK) and purified using the RNeasy Mini kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. RNA pellets were rehydrated in nuclease-free water (Qiagen, Crawley, UK). Total RNA content and purity was determined by 260/280 nm ratio using the NanoDrop® ND-1000 (Saveen &Werner AB, Limhamn, Sweden). All samples used had ratios higher than 1.9. In addition, nine random samples from each experiment were checked for total RNA integrity using microcapillary electrophoresis (Agilent 2100 Bioanalyzer; Agilent Technologies, Waldbronn, Germany), as described for quality assurance of the extracted RNA [4].
Quantitative Real-Time PCR
Complementary DNA (cDNA) fragments were obtained using the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions, where 1 µg of total RNA was used for reverse transcription with both oligo(dT) and random hexamers as primers. Real-time PCR was performed using the Rotor-Gene 3000 (Corbett Life Science, Sydney, Australia) with the iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. IL-23 primers (forward: 5’-TGT GGA TCT ACC AAG AGA AGA GG and reverse: 5’-AGG ACT GAC TGT TGT CCC TGA), were designed to amplify a 110 bp cDNA fragment corresponding to nucleotides 168-277 of the pig IL-23 alpha subunit p19 mRNA sequence [GenBank Accession number NM_001130236]. Hypoxanthine phosphoribosyl-transferase (HPRT) primers (forward: 5’-GTG ATA GAT CCA TTC CTA TGA CTG TAG A and reverse: 5’-TGA GAG ATC ATC TCC ACC AAT TAC TT [GenBank Accession number U69731]; [22]) and cyclophilin A primers (forward: 5’-TGC TTT CAC AGA ATA ATT CCA GGA TTT A and reverse: 5’-GAC TTG CCA CCA GTG CCA TTA [GenBank Accession number AY266299]) were used to amplify cDNA fragments of 104 and 77 bp respectively. The reaction volume was set to 25 µL and the concentration of each primer was 0.2 µM. 1 µL cDNA was added to each reaction. Duplicate samples were submitted to amplification as follows: 95 °C for 2 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Nonspecific amplification was eliminated by generating dissociation curves. Amplification products were randomly analyzed by agarose gel electrophoresis. Reactions without template added were used as negative controls. Efficiency (E) was calculated for each amplicon in a randomly chosen sample (infundibulum; SP treatment) giving efficiencies of 111% (IL-23), 96% (HPRT) and 95% (cyclophilin A).
For optimal comparison between runs the threshold was adjusted to obtain the same Ct value for an inter-assay reference sample included in each run. IL-23 mRNA expression was normalized to HPRT and cyclophilin A mRNA expression by calculating the copy number for IL-23 in relation to the geometric mean value for the copy numbers for HPRT and cyclophilin A according to: 2Ct for IL-23 / geometric mean of 2Ct for HPRT and 2Ct for cyclophilin A
The normalized expression was used for statistical comparison between treatment groups. The results are presented as the inverted ratio (1/ratio) of the normalized expression [6].
Immunohistochemistry
Immunohistochemical analysis was performed on both oviductal (isthmus and infundibulum) and uterine samples. Transverse (oviduct) or longitudinal (uterus) sections of about 7 µm were cut on a cryostat and placed on SuperFrost®Plus microscope slides (Menzel GmbH & Co KG., Braunschweig, Germany). Sections were air-dried for about 30 min and rehydrated in phosphate-buffered saline (PBS) for 5 min. Sections were blocked in 5% goat serum for 30 min and incubated overnight at 4°C with a rabbit anti-human IL-23 antibody (H-113; Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted to 0.2 µg/ml. The amino acid sequence showed 91.2% identity with the corresponding porcine protein sequence; sc-50303. Negative controls included sections incubated in the absence of the primary antibody and sections incubated with a control rabbit IgG (ChromPure Rabbit IgG [011-000-003], Jackson ImmunoResearch Laboratories Inc., West Grove, PA) diluted to a concentration exceeding that of the primary IL-23 antibody. After washing in PBS, IL-23-like immunolabelling was detected using the Vectastain® Elite ABC kit for Mouse IgG (PK-6102; Vector Laboratories Inc., Burlingame, CA). A biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories Inc., Burlingame, CA) diluted 1/1000 was used instead of the biotinylated anti-mouse IgG as a negative control. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxidase in methanol. For visualization, 3, 3’-diaminobenzidine tablets (D-5905; Sigma-Aldrich Inc., Saint Louis, MO) was used and sections were counterstained with Mayer’s Hematoxylin. Slides were mounted in Kaiser’s glycerol gelatine (109242; Merck KGaA, Darmstadt, Germany).
For semi-quantification of IL-23 immunolabelling, photographs were taken in a Nikon-FXA photomicroscope (Nikon Corporation, Tokyo, Japan) using the ×20 objective with identical exposure settings for all photomicrographs taken. Photomicrographs were coded and examined by a group of three persons. The intensity of the immunolabelling of endometrium and endosalpinx was estimated according to a scoring system from 0-4, where 0 corresponds to the intensity of the negative controls and 4 to very high intensity.
Statistical analyses
Quantitative real-time PCR results are presented as mean values ± standard error of the mean (sem). Normalized mRNA expression data and IL-23-like immunolabelling intensity scores were statistically analyzed using the SAS statistical package (version 9.1.3, SAS Institute, Inc., 2002-2003, Cary, NC). A natural log transformation was applied to the normalized copy number to achieve the assumption required for analysis of variance. Differences in mean ratios were tested using analysis of variance (The Mixed Procedure). The statistical model included the fixed effect of treatment (SP, spermatozoa, extender and catheter only) and tissue (isthmus, infundibulum and endometrium). It also included analysis of the interaction between each group and tissue as well as the random effect of gilts nested within groups. The Bonferroni t-test was used to compare least-square mean values between experimental groups when an overall significance for the effect was found. A value of p ≤ 0.05 was considered as statistically significant.
For statistical analysis of IL-23-like immunolabelling scores the NPAR1WAY procedure was used for standard analysis of variance. The Kruskal-Wallis test was used for one-way analysis of variance whereas differences in mean scores were analyzed using the Wilcoxon rank sum test for non-parametric data. A value of p ≤ 0.05 was considered as statistically significant.