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  • Studies were reviewed by University of Edinburgh Animal Research Ethics Committee and conducted under Project Licence approved by the UK Home Office. All reagents and chemicals were obtained from Sigma-Aldrich (Dorset, United Kingdom), unless stated otherwise. Progesterone was measured in weekly plasma samples by radioimmunoassay as described previously [20]. This was used to confirm anovulation prior to diathermy and assess the effect of ovarian diathermy on the induction of ovulation. Scottish Greyface ewes (Ovis aries) of comparable body condition score were cycle-synchronised using progesterone sponges and mated with Texel rams. Pregnant ewes received intramuscular injections of 100 mg testosterone propionate (TP; AMS Biotechnology Ltd, Abingdon, United Kingdom) in vegetable oil twice weekly, from day (d) 30 to 100 of gestation (d147) and delivered normally. After weaning, the resulting female offspring were housed together in spacious pens and hay was available ad libitum, with Excel Ewe Nuts (0.5–1.0 kg/day; Carrs Billington, Lancashire, UK) and Cystalayx Extra High Energy Lick (Caltech Solway Mills, Cumbria, UK). These offspring (n = 12) were maintained until they were adults in their second breeding season (22 months of age) after confirmation of anovulation by serial progesterone assessment for eight weeks prior to randomisation. The sheep were then randomly divided into three PCOS cohorts (Table 1). The acute cohort (n = 4) were used to investigate the early cellular, molecular and vascular effects of diathermy on the ovary. They underwent mini-laparotomy, ovarian assessment and unilateral ovarian diathermy, allowing the other ovary to serve as an internal control. Twenty four hours later they underwent mini-laparotomy, ovarian assessment and ovarian collection under terminal anaesthesia. The chronic cohort (n = 8) studied the later effects of diathermy on the ovary. A treatment group (n = 4) underwent mini-laparotomy, ovarian assessment and bilateral ovarian diathermy whilst the control group (n = 4) underwent mini-laparotomy and ovarian assessment without diathermy. Five weeks later they underwent mini-laparotomy, ovarian assessment and ovarian collection under terminal anaesthesia. The study was completed in the last month of the breeding season when ovulation would still be expected. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0111280.t001 Summary of treatment and investigation including contrast enhances ultrasound (CEUS) and reverse transcription polymerase chain reaction (RT-PCR). A mini-laparotomy was performed with sterile technique under general anaesthesia, induced using isoflourane (Isoflo, Abbott Animal Health, Maidenhead, UK). A midline incision exposed the ovaries that were stabilised with a clamp to the uterine fundus. Ovarian diathermy was performed using a needle probe with monopolar coagulation current (Surgitran, STW-100). The coagulation current was applied to the ovary at four separate sites of approximately 4 mm in depth for 10 seconds. Ovarian blood flow and ovarian artery resistance index were assessed prior to and after diathermy. Following surgery, all sheep received antibiotics (Penicillin and Dihydrostreptomycin), analgesics and they were carefully monitored during their rapid recovery. In vivo measurement: Contrast enhanced ultrasound (CEUS) of ovarian capillary bed: All ultrasound imaging was undertaken using a Philips iU22 ultrasound scanner (Philips Medical Systems, Bothwell, WA, USA) with linear array transducers L9-3 and L15-7. Using microbubbles along with ultrasound scanning provides a novel method to assess the capillary bed of the ovary. A well-established house contrast agent [21] was utilised. Briefly decafluorobutane microbubbles were prepared by a standard sonication protocol from an aqueous micellar dispersion of phosphatidylcholine (2 mg/ml; Avanti Lipids) and PEG stearate (2 mg/ml; Stepan Kesso). Following two days incubation under fluorocarbon atmosphere in the refrigerator, microbubbles floated to the top of the reaction vessel forming a thick cake. Infranatant containing micellar lipid was removed and replaced with degassed perfluorocarbon-saturated saline. Larger bubbles (in excess of 8–10 µm size) were removed from the preparation by flotation in normal gravity. Microbubbles in aqueous saline were then packaged in glass vials with Teflon-lined rubber stoppers and sealed under perfluorocarbon atmosphere. This preparation has a concentration of 109 microbubbles per ml with a 2 µm average size. A bolus (0.2 ml volume) was injected into a jugular vein catheter followed by a 10 ml saline flush to ensure all contrast agent was administered. This protocol provides good contrast in the ovary while avoiding signal saturation at peak contrast. CEUS was carried out in sheep destined for the acute analysis. The wash in time (WIT) was recorded for contrast before diathermy, immediately after diathermy and 24 hours after diathermy in triplicate. This is considered a reproducible measurement in the literature [22]–[24]. Details of the protocol for calculating the WIT under these conditions have been reported elsewhere [24]. In vivo measurement: Ovarian Blood Flow: The whole ovary was scanned in the longitudinal plane in colour Doppler mode with fixed thresholding and video images stored. Doppler scans were replayed back on the quantification software (Q-LAB v6, Philips Healthcare, Andover, MA, USA) and three images per ovary with the greatest area of blood flow were chosen for quantification by two observers blinded to the treatment groups. Using Image J software (http://rsbweb.nih.gov/ij/) the area of total Doppler signal flow for each image was quantified and averaged per ovary. In vivo measurement: Resistance Index: The ovarian artery was identified using colour Doppler assessment with an L15-7 ultrasound probe. Resistance index (RI) of the ovarian artery was assessed using automatic RI measurements calculated on the Philips iU22 Ultrasound system backed up with manual measurement confirmation. A continuous wave of at least five peaks was obtained and three separate measurements were taken to give an average value. This was repeated three times and the mean of the averaged values per ovary used for analysis. At the end of the study ewes were sacrificed. Ovaries were collected and cut longitudinally into two sections (two thirds and one third of the ovary). The smaller section was snap frozen and stored at −80°C for subsequent dissection of an area of stroma (1 mm3), from the inner aspect of the outer third at least 1 mm from the ovarian surface, with no visible antral follicles, for RNA extraction and gene analysis studies. The larger, two third portion of the ovary was fixed in Bouins solution for 24 hours before transferral to 70% ethanol for paraffin wax embedding, sectioning and immunohistochemical study. Ovarian sections (5 µm) from the middle of the ovary were analysed. A representative section with maximal ovarian diameter, informed by our previous study on the assessment of follicle number in the ewe [20], underwent haematoxylin and eosin (H&E) staining for subsequent antral follicle counting. Immunohistochemical analysis was also performed for markers of cell proliferation (Ki67) and apoptosis (activated caspase 3). Ovarian tissue sections were dewaxed and rehydrated. Sections then underwent antigen retrieval in a decloaking chamber (Biocare Medical, Concord, California) containing sodium citrate retrieval buffer (0.01M, pH 6.0). Peroxidase quenching and blocking steps were performed via incubation in 3% H2O2 for 10 minutes, avidin and biotin blocking (Vector Laboratories Ltd, Peterborough, United Kingdom) and finally 5% BSA diluted in 20% normal serum from the host species of the secondary antibody. Primary antibodies, diluted in appropriate serum were applied to sections overnight at 4°C. After washing, secondary antibody was applied to slides for 1 hour, followed by Vectastain ABC Elite tertiary complex (PK-1600 series; Vector Laboratories) for 1 hour. Binding was visualised with 3,3′-diaminobenzidine (Dako, Cambridge, United Kingdom) for 30 seconds. Sections were counterstained with hematoxylin and mounted. Negative controls consisted of either primary antibody incubated with a blocking peptide or, in the absence of a specific blocking peptide, non-immune serum of equivalent immunoglobulin concentrations. Two independent examiners, blinded to treatment, counted the number of follicles with a clear fluid-filled antrum (≥500 µm) from a mid-section of the ovary [20]. The average antral follicle count per ovarian section was recorded. Immunohistochemical staining of whole ovary sections stained for proliferation (Ki67) and atresia (activated caspase 3) were blindly examined by two independent expert examiners. Each antral follicle was examined and staining was divided into two classifications, positive (clearly positive immunostaining present in multiple cells) and negative (scant/absent immunopositive cells). Number of follicles per classification was used for proportional analysis. Quantitative Real Time (qRT) PCR: RNA was extracted from tissue using RNeasy mini spin columns following manufacturer’s protocol and concentration measured using NanoDrop 1000 Spectrophotometer. Complimentary DNA (cDNA) was synthesised from 200 ng RNA in accordance with manufacturer’s protocol (Applied Biosystems, California, USA). Subsequently, qRT-PCR was performed using SYBR Green as previously described [25]. Primer3 Input version 0.4, online software, was used to design forward and reverse primers (Table 2) from DNA sequences obtained from Ensembl Genome Browser, sequences were checked for specificity using Basic Local Alignment Search Tool and validity confirmed as previously described [26]. Real-time PCR reactions were carried out in duplicate 10 µl reactions, negative controls consisted of cDNA reaction without reverse transcriptase and a reaction replacing cDNA with nuclease-free water. Melt curve analysis revealed a single amplicon in all cases. GAPDH has been reported as a suitable internal control for ovarian stromal gene expression [27] and target gene expression was analysed relative to GAPDH and quantified using the ΔCt method. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0111280.t002 List of the primer pairs used in SYBR Green quantitative real-time PCR. Proportional analysis was conducted using a Fishers exact test. The means of two groups were compared with non-paired or paired t-test where appropriate when the data was parametric with equal variance and the Mann Whitney test when not. Where the distribution was not normal logarithmic transformation was used prior to statistical testing. A P value of less than 0.05 was considered statistically significant.
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