See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/282875300 Peri-ovulatory endocrine regulation of the prostanoid pathways in the bovine uterus at early dioestrus Article  in  Reproduction Fertility and Development · October 2015 DOI: 10.1071/RD15269 CITATION 1 READS 157 9 authors, including: Some of the authors of this publication are also working on these related projects: NADPH oxidase-derived ROS role in growth factor signaling of uterine smooth muscle cells View project Targeting Renal Microvascular Oxidative Stress in Cardiovascular & Metabolic Disease View project Milena L Oliveira University of São Paulo 13 PUBLICATIONS   116 CITATIONS    SEE PROFILE Guilherme Pugliesi Federal University of Minas Gerais 92 PUBLICATIONS   443 CITATIONS    SEE PROFILE Veerle Van Hoeck University of Antwerp 56 PUBLICATIONS   590 CITATIONS    SEE PROFILE Fernando Silveira Mesquita Universidade Federal do Pampa - Unipampa 59 PUBLICATIONS   336 CITATIONS    SEE PROFILE All content following this page was uploaded by Craig E Wheelock on 19 October 2015. The user has requested enhancement of the downloaded file. https://www.researchgate.net/publication/282875300_Peri-ovulatory_endocrine_regulation_of_the_prostanoid_pathways_in_the_bovine_uterus_at_early_dioestrus?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_2&_esc=publicationCoverPdf https://www.researchgate.net/publication/282875300_Peri-ovulatory_endocrine_regulation_of_the_prostanoid_pathways_in_the_bovine_uterus_at_early_dioestrus?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_3&_esc=publicationCoverPdf https://www.researchgate.net/project/NADPH-oxidase-derived-ROS-role-in-growth-factor-signaling-of-uterine-smooth-muscle-cells?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_9&_esc=publicationCoverPdf https://www.researchgate.net/project/Targeting-Renal-Microvascular-Oxidative-Stress-in-Cardiovascular-Metabolic-Disease?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_9&_esc=publicationCoverPdf https://www.researchgate.net/?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_1&_esc=publicationCoverPdf https://www.researchgate.net/profile/Milena_Oliveira6?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_4&_esc=publicationCoverPdf https://www.researchgate.net/profile/Milena_Oliveira6?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_5&_esc=publicationCoverPdf https://www.researchgate.net/institution/University_of_Sao_Paulo?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_6&_esc=publicationCoverPdf https://www.researchgate.net/profile/Milena_Oliveira6?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_7&_esc=publicationCoverPdf https://www.researchgate.net/profile/Guilherme_Pugliesi?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_4&_esc=publicationCoverPdf https://www.researchgate.net/profile/Guilherme_Pugliesi?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_5&_esc=publicationCoverPdf https://www.researchgate.net/institution/Federal_University_of_Minas_Gerais?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_6&_esc=publicationCoverPdf https://www.researchgate.net/profile/Guilherme_Pugliesi?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_7&_esc=publicationCoverPdf https://www.researchgate.net/profile/Veerle_Van_Hoeck?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_4&_esc=publicationCoverPdf https://www.researchgate.net/profile/Veerle_Van_Hoeck?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_5&_esc=publicationCoverPdf https://www.researchgate.net/institution/University_of_Antwerp?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_6&_esc=publicationCoverPdf https://www.researchgate.net/profile/Veerle_Van_Hoeck?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_7&_esc=publicationCoverPdf https://www.researchgate.net/profile/Fernando_Mesquita3?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_4&_esc=publicationCoverPdf https://www.researchgate.net/profile/Fernando_Mesquita3?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_5&_esc=publicationCoverPdf https://www.researchgate.net/profile/Fernando_Mesquita3?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_7&_esc=publicationCoverPdf https://www.researchgate.net/profile/Craig_Wheelock?enrichId=rgreq-e995806793bb81ecb98f0f1c4f3cfc71-XXX&enrichSource=Y292ZXJQYWdlOzI4Mjg3NTMwMDtBUzoyODYzNTU1OTU3NzYwMDFAMTQ0NTI4MzkwMjM3MQ%3D%3D&el=1_x_10&_esc=publicationCoverPdf Peri-ovulatory endocrine regulation of the prostanoid pathways in the bovine uterus at early dioestrus Milena Lopes OliveiraA, Fabio Luiz D’AlexandriA, Guilherme PugliesiA, Veerle Van HoeckA, Fernando Silveira MesquitaB, Claudia M. B. MembriveC, João Alberto NegrãoD, Craig E. WheelockE and Mario BinelliA,F ADepartment of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo – Avenida Duque de Caxias Norte, 225, 13630-000, Pirassununga, SP, Brazil. BUniversidade Federal do Pampa, School of Veterinary Medicine – BR472, Km592, 97508-000, Uruguaiana, RS, Brazil. CUniversidade Estadual Paulista ‘Julio de Mesquita Filho’ – Rodovia Comandante João Ribeiro de Barros, Km65, 17900-000, Dracena, SP, Brazil. DCollege of Animal Science and Food Engineering, University of São Paulo – Avenida Duque de Caxias Norte, 225, 13630-000, Pirassununga, SP, Brazil. EDepartment of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet – SE-171 77 Stockholm, Sweden. FCorresponding author. Email: binelli@usp.br Abstract. We hypothesised that different endocrine profiles associated with pre-ovulatory follicle (POF) size would impact on uterine prostanoid pathways and thereby modulate the histotroph composition. Beef cows (n¼ 15 per group) were hormonally manipulated to have small (SF-SCL group) or large (LF-LCL group) pre-ovulatory follicles (POF) and corpora lutea (CL). Seven days after induction of ovulation, animals were slaughtered and uterine tissues and flushings were collected for quantification of prostanoids. The POF and CL size and the circulating progesterone concentrations at Day 7 were greater (P, 0.05) in the LF-LCL cows than in the SF-SCL group, as expected. The abundance of 5 out of 19 genes involved in prostanoid regulation was different between groups. Transcript abundance of prostaglandin F2a, E2 and I2 synthases was upregulated (P, 0.05) and phospholipase A2 was downregulated (P, 0.05) in endometrium of the LF-LCL group. No difference (P. 0.1) in prostanoid concentrations in the endometrium or in uterine flushings was detected between groups. However, prostaglandin F2a and E2 concentrations in the uterine flushings were positively correlated with the abundance of transcripts for prostaglandin endoperoxide synthase 2 (0.779 and 0.865, respectively; P, 0.002). We conclude that endometrial gene expression related to prostanoid synthesis is modulated by the peri- ovulatory endocrine profile associated with POF size, but at early dioestrus differences in transcript abundance were not reflected in changes in prostanoid concentrations in the uterine tissue and fluid. Additional keywords: endometrium, oestrogen, physiology, prostaglandins. Received 2 December 2014, accepted 16 August 2015, published online 14 October 2015 Introduction A significant proportion of bovine females fail to become pregnant after insemination (Diskin et al. 2012; Pohler et al. 2012) and this has a negative economic impact on beef cattle operations. This high proportion of non-pregnant animals is mainly caused by early embryo loss (Diskin and Sreenan 1980; Diskin andMorris 2008). Therefore, necessary improvements in the reproductive efficiency depend on a greater understanding of the endocrine, cellular and molecular mechanisms involved in reproductive events during early dioestrus. The oviductal and uterine environments play a relevant role during the establishment and maintenance of pregnancy (Bauersachs et al. 2003; El-Sayed et al. 2006; Ulbrich et al. 2013). Indeed, pre- vious studies determined that specific transcriptomic profiles at early dioestrus are necessary for adequate uterine receptivity (Forde et al. 2009; Mansouri-Attia et al. 2009; Walker et al. 2012; Beltman et al. 2014; Binelli et al. 2015; Mesquita et al. 2015). It is known that the timing and magnitude of oestradiol (E2) exposure during pro-oestrus and oestrus, followed by progesterone (P4) at dioestrus, modulate gene expression in the endometrium and histotroph composition and function (Forde et al. 2009; Bridges et al. 2012; Ramos et al. 2015). In this CSIRO PUBLISHING Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD15269 Journal compilation � CSIRO 2015 www.publish.csiro.au/journals/rfd context, Mesquita et al. (2014) also showed that the size of the pre-ovulatory follicle (POF) alters the peri-ovulatory endocrine milieu (i.e. the concentrations of E2 in pro-oestrus and P4 in dioestrus) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might be modulated in response to peri-ovulatory endocrine changes. Several molecules and associated pathways have been proposed as key factors to determine uterine receptivity and the endocrine regulation of these pathways has been studied in detail by our group (Ramos et al. 2014, 2015; Araújo et al. 2015; França et al. 2015) and others (Bauersachs et al. 2006). However, the search continues for regulatory signals that might be involved in the critical processes of maternal receptivity in order to understand and subsequently tackle the possible causes for high rates of early embryonic death in beef cattle. In this context, reports have focussed on unravelling the complex role of the endometrial eicosanoid lipid mediators in the control of a range of reproductive processes (Weems et al. 2006). Prostanoids are well-described eicosanoids, which exert pivotal roles in regulation of reproductive processes such as ovulation, implantation, luteolysis and parturition in mammals (Lim et al. 1997; Wiltbank and Ottobre 2003). However, prostaglandin (PG) synthesis pathways are also important and active before maternal recognition of pregnancy in ruminants. Previous studies have shown that bovine embryos at morula and blastocyst stages were susceptible to elevated prostaglandin F2a (PGF2a) concentrations in the uterine lumen, which could negatively influence embryo viability and pregnancy rates (Schrick et al. 1993; Buford et al. 1996; Seals et al. 1998; Hockett et al. 2004). Regarding prostaglandin E2 (PGE2), the expression of its main synthase (prostaglandin E synthase (PTGES)) was downregulated in the endometrium of heifers with a retarded embryo at Day 7 after oestrus (Beltman et al. 2010), indicating that lack of PGE2 embryotrophic stimulus (Arosh et al. 2004; Ulbrich et al. 2009) could have contributed to the decreased fertility in these beef heifers. In addition, PGE2 is known to stimulate embryo implantation, luteal function and to modulate the uterine immune response and embryo develop- ment mainly by exerting anti-inflammatory effects (Arosh et al. 2004; Cong et al. 2006; Mosher et al. 2012; Vilella et al. 2013). Prostaglandin I2 (PGI2) improves the developmental compe- tence of embryos, as the supplementation of in vitro culture medium with a PGI2 analogue improved embryonic quality by increasing the proportion of bovine embryos that developed to the expanded blastocyst stage (Song et al. 2009). Expression of genes involved in prostaglandin synthesis was reported by Dorniak et al. (2011). These authors concluded that PGF2a and PGE2 are important regulators of conceptus elongation and mediators of endometrial responses to P4 in sheep. Therefore, because of critical effects of prostaglandins on embryo devel- opment during early dioestrus, deregulation of their biosynthesis may be one of the mechanisms associated with early embryonic loss in cattle. Herein, we propose that prostanoids are a possible class of endocrine-modulated molecules that are important for embryo receptivity and thus female fertility at early dioestrus. In the present study, we are the first to evaluate the endocrine influences on prostanoid pathways in Day-7 endometrial tissue and uterine flushings; a timing that coincides with the moment of embryo reception by the maternal uterus. Therefore, we used a bovine fertility model as previously described by Mesquita et al. (2014, 2015), Ramos et al. (2014, 2015), Araújo et al. (2015) and França et al. (2015) and associated with fertility (Pugliesi et al. 2015) in order to evaluate whether peri-ovulatory variations in circulating steroids, positively associated with the ovulatory follicle size, regulate: (1) the expression of endome- trial genes involved in the synthesis, transport, signalling and catabolism of eicosanoids and (2) the concentration of eicosa- noids in endometrial tissue and uterine secretions. Materials and methods Animal procedures This study was conducted at the Universidade de São Paulo, Pirassununga, São Paulo, Brazil. Animal procedures were approved by the Ethics and Animal Handling Committee of the School of Veterinary Medicine and Animal Science of the University of São Paulo with protocol number 2287/2011. Thirty multiparous Nelore cows (Bos taurus indicus) without reproductive abnormalities and with body-condition scores between 3 and 4 (0 being emaciate, 5 being obese) were kept on grazing conditions supplemented with sugarcane or corn silage (or both), concentrate and mineral salt. Animals received water ad libitum. In order to form two groups of cows with different POF sizes and subsequent corpus luteum (CL) volume and P4 concentra- tion, a hormonal protocol was used in all cows to manage endocrine patterns of the peri-ovulatory period as described by Mesquita et al. (2014). The model was based on the pharmaco- logical control of follicle growth to result in a group exhibiting larger (LF-LCL group) or smaller (SF-SCL group) POF and CL and consequently resulting in different circulating P4 concen- trations during early dioestrus. To reach this goal, cows (n¼ 15 per group) received two intramuscular (i.m.) doses of prosta- glandin F2a (PGF; 0.5mg; sodium cloprostenol; Sincrocio; Ourofino, Cravinhos, Brazil) 14 days apart. Following this pre-synchronisation procedure, ovaries were visualised using transrectal ultrasound scanning in order to confirm the presence of a PGF-responsible CL 10 days after the second PGF admin- istration, (Day �10 of the experiment; D–10). On D–10, all cows were treated with 2mg of oestradiol benzoate (Sincrodiol; Ourofino) and received a P4 intravaginal releasing device (1 g; Sincrogest; Ourofino) to stimulate recruitment of a new follicular wave. Females assigned to the larger POF and subse- quent larger CL group (LF-LCL) additionally received a PGF injection on D–10 (0.5mg; sodium cloprostenol; Sincrocio; Ourofino) to induce CL regression during follicle development, whereas cows assigned to the smaller POF followed by smaller CL group (SF-SCL) did not. Sixty hours before the induction of ovulation, P4 devices were removed and a PGF dose (0.5mg; Cloprostenol) was administered i.m. to cows in the LF-LCL group, whereas cows in the SF-SCL group received the same treatment 12 h later (D–2.5 and D–2, respectively). Ovulation was induced onDay 0 by an i.m. administration of gonadotrophin- releasing hormone (GnRH; 10mg buserelin acetate, Sincroforte; B Reproduction, Fertility and Development M. L. Oliveira et al. Ourofino). Seven days after induction of ovulation, cows that ovulated in response to GnRH within 48 h (n¼ 11 cows in the SF-SCL group and 12 cows in LF-LCL group) were slaughtered and their reproductive tracts were collected for further analysis. More details about the animal model are available in previous publications by our group (Mesquita et al. 2014; França et al. 2015). Ultrasonography The ovaries were evaluated by transrectal ultrasonography on D–10, D–6 and every 24 h, starting on D–2 until D7, using both an Aloka SSD-500 attached to a 5-MHz linear probe (Hitachi Aloka, Tokyo, Japan) and an Esaote MyLab 30 attached to a multi-frequency probe (Esaote, Genoa, Italy) set between 6 and 7.5MHz. Ultrasonography was performed to evaluate the presence and size of dominant follicles and CL. The maximum diameter and perpendicular diameter of the largest ovarian follicle were measured using a B-mode still image and an averaged diameter was calculated. Ovulation was defined as the disappearance of the largest ovarian follicle followed by the presence of a new CL at the same location. For evaluation of size of the CL, the maximum CL area was determined using a B-mode still image and the tracing function. For CL with an anechoic fluid-filled cavity, the area of the cavity was subtracted from the total area (Kastelic et al. 1990; Pugliesi et al. 2014). Sample collection and processing At D7 the animals were slaughtered for collection of the reproductive tract. The reproductive tissues were transported on ice to the laboratory within 15min. The uterine horn ipsilateral to the ovulation was flushed with 20mL of phosphate-buffered saline (PBS). The uterine flushing was centrifuged at 3000g for 30min at 48C and the supernatant was stored at �808C for quantification of eicosanoids. After the flushing, the ipsilateral uterine horn was dissected and fragments of the intercaruncular area were taken. This region was chosen because most endo- metrial glands responsible for secretion of histotroph are found in this region on the uterine tissue (Dhaliwal et al. 2002). The uterine samples were stored at�808C for quantification of RNA and prostanoid metabolites. Quantification of progesterone concentrations Blood samples were taken from the jugular vein on the day of slaughter (D7). Plasma was removed after blood centrifugation at 1500g for 30min at 48C. P4 plasma concentrations were measured in the samples using a commercial kit (Coat-A-Count; SiemensMedical Solutions Diagnostics, Los Angeles, CA, USA), previously validated for bovine plasma samples (Garbarino et al. 2004). The intra- and inter-assay CV and sensitivity for P4, were 0.3%, 7.0% and 0.076 ngmL�1, respectively. Transcript quantification by real-time reverse transcription polymerase chain reaction (PCR) Approximately 30mg of endometrial tissue was macerated in liquid nitrogen (SF-SCL, n¼ 11; LF-LCL, n¼ 12) and sub- mitted to total RNA extraction using the RNeasy Mini columns kit (Qiagen Laboratories, Valência, CA, USA) according to the manufacturer’s instructions. The RNA concentration was mea- sured spectrophotometrically (NanoDrop; Thermo Scientific, Wilmington, MA, USA). Before the reverse transcription (RT), the RNA samples were treatedwithDNase I (deoxyribonuclease I, Pure Link Genomic DNA Purification; Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions. Briefly, the treatment with DNase was done at room temperature using 1.0mg of RNA in a 10-mL reaction volume. After 15min of incubation, 1.0mL of ethylenediamine tetraacetic acid (EDTA, 25mM; Invitrogen) was added and warmed to 658C for 10min. Synthesis of cDNA was performed using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies Corpora- tion, Frederick, MD, USA). A master mix (9.0 mL) was added to the 11.0 mL of the treated samples. The samples were incubated at 258C for 10min and then at 378C for 2 h, followed by an enzymatic inactivation period at 858C for 5min. The primers were designed using the Oligo Analyzer 3.1 software (Integrated DNA Technologies, Inc., Coralville, IA, USA, http://www.idtdna.com/calc/analyzer, accessed 15 June 2012) and Software Primer Express 3.0.1 (Life Technologies, Frederick, MA, USA) or were obtained from previous reports. The qPCR reactions were performed using SYBR Green Chem- istry for the amplification analysis in a thermocycler (Step One Plus Real Time System; Life Technologies, Frederick, MA, USA). The thermocycler was programmed to start in a holding stage (958C for 10min), followed by 40 cycles. Each cycle had a denaturation step (958C for 15 s) and an annealing phase (608C for 1min). A dissociation (‘melting’) curve was obtained immediately after the amplification and thenmaintained at 958C for 15min, at 608C for 1min and then at 958C for 15min. The criteria for validation of primers were: amplification efficiency between 85 and 110%, absence of amplification of the negative control, a single peak in the melting curve and the smallest cycle threshold. Standard curves for each primer were validated using five progressive dilutions and using duplicates. This was obtained using a pool of endometrial cDNA samples in dilutions of 1 : 20, 1 : 40, 1 : 80, 1 : 160 and 1 : 320 (cDNA :H2O). Deter- mination of PCR efficiency and Cq (quantification cycle) values per sample was performed with LinReg PCR software (http:// linregpcr.nl/, verified 28 August 2015). Quantification was obtained after normalisation of the target gene expression values (Cq values) by the endogenous control expression in triplicate values of peptidylprolyl isomerase A (cyclophilin A, PPIA), using the equation described by Pfaffl (2001) and expressed as a ratio of target gene-to-endogenous control. PCR products of reactions using the primers designed were submitted to electro- phoresis and sequencing. Details of primers are provided in Table 1 and the validation data is given in Table S1, available as Supplementary Material to this paper. Quantification of prostanoid abundance Liquid chromatography –mass spectrometry (LC–MS/MS) The prostanoid concentrations were measured in the endo- metrial tissue and in the uterine flushings from a subgroup of cows selected randomly from each experimental group (n¼ 4–6 samples per group). The oxylipin analysis was basically per- formed as described by Lundström et al. (2013) using the Uterine prostanoid pathways at early dioestrus Reproduction, Fertility and Development C http://www.idtdna.com/calc/analyzer http://linregpcr.nl/ http://linregpcr.nl/ T a b le 1 . P ri m er n a m e, fo rw a rd (F ) a n d re v er se (R ) se q u en ce , re p re se n ta ti v e id en ti fi ca ti o n n u m b er a n d a m p li co n si ze o f g en es fr o m th e p ro st a n o id sy n th es is a n d si g n a ll in g p a th w a y G en e sy m b o l G en e n am e S eq u en ce 5 0 – 3 0 ID S o ft w ar e/ re fe re n ce A S iz e (b p ) A K R 1 B 1 A ld o – k et o re d u ct as e fa m il y 1 , m em b er B 1 F -A T A C A A G C C G G C G G T T A A C N M _ 0 0 1 0 1 2 5 1 9 U lb ri ch et a l. 2 0 0 9 1 8 8 R -T G T C T G C A A T C G C T T T G A T C A K R 1 C 3 A ld o – k et o re d u ct as e fa m il y 1 , m em b er C 3 F - G A C T C A G T T C T T T G T G C C A T T G C N M _ 0 0 1 0 3 8 5 8 4 .1 P ri m er E x p re ss 1 5 4 R -T C A G T T C A A A G T C A A A C A C C T G T A T G A K R 1 C 4 A ld o – k et o re d u ct as e fa m il y 1 , m em b er C 4 F - T C C T G T C C T G G G A T T T G G A A C C T T N M _ 1 8 1 0 2 7 .2 P ri m er Q u es t 1 6 6 R -A T C G G C A A T C T T G C T T C G A A T G G C C B R 1 C ar b o n y l re d u ct as e 1 F - T T G C C T T C A A G A C T G C T G A C A N M _ 0 0 1 0 3 4 5 1 3 .1 P ri m er E x p re ss 1 5 3 R -C A C T G A C A A A G C T G G A T A C A T T C A C H P G D H y d ro x y p ro st ag la n d in d eh y d ro g en as e F - T G A T C A G T G G A A C C T A C C T G G N M _ 0 0 1 0 3 4 4 1 9 U lb ri ch et a l. 2 0 0 9 1 8 3 R -T G A G A T T A G C A G C C A T C G C P P IA P ep ti d y lp ro ly l is o m er as e A (c y cl o p h il in A ) F - G C C A T G G A G C G C T T T G G N M _ 1 7 8 3 2 0 .2 B et te g o w d a et a l. 2 0 0 6 6 9 R -C C A C A G T C A G C A A T G G T G A T C T P L A 2 G 1 0 P h o sp h o li p as e A 2 , g ro u p X , tr an sc ri p t v ar ia n t F - T G T G C C C G A A G G T A G G G C T G T T X M _ 8 6 4 9 5 0 .4 U lb ri ch et a l. 2 0 0 9 1 3 8 R -G G C G A G G G C C A A C A C A G T C A A T P T G D R P ro st ag la n d in D 2 re ce p to r F - T T C A G C A C A G C A A C A A G C T C A C A G N M _ 0 0 1 0 9 8 0 3 4 .1 P ri m er Q u es t 1 1 5 R -A T C T T A C C A T C T C C A C C A A G G G C A P T G D S P ro st ag la n d in D 2 sy n th as e F -A G G T C A A G G A A C A C T T C A C C A C C T N M _ 1 7 4 7 9 1 .4 P ri m er Q u es t 8 2 R -T T G T C A G T C T T C G G C A G G A A C A C A P T G E R 2 P ro st ag la n d in E re ce p to r 2 F - C T A C T T G C C T T T T C C A T G A C C N M _ 1 7 4 5 8 8 U lb ri ch et a l. 2 0 0 9 2 1 0 R -G A T G A A G C A C C A C G T C C C P T G E R 4 P ro st ag la n d in E re ce p to r 4 F - C G A T G A G T A T T G A G C G C T A C C N M _ 1 7 4 5 8 9 U lb ri ch et a l. 2 0 0 9 2 3 7 R -A G C C C G C A T A C A T G T A G G A G P T G E S P ro st ag la n d in E sy n th as e F - G C T G C G G A A G A A G G C T T T T G C C N M _ 1 7 4 4 4 3 .2 P ri m er Q u es t 1 0 1 R -G G G C T C T G A G G C A G C G T T C C P T G E S 2 P ro st ag la n d in E sy n th as e 2 F - G T G G G C G G A C G A C T G G T T G G N M _ 0 0 1 1 6 6 5 5 4 .1 P ri m er Q u es t 1 9 2 R -C G G A G G T G G T G C C T G C G T T T P T G E S 3 P ro st ag la n d in E sy n th as e 3 F - C A G T C A T G G C C A A G G T T A A C A A A N M _ 0 0 1 0 0 7 8 0 6 .2 P ri m er E x p re ss 1 5 0 R -A T C A C C A C C C A T G T T G T T C A T C P T G S 1 P ro st ag la n d in en d o p er o x id e sy n th as e 1 F - C A C C C G C T C A T G C C C G A C T C N M _ 0 0 1 1 0 5 3 2 3 .1 P ri m er Q u es t 1 5 5 R -T T C C T A C C C C C A C C G A T C C G G P T G S 2 P ro st ag la n d in en d o p er o x id e sy n th as e 2 F - C C A G A G C T C T T C C T C C T G T G N M _ 1 7 4 4 4 5 .2 P ri m er Q u es t 1 6 1 R -G G C A A A G A A T G C A A A C A T C A P T G IS P ro st ag la n d in I2 sy n th as e F -A A G A T G G G A A G C G A C T G A A G N M _ 1 7 4 4 4 4 .1 P ri m er Q u es t 1 3 6 R -A T C A G C T C C A G G T C A A A C T G S L C O 2 A 1 S o lu te ca rr ie r o rg an ic an io n tr an sp o rt er fa m il y , m em b er 2 A 1 F - T G T G G A G A C G A T G G G A T T G A N M _ 1 7 4 8 2 9 .3 P ri m er E x p re ss 1 5 0 R -G G G A C A C G G G C C T G T C T T T B X A 2 R T h ro m b o x an e A 2 re ce p to r F - T G T C C T T C C T G C T C A A C A C C A T C A N M _ 0 0 1 1 6 7 9 1 9 .1 P ri m er Q u es t 1 4 1 R -A A A T G C T G G C C A C C A C C A T A A T G C T B X A S 1 T h ro m b o x an e A sy n th as e 1 F - T C A C C A A C A C T C T C T C T T T C G C C A N M _ 0 0 1 0 4 6 0 2 7 .1 P ri m er Q u es t 9 4 R -T C C T T G C T G A A A C A G T C C A C C T C T A S o ft w ar e u se d fo r al ig n m en t o f p ri m er se q u en ce s o r li te ra tu re re fe re n ce . D Reproduction, Fertility and Development M. L. Oliveira et al. LC–MS/MS approach and is only briefly described here. The analytical standards and deuterated surrogates were obtained from Cayman Chemical (Ann Arbor, MI, USA), Larodan Fine Chemicals AB (Malmö, Sweden) or Biomol International (Plymouth Meeting, PA, USA). The oxylipins were extracted from 2mL of uterine flushing using Waters Oasis-HBL car- tridges (Waters, Milford, MA, USA) preconditioned with wash solution (H2O :MeOH; 95 : 5, in 0.1% acetic acid). The uterine flushing aliquots, 200mL of wash solution, 10 mL of surrogate standards (400 nM per standard in MeOH), 10 mL anti-oxidant and enzyme inhibitor solution (0.2mgmL�1 of butylated hydro- xytoluene (BHT), EDTA, thiamine pyrophosphate and indo- methacin) were applied to the cartridge, rinsed with wash solution, eluted with 500mL of methanol and then with 1.5mL of ethyl acetate and collected into polypropylene tubes contain- ing 6mL of 30% glycerol in methanol. The solvent was stripped and the sample was suspended in 50mL of methanol containing the technical standard 1-cyclohexyl-dodecanoic acid urea (CUDA; 800 nM). The samples were then centrifuged at 10 000g for 30min at 48C and the supernatants were stored at �208C until analysis. Oxylipin profiling was performed using 10-mL sample injections on a Waters ACQUITY UPLC system via a 2.1� 150mm, 1.7-mm Waters Acquity BEH column maintained at 608C coupled to an XEVO TQ triple quadrupole mass spectrometer (Waters). The samples were maintained at 48C before injection. Solvents A (0.1% acetic acid in water) and B (acetonitrile : methanol : acetic acid, 88 : 12 : 0.1) were used in the following gradient: 15% B for 0.74min, 30% B at 1.5min, 47% B at 3.5min, 54% B at 6min, 60% B at 10.5min, 70% B at 15min, 80% B at 16min, 100% B from 17 to 19min, 30% B from 19.3 to 21min. The oxylipins detected above the limit of quantitation (LOQ) were quantified, recalculated based on the original uterine flushing concentrations and normalised to the uterine flushing recovery (V[recovered volume]/V[instilled volume]). The normalisation to uterine flushing recovery did not affect the overall trends in the samples. For the endometrial tissue, the samples were previously extracted with organic solvents before solid-phase extraction. For this step, 100mg of cryo-pulverised endometrial tissue was added to amber vials (2mL, polytetrafluoroethylene (PTFE) caps; National Scientific Co., Rockwood, TN, USA) prepared with 5mL of BHT–EDTA (0.2mgmL�1 in 1 : 1 MeOH :H2O) and 20mL of the surrogate standards (1000 nM per standard in MeOH). Then, 500mL of MeOH was added and the vials were capped and then briefly vortexed. The samples were then centrifuged at 3.000g and 08C for 5min. The supernatant was collected and saved. Then, 350mL of isopropyl alcohol (IPA) was added to the remaining tissue. The samples were treated in an identical manner using methanol and the IPA extract was added to the MeOH fraction. The remaining tissue was mixed with 350mL of cyclohexane. The cyclohexane extract was treated as described above and the supernatant was pooled with IPA and MeOH. The combined fraction was dried at reduced pressure (Genevac Inc., Stone Ridge, NY, USA) for ,1 h. The dried samples were reconsti- tuted in 200mL of MeOH : toluene (1 : 1) and 100mL of a sub- aliquot of the extract was loaded into the solid phase extraction (SPE) cartridges and extracted as described above for the uterine flushing. Enzyme-linked immunosorbent assay (ELISA) Because a reduced number of samples was used for mass spectrometry and considering that PGE2 and PGF2a are the most important prostanoids in the uterus, enzyme-linked immu- nosorbent assays (ELISAs) to measure the concentrations of PGE2 and PGF2a in uterine flushing samples were validated using commercial kits for PGE2 and PGF2a (both fromCayman Chemical Co.). Initially, a uterine flushing pool was treated with activated charcoal to remove prostaglandins (Turzillo and Fortune 1990). Briefly, 500mg of activated charcoal was added for each mL of uterine flushing and this mixture was incubated for 45min and then centrifuged at 12 000g and 48C for 1 h. The supernatant was filtered and stored at�808C. This prostaglandin- freematrixwas used only to prepare PGE2 and PGF2a standards from 15.6 to 1000 pgmL�1 and from 7.8 to 500 pgmL�1, respectively. The matrix volume added to each standard was equal at each standard-curve point (five points were used in each curve). The standard curves were compared with the curves produced using only the manufacturer’s enzyme immunoassay (EIA) buffer and validated by parallelism. All of the assay- specific reagents were prepared as described and suggested by the manufacturers. After the plate setup and incubations, the absorptions were read spectrophotometrically at a wavelength of 414 nm (Labsystems Multiskan – MS; Thermo Fisher Scien- tific, Waltham, MA, USA). After validation, the concentrations of PGE2 and PGF2a in the uterine flushings collected from a subset of cows in the LF-CL group (n¼ 10) and the SF-SCL group (n¼ 10) were assayed in duplicate. Concentrations were calculated in reference to a regression equation generated from a standard curve preparedwith increasing concentrations of PGE2 or PGF2a, diluted in prostaglandin-free uterine flushing. Statistical analyses Outlying observations greater than two standard deviation ranges from the mean were not used in the statistical analyses. The data were tested for normality of the residues using the Shapiro–Wilk test and for homogeneity of variance using the F-max test and natural log-transformed if needed. The ovarian and endocrine variables were analysed by one-way ANOVA to test the effect of the treatment using the PROC GLM proce- dure of the SAS software (Version 9.2; SAS Institute, Cary, NC, USA). The eicosanoid concentrations and relative gene expression levels were analysed through non-paired Students’ t-test. Pearson’s correlation coefficients were calculated bet- ween P4 concentrations, POF and CL size or P4 concentrations and abundance of transcripts and concentrations of prostanoids in the uterus, and between abundance of transcripts and con- centrations of prostanoids in the endometrial tissues or uterine flushings. A probability of P# 0.05 indicated that an effect was significant and a probability of P. 0.05 to P# 0.1 indicated that significance was approached. Results Ovarian responses and circulating P4 concentrations: animal model The hormonal treatments successfully resulted in two groups of cows with distinct ovarian characteristics, as previously Uterine prostanoid pathways at early dioestrus Reproduction, Fertility and Development E described by Mesquita et al. (2014). More specifically, cows assigned to the LF-LCL group had larger POF diameters when compared with animals from the SF-SCL group (12.7� 0.3mm and 11.2� 0.4mm, respectively; P, 0.05). Furthermore, the larger POFs resulted in larger (2.5� 0.31 vs 1.6� 0.1 cm3; P, 0.05) and heavier (2.9� 0.4 vs 2.0� 0.1 g; P, 0.05) cor- pora lutea on Day 7 after induction of ovulation. Mean P4 concentrations were higher in cows from the LF-LCL group when compared with cows from the SF-SCL group (4.4� 0.4 vs 3.5� 0.3 ngmL�1; P, 0.05; Fig. 1). Transcript abundance in endometrial tissue Gene expression analyses showed that 5 out of 19 analysed genes displayed a significantly modulated expression in response to the differential peri-ovulatory endocrine profiles (Table 2; Fig. 2). More specifically, the expression of phos- pholipase A2 (PLA2G10), encoding an enzyme that releases arachidonic acid for eicosanoid synthesis, was decreased (fold change (fc) 0.58; P, 0.05) in the endometrium of cows belonging to the LF-LCL group when compared with the SF-SCL group. The abundance of prostaglandin E synthase (PTGES) transcripts in the LF-LCL group was higher than in the SF-SCL group (fc 1.32; P, 0.05). The latter prostaglandin synthase E2 enzyme converts prostaglandin H2 (PGH2) into PGE2. Furthermore, the gene expression levels of aldo–keto reductase family 1, member C4 (AKR1C4) and aldo–keto reductase family 1, member C3 (AKR1C3), involved in PGF2a synthesis, were upregulated (fc 1.65; P, 0.05 and fc 1.84; P, 0.05, respectively) in the LF-LCL group compared with the SF-SCL counterparts. Also the expression of prostaglandin Table 2. Mean± s.e.m. of relative transcript abundance of target genes involved in prostanoid biosynthesis, signalling and catabolism in endometrial tissue atDay 7 after ovulation induction in cowswith large pre-ovulatory follicle (POF) and large corpus luteum (LF-LCL,n5 11) and cowswith small POF and small corpus luteum (SF-SCL, n 5 10) NS, no significant difference Gene symbol Gene name LF-LCL SF-SCL Fold change LF-LCL/SF-SCL P value Prostanoid synthesis PLA2G10 Phospholipase A2, group X, transcript variant 0.07� 0.08 0.12� 0.01 0.58 0.04 PTGS1 Prostaglandin endoperoxide synthase 1 0.002� 0.0003 0.001� 0.0001 1.32 NS PTGS2 Prostaglandin endoperoxide synthase 2 0.001� 0.0002 0.001� 0.0003 1.00 NS PTGES Prostaglandin E synthase 0.004� 0.0004 0.003� 0.0002 1.32 0.05 PTGES2 Prostaglandin E synthase 2 0.01� 0.0006 0.01� 0.0005 1.06 NS PTGES3 Prostaglandin E synthase 3 0.22� 0.02 0.2� 0.03 1.20 NS AKR1B1 Aldo–keto reductase family1, member B1 0.34� 0.02 0.3� 0.03 1.09 NS AKR1C4 Aldo–keto reductase family1, member C4 0.007� 0.001 0.004� 0.0005 1.65 0.04 AKR1C3 Aldo–keto reductase family1, member C3 0.004� 0.0008 0.002� 0.0003 1.84 0.02 CBR1 Carbonyl reductase 1 0.018� 0.002 0.01� 0.002 1.25 0.07 PTGDS Prostaglandin D2 synthase 0.06� 0.009 0.06� 0.006 1.06 NS PTGIS Prostaglandin I2 synthase 0.036� 0.008 0.029� 0.007 1.22 0.04 TBXAS1 Thromboxane A synthase 1 0.001� 0.0002 0.001� 0.0002 1.08 NS Prostanoid transporter SLCO2A1 Solute carrier organic anion transporter family, member 2A1 0.003� 0.0007 0.002� 0.0005 1.22 NS Prostanoid receptors PTGER2 Prostaglandin E receptor 2 0.001� 0.0001 0.001� 0.0001 0.92 NS PTGER4 Prostaglandin E receptor 4 0.006� 0.001 0.006� 0.0009 1.09 NS PTGDR Prostaglandin D2 receptor 0.0007� 0.0001 0.0006� 0.0001 1.03 NS TBXA2R Thromboxane A2 receptor 0.001� 0.0001 0.0004� 0.0001 3.39 NS Prostanoid Catabolism HPGD Hydroxyprostaglandin dehydrogenase 0.17� 0.017 0.1441� 0.0139 1.20 NS 7 5 6 4 3 2 1 0 LF-LCL SF-SCL P ro ge st er on e on d ay 7 ( ng m L� 1 ) Fig. 1. Box plot showing the mean (diamond), median (continuous horizontal line) and individual values (dots) for progesterone concentrations (ngmL�1) on Day 7 after induction of ovulation in cows treated to achieve a large pre-ovulatory follicle and corpus luteum (LF-LCL group; n¼ 12) and cows treated to achieve a small pre-ovulatory follicle and corpus luteum (SF-SCL group; n¼ 11). F Reproduction, Fertility and Development M. L. Oliveira et al. I synthase (PTGIS), an enzyme that converts PGH2 into PGI2, was greater (fc 1.22; P, 0.05) in the endometrium from the LF-LCL group than in the SF-SCL group. The transcript abundance of carbonyl reductase 1 (CBR1), which uses PGE2 as a substrate for the synthesis of PGF2a, tended to be upregulated in the LF-LCL compared with the SF-SCL tissue (fc 1.25; P¼ 0.07). No difference (P. 0.1) between groups was detected for the transcripts of the main gene related to PGF2a synthesis (aldo–keto reductase family 1, member B1 (AKR1B1); Fig. 2). Eicosanoid abundance in endometrial tissue and in uterine flushings LC–MS/MS data did not (P. 0.1) reveal significant changes in the concentrations of prostanoids between the LF-LCL and SF-SCL endometrial tissue or uterine fluid (Tables 3 and 4). Moreover, concentrations of PGE2 and PGF2a were also mea- sured in a large number of samples (n¼ 10 cows per group) using ELISA techniques in the uterine flushing samples. Con- sistently, no differences (P. 0.1) in either PGE2 or PGF2a concentrations were observed in the Day-7 uterine flushings when comparing LF-LCL versus SF-SCL treatments (Fig. 3). Correlations between P4 concentrations, POF and CL size Among the ovarian variables analysed, a significant positive correlation with the P4 concentrations was observed for CL diameter (0.599; P¼ 0.004), CL area (0.579; P¼ 0.006), CL volume (0.554; P¼ 0.009) and CL weight (0.554; P¼ 0.01). A tendency for positive correlation was also observed between Table 3. Mean ± s.e.m. of eicosanoid metabolite concentrations (pg mg21) in the endometrial tissue at Day 7 after ovulation induction in cows with large pre-ovulatory follicle (POF) and large corpus luteum (LF-LCL, n 5 4) and cows with small POF and small corpus luteum (SF-SCL, n 5 5) NS, no significant difference Eicosanoid metabolite (pg mg�1) LF-FCL SF-SCL Fold change LF-LCL/SF-SCL P value Prostaglandin E2 9.72� 2.51 9.50� 1.41 1.02 NS 8-Iso-prostaglandin E2 1.37� 0.34 1.15� 0.58 1.19 NS Prostaglandin F2a 14.7� 1.6 17.6� 2.4 0.83 NS 6-Keto-prostaglandin 1a 53.0� 1.1 76.0� 23.0 0.70 NS 11b-Prostaglandin F2a 1.14� 0.44 0.94� 0.25 1.21 NS Prostaglandin D2 90.6� 16.1 86.9� 18.3 1.04 NS D-12 Prostanglandin J2/prostaglandin J2 0.08� 0.01 0.12� 0.02 0.72 NS Thromboxane B2 0.56� 0.12 0.48� 0.08 1.16 NS 0.0035 0.0030 0.0025 0.0020 0.0015 0.0010 0.0015 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0 LF-LCL SF-SCL LF-LCL SF-SCL LF-LCL SF-SCL PTGES1 PTGS2 AKR1B1 PLA2G10 LF-LCL SF-SCL m R N A r el at iv e ab un da nc e Fig. 2. Box plot showing the mean (diamond), median (continuous horizontal line) and individual values (dots) for relative abundance of mRNA for PTGS2, PLA2G10, PTGES1 and AKR1B1 on Day 7 after induction of ovulation in cows treated to achieve a large pre-ovulatory follicle and corpus luteum (LF-LCL group; n¼ 12) and cows treated to achieve a small pre- ovulatory follicle and corpus luteum (SF-SCL group; n¼ 11). Uterine prostanoid pathways at early dioestrus Reproduction, Fertility and Development G P4 concentrations on D7 and POF size (0.414; P¼ 0.062). Also, a positive correlation was detected between POF size and CL diameter (0.613; P¼ 0.002), area (0.614; P¼ 0.002), volume (0.612; P¼ 0.002) and weight (0.697; P¼ 0.001). Correlation between POF size and P4 concentrations with the abundance of transcripts or concentrations of prostanoids in the uterus There was no significant correlation between POF size and P4 concentrations and the abundance of transcripts for the enzymes involved in prostanoid synthesis, nor between POF size and P4 concentrations and concentrations of uterine meta- bolites analysed. Correlation between abundance of transcripts and concentrations of prostanoids in the endometrial tissues and uterine flushings There were significant correlations between abundance of transcripts and concentrations of prostanoids in the endometrial tissues and uterine flushings. The transcript for prostaglandin endoperoxide synthase 2 (PTGS2) was positively correlated with the concentrations of PGE2 (0.865; P¼ 0.0001) and PGF2a (0.779; P¼ 0.002) measured by ELISA. In contrast, the abundance of PTGS2was negatively correlated with the PGF2a concentrations in the endometrial tissue (�0.936; P¼ 0.002) measured byMS/MS. In addition, no significant correlation was observed between prostaglandin synthase and its metabolites. The abundance of CBR1 transcript, the gene encoding the enzyme responsible for PGE2 conversion to PGF2a, was posi- tively correlated with PGF2a concentrations in the uterine fluid (0.668; P¼ 0.005). No difference (P. 0.1) in the ratio between PGF2a and PGE2 (PGF2a : PGE2) was detected between the LF-LCL (3.68) and SF-CL (4.97) groups. Discussion The quality of the preimplantation uterine environment encompasses a variety of aspects that potentially affect early embryo survival. Hormonal variations during each bovine oestrous cycle induce uterine changes that are crucial for its receptivity to the embryo, as indicated by the increased preg- nancy rates in cows with higher circulating P4 concentrations at Day 7 after insemination (McNeill et al. 2006; Peres et al. 2009). In the present study, we were the first to evaluate the endocrine influences on prostanoid pathways during early dioestrus, which coincides with the moment of embryo reception by the maternal uterus and may consequently interfere with embryo survival. Considering that the POF size is positively associatedwith its capacity to secrete E2 and subsequent CL size and P4 secretion (Vasconcelos et al. 2001; Carter et al. 2008; Peres et al. 2009), we used an experimental model based on the modulation of 2200 1800 1400 1000 600 400 300 200 100 0 200 0 LF-LCL SF-SCL LF-LCL SF-SCL C on ce nt ra tio n of P G E 2 on d ay 7 (p g m L� 1 ) C on ce nt ra tio n of P G F 2α o n da y 7 (p g m L� 1 ) Fig. 3. Box plot showing the mean (diamond), median (continuous horizontal line) and individual values (dots) for PGF2a and PGE2 concen- trations (pg mL�1) in uterine flushings at Day 7 after induction of ovulation in cows treated to achieve a large pre-ovulatory follicle and corpus luteum (LF-LCL group; n¼ 10) and cows treated to achieve a small pre-ovulatory follicle and corpus luteum (SF-SCL group; n¼ 10). Table 4. Mean ± s.e.m. of concentrations (pg mL21) of prostanoid metabolites in uterine flushings at Day 7 after ovulation induction in cows with large pre-ovulatory follicle (POF) and large corpus luteum (LF-LCL, n 5 6) and cows with small POF and small corpus luteum (SF-SCL, n 5 5) NS, no significant difference Prostanoid metabolite LF-LCL SF-SCL Fold changeLF-LCL/SF-SCL P value Prostaglandin E2 115.2� 28.0 75.4� 36.6 1.53 NS 8-Iso-prostaglandin E2 7.1� 3.1 3.8� 0.3 1.85 NS Prostaglandin F2a 675.3� 154.8 664.4� 249.4 1.02 NS Prostaglandin D2 53.1� 14.9 40.7� 21.4 1.31 NS Prostaglandin J2/D Prostaglandin J2 1.9� 0.2 1.9� 0.2 1.03 NS 15-Deoxy-D-12.14 prostaglandin J2 7.6� 0.7 8.4� 0.8 0.91 NS 6-Keto-prostaglandin F1a 508.8� 118.7 507.4� 164.5 1.00 NS Thromboxane B2 9.75� 5.1 18.1� 8.8 0.54 NS H Reproduction, Fertility and Development M. L. Oliveira et al. follicle growth and CL size, as has been previously described by our group (Mesquita et al. 2014, 2015; França et al. 2015; Ramos et al. 2014). In the present study, positive correlations between P4 concentrations on D7 and POF and CL size were observed. This confirmed that our experimental model not only modulated POF growth but efficiently altered CL growth and function, based on the P4 concentrations during early dioestrus. However, there was no significant correlation between POF size and P4 concentrations with the transcripts involved in the synthesis of PGE2 and PGF2a. Based on the present results, at Day 7 after induction of ovulation the expression of several enzymes responsible for prostaglandin synthesis was upregulated in the endometrial tissue of cows that ovulated larger follicles compared with the tissue from cows ovulating small follicles and consequently small CLs. Using LC–MS/MS and ELISA techniques, the relevance of the differently expressed genes have been studied in detail, by eicosanoid identification and quantification in the Day-7 endometrial tissue and associated uterine flushings from cows ovulating large or small POF. Interestingly, no differences in concentrations of prostanoids could be observed either in endometrial tissue or in associated uterine flushings when comparing the experimental groups. In Fig. 4, an overview of the prostanoid metabolic pathway and the modulation of gene expression in the LF-LCL group is provided. When focusing on the genes involved in each eicosanoid synthesis pathway, the endometrium of the cows in the LF-LCL group apparently supported synthesis of PGF2a. The expression of AKR1C3, AKR1C4, CBR1, PTGES and PTGIS was upregu- lated in cowswith larger POF and CL. Indeed, expression of two enzymes belonging to the aldo–keto reductase family (AKR), which convert PGH2 into PGF2a (Dozier et al. 2008; Bresson et al. 2011; Phillips et al. 2011), was stimulated in the LF-LCL endometrial tissue compared with the SF-SCL counterparts. This effect on expression of prostaglandin synthases may be caused by the combined effect of pre-ovulatory E2 and post- ovulatory P4 modulated by differential POF growth, as correla- tions between P4 concentrations alone and AKR enzymes and PTGES were not detected. Despite the fact that abundance of mRNAs for AKR1C3 and AKR1C4 was upregulated in cows with larger POF and CL, the concentrations of PGF2awere not increased in the endometrium and uterine flushings. Additional support for this mismatch was that a positive correlation between PGF2a synthases and PGF2a concentrations in uterine tissue and flushings were not detected. Several possible explanations should be taken into consider- ation. The first intuitive reason for this inconsistency is that the synthesis of PGF2a is not dependent only on the conversion of PGH2 by the PGF synthases but also on the expression of other mediators, such as the cyclo-oxygenases (PTGS1 and PTGS2) PGE2 PGE2 PGH2 PTGER1 PTGER2 PTGES PTGES2 PTGES3 PTGD2 PTGS1 AA PLA2G10 PTGS2 TBXAS1 AK1B1 PGI2 CBR1 AKR1C4 AKR1C3 PTGER3 PTGER4 PTGFR PTGDR PTGIR TBXAR2 PGF2α PGF2α PGF2α PGD2 PGD2 15-Δ-PGJ2 Iso-PGF1α TXA2 PGI2 PGE2 PGD2 HPGD PGl2 PGE2 PGD2 SLCO2A1 Other metabolites NUCLEUS P P A R PGF2αTXA2 Fig. 4. Biosynthesis, metabolism and regulation pathways of prostanoids in the endometrium. The enzyme phospholipase A2 (PLA2) releases arachidonic acid (AA) frommembrane phospholipids. The AA ismetabolised by cyclo-oxygenases 1 and 2 (PTGS2 and PTGS1) to PGH2, which is the precursor of all prostanoids. Specific synthases convert PGH2 into PGE2 (PTGES1, PTGES2 and PTGES3), PGF2a (AKR1B1, AKR1C3, AKR1C4 and CBR1), PGD2 (prostaglandin D2 synthase (PTGDS)), PGI2 (PTGIS) and TXA2 (thromboxane A synthase 1 (TBXAS1)). Once synthesised, the transport of prostaglandins through the plasma membrane is done bi-directionally, passively or facilitated by membrane carrier protein (solute carrier organic anion transporter family, member 2A1 (SLCO2A1)). Once outside the cell, prostaglandins can specifically bind to their membrane receptors PTGER1–4, PTGFR, PTGIR, prostaglandin D2 receptor (PTGDR) and thromboxaneA2 receptor (TBXAR2). PGI2 and PGD2 promote their biological effects by signalling to nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) family. Prostaglandins can be inactivated by the enzyme hydroxyprostaglandin dehydrogenase (HPDG), which converts prostaglandins to other metabolites. Up and down black arrows in the enzyme symbols indicate the up and downregulated genes, respectively, in the Day-7 endometrial tissue in cows that ovulated larger follicles and had larger corpus luteum (LF-LCL) compared with cows with smaller follicles and smaller corpus luteum. (Adapted from Fortier et al. 2008). Uterine prostanoid pathways at early dioestrus Reproduction, Fertility and Development I to convert the arachidonic acid (AA) into PGH2. In this regard, the production of PGH2 by the cyclo-oxygenase PTGS1 (con- stitutive) and PTGS2 (regulatory) is considered the rate-limiting step of prostaglandin biosynthesis in the endometrium (Smith et al. 2000; Parent et al. 2003). This was supported in the present study by the strong positive correlations betweenPTGS2 and the concentrations of PGF2a and PGE2 in the uterine flushings. Secretion of PGF2a by endometrial explants is also correlated with their PTGS2 content, suggesting that the increase in the ability of the uterus to produce prostaglandin during the luteal phase of the oestrous cycle is due to the increase in PTGS2 levels (Charpigny et al. 1997). In addition, the expression of PTGS2 increases 70–100 times before PGF2a elevation at parturition, whereas PGF2a synthase (AKR1B1) increases only 2.6 times (Schuler et al. 2006). Steroid hormones may modulate the expression of PTGS2 in endometrial cells (Madore et al. 2003), but the significant increase in P4 concentrations in cows with large POF and CL did not result in altered expression of this gene in the present study. A second consideration is that the abundance ofAKR1B1was also similar between cows with large and small POF and CL. AKR1B1 is considered to be the main synthase enzyme in the ARK family responsible for PGF2a biosynthesis in the human and bovine endometrium (Madore et al. 2003; Bresson et al. 2011) and its expression is positively associated with PTGS2 abundance (Charpigny et al. 1997; Xiao et al. 1998; Schuler et al. 2006). Consequently, the similar abundance of transcripts forPTGS2 andAKR1B1 in cowswith large or small POF andCL and the absence of a significant correlation between P4 con- centrations and the abundance of these transcripts may be the main explanations for the lack of difference in PGF2a concen- trations in the endometrium and uterine flushings between groups. Therefore, the upregulation of AKR1C3 and AKR1C4 in cows with large POF and CL was possibly a response to the greater P4 concentrations on D7, as enzymes in the AKR family have a double function of prostaglandin synthesis and P4 catabolism (Pelletier et al. 1999; Madore et al. 2003; Ito et al. 2006). This result is also a novel finding, as a previous study reported that AKR1C familymembers were not expressed in the bovine endometrium during dioestrus (Madore et al. 2003). Similarly, the concentrations of PGE2 were not increased in consequence of the greater abundance of PTGES1 transcript in cows with larger POF and CL. This mismatch between a synthase and its prostanoid may also be caused by the absence of the modulation of PTGS2 by the different peri-ovulatory endocrine profiles. In line with this, Arosh et al. (2002) sug- gested that the increased PGE2 production in endometrial cells is mainly caused by the associative upregulation of PTGES1 with PTGS2. In addition, lower levels of PGE synthase and PTGS2 in the bovine endometriumwere detected between Days 1 and 12 of the oestrous cycle (Arosh et al. 2002), indicating a limited capacity of the uterus to secrete PGE2 during early and mid dioestrus. This also suggested that the cyclo-oxygenases might be the key component monitoring final prostaglandin concentrations in the bovine endometrium at early dioestrus. Furthermore, there was an increased abundance of CBR1 tran- scripts in the LF-LCL group. This enzyme uses PGE2 as a substrate for the synthesis of PGF2a (Kankofer and Wierciński 1999; Asselin and Fortier 2000; Kankofer et al. 2002). There- fore, part of the PGE2 converted by PTGES1 could be instantly transformed into PGF2a by CBR1 activity. The concentrations of PGF2a were greater than the concentrations of PGE2 in the uterine flushings and in the endometrial tissue on D7 of the oestrous cycle. Thus, at least part of this abundance of PGF2a may be caused by the conversion of PGE2 into PGF2a through CBR1, as indicated by the positive correlation between PGF2a and CBR1 in the uterine flushings. A third consideration is related to the gene expression results of the PLA2G10 enzyme. This phospholipase comes into view as a potential regulator of eicosanoid homeostasis, as its downregulated expression in the LF-LCL compared with SF-SCL tissue might result in a limited substrate provision towards effective production of prostaglandins. In this regard, the PLA2 acts on the release of AA, the primary precursor of prostanoids (Godkin et al. 2008). Another consideration regarding the mismatch between gene expression and prostaglandin concentrations is that our results are primarily based on the transcript abundance data. It is not clear whether all transcripts will be translated or even post-translationally modified (Robert 2010). A previous report (Ulbrich et al. 2009) documented a similar mismatch when comparing eicosanoid transcripts and metabolite concentrations in the uterus, although in a reverse way and during a later time window during dioestrus. Finally, post-transcriptional effects regulating activities should be considered as well. The impor- tance of the latter assumption has been recently emphasised by Walker et al. (2013); DNA methylation is involved in early pregnancy events, which might point towards potential post- transcriptional alterations. The complete role of prostaglandins in the fertility of cows still needs to be elucidated, but during early embryo develop- ment the evidence is that specific prostanoids are needed for adequate embryonic viability during early dioestrus. Previous research has revealed that development of bovine embryos is impaired by increased PGF2a levels (Scenna et al. 2004, 2005) and is stimulated by PGE2 (Arosh et al. 2004; Ulbrich et al. 2009). Prostaglandins are also essential for elongation of the conceptus, as intrauterine infusions of a selective PTGS2 inhibitor prevented conceptus elongation in early-pregnant sheep (Simmons et al. 2010; Dorniak et al. 2011). In the present study, a bovine model was used in order to screen for endocrine preparation of maternal receptivity without the presence of the embryo. Considering the previous studies and our working model where cows with large POF and CL had an 80% increase in pregnancy rates (Pugliesi et al. 2015), the expectation was that cows in the LF-LCL group could stimulate PGE2 synthesis and inhibit PGF2a in the endometrium. However, as the concen- trations of PGE2 and PGF2awere correlated onlywith abundance of transcripts for PTGS2, the study of other important metabolic pathways in uterine tissue at early dioestrus are indicated to understand the positive effects of greater steroid concentrations during the peri-ovulatory period on bovine fertility. In conclusion, the peri-ovulatory endocrine changes associ- ated with the size of the POF regulate transcript abundance of genes belonging to prostanoid synthesis pathways in the bovine endometrium at early dioestrus (at Day 7 after induction J Reproduction, Fertility and Development M. L. Oliveira et al. of ovulation). Specifically, cows that ovulated larger follicles have increased abundance of AKR1C4, AKR1C3, PTGIS, PTGES and CBR1 transcripts in the endometrium, whereas the expression of PLA2G10 was reduced. These changes in tran- scription do not result in modifications in the prostanoid con- centrations in the endometrium nor in the uterine flushings, which probably result from the lack of modulation of PTGS2, the regulatory rate-limiting enzyme in prostaglandin biosynthe- sis. Indeed, the abundance of transcripts forPTGS2 is highly and positively correlated with PGF2a and PGE2 concentrations in the uterine flushings. Although the concentrations of prosta- noids are not affected by the peri-ovulatory endocrine profiles at this time point, these novel results characterising the prostanoid concentrations at early dioestrus point towards maintenance of homeostasis at the time of early embryo development. Acknowledgements This work was supported by LFEM (Projects #: 204 and 206), CNPq (481199/2012–8) and FAPESP (2011/03226–4). The authors thank S. C. Scolari, R. Ramos, M. Sponchiado, M. França, Everton Lopes and Estela R. Araújo for technical assistance, the administration of the Pirassununga campus of the University of São Paulo and CAPES (Coordination for the Improvement of Higher Education Personnel), Brazil for a scholarship to the first author. References Araújo, E. R., Sponchiado,M., Pugliesi, G., Van Hoeck, V., Mesquita, F. S., Membrive, C. M., and Binelli, M. (2015). Spatio-specific regulation of endocrine-responsive gene transcription by peri-ovulatory endocrine profiles in the bovine reproductive tract. Reprod. Fertil. Dev. doi:10.1071/RD14178 Arosh, J. A., Parent, J., Chapdelaine, P., Sirois, J., and Fortier, M. A. (2002). Expression of cyclo-oxygenases 1 and 2 and prostaglandin E synthase in bovine endometrial tissue during the oestrous cycle. Biol. Reprod. 67, 161–169. doi:10.1095/BIOLREPROD67.1.161 Arosh, J. A., Banu, S. K., Chapdelaine, P., Madore, E., Sirois, J., and Fortier, M. A. (2004). Prostaglandin biosynthesis, transport and signalling in corpus luteum: a basis for autoregulation of luteal function. Endocrinol- ogy 145, 2551–2560. doi:10.1210/EN.2003-1607 Asselin, E., and Fortier, M. A. (2000). Detection and regulation of the messenger for a putative bovine endometrial 9-keto-prostaglandin E(2) reductase: effect of oxytocin and interferon-tau. Biol. Reprod. 62, 125–131. doi:10.1095/BIOLREPROD62.1.125 Bauersachs, S., Blum, H., Mallok, S., Wenigerkind, H., Rief, S., Prelle, K., and Wolf, E. (2003). Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcrip- tomics approach. Biol. Reprod. 68, 1170–1177. doi:10.1095/BIOLRE PROD.102.010660 Bauersachs, S., Ulbrich, S. E., Gross, K., Schmidt, S. E., Meyer, H. H., Wenigerkind, H., Vermehren, M., Sinowatz, F., Blum, H., and Wolf, E. (2006). Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity. Reproduction 132, 319–331. doi:10.1530/REP.1.00996 Beltman, M. E., Forde, N., Furney, P., Carter, F., Roche, J. F., Lonergan, P., and Crowe, M. A. (2010). Characterization of endometrial gene expres- sion and metabolic parameters in beef heifers yielding viable or non-viable embryos on D 7 after insemination. Reprod. Fertil. Dev. 22, 987–999. doi:10.1071/RD09302 Beltman, M. E., Mullen, M. P., Elia, G., Hilliard, M., Diskin, M. G., Evans, A. C., and Crowe, M. A. (2014). Global proteomic characterisation of uterine histotroph recovered from beef heifers yielding good-quality and degenerate Day-7 embryos. Domest. Anim. Endocrinol. 46, 49–57. doi:10.1016/J.DOMANIEND.2013.10.003 Binelli, M., Scolari, S. C., Pugliesi, G., Van Hoeck, V., Gonella-Diaza, A. M., Andrade, S. C., Gasparin, G. R., and Coutinho, L. L. (2015). The transcriptome signature of the receptive bovine uterus determined at early gestation. PLoS One 10, e0122874. doi:10.1371/JOURNAL. PONE.0122874 Bresson, E., Boucher-Kovalik, S., Chapdelaine, P., Madore, E., Harvey, N., Laberge, P. Y., Leboeuf, M., and Fortier, M. A. (2011). The human aldose reductase AKR1B1 qualifies as the primary prostaglandin F synthase in the endometrium. J. Clin. Endocrinol. Metab. 96, 210–219. doi:10.1210/JC.2010-1589 Bridges, G. A.,Mussard,M. L., Pate, J. L., Ott, T. L., Hansen, T. R., andDay, M. L. (2012). Impact of pre-ovulatory oestradiol concentrations on conceptus development and uterine gene expression. Anim. Reprod. Sci. 133(1–2), 16–26. doi:10.1016/J.ANIREPROSCI.2012.06.013 Buford, W. I., Ahmad, N., Schrick, F. N., Butcher, R. L., Lewis, P. E., and Inskeep, E. K. (1996). Embryotoxicity of a regressing corpus luteum in beef cows supplemented with progestogen. Biol. Reprod. 54, 531–537. doi:10.1095/BIOLREPROD54.3.531 Carter, F., Forde, N., Duffy, P., Wade, M., Fair, T., Crowe, M. A., Evans, A. C., Kenny, D. A., Roche, J. F., and Lonergan, P. (2008). Effect of increasing progesterone concentration from Day 3 of pregnancy on subsequent embryo survival and development in beef heifers. Reprod. Fertil. Dev. 20, 368–375. doi:10.1071/RD07204 Charpigny, G., Reinaud, P., Tamby, J. P., Créminon, C., Martal, J., Maclouf, J., and Guillomot,M. (1997). Expression of cyclo-oxygenase-1 and -2 in ovine endometrium during the oestrous cycle and early pregnancy. Endocrinology 138, 2163–2171. Cong, J., Diao, H. L., Zhao, Y. C., Ni, H., Yan, Y. Q., and Yang, Z. M. (2006). Differential expression and regulation of cyclo-oxygenases, prostaglandin E synthases and prostacyclin synthase in rat uterus during the peri-implantation period. Reproduction 131(1), 139–151. doi:10.1530/REP.1.00861 Dhaliwal, G. S., Murray, R. D., Rees, E. M., Howard, C. V., and Beech, D. J. (2002). Quantitative unbiased estimates of endometrial gland surface area and volume in cycling cows and heifers. Res. Vet. Sci. 73, 259–265. doi:10.1016/S0034-5288(02)00098-X Diskin, M. G., and Morris, D. G. (2008). Embryonic and early fetal losses in cattle and other ruminants. Reprod. Domest. Anim. 43, 260–267. doi:10.1111/J.1439-0531.2008.01171.X Diskin, M. G., and Sreenan, J. M. (1980). Fertilisation and embryonic mortality rates in beef heifers after artificial insemination. J. Reprod. Fertil. 59, 463–468. doi:10.1530/JRF.0.0590463 Diskin,M. G., Parr,M. H., andMorris, D. G. (2012). Embryo death in cattle: an update. Reprod. Fertil. Dev. 24, 244–251. doi:10.1071/RD11914 Dorniak, P., Bazer, F.W., and Spencer, T. E. (2011). Prostaglandins regulate conceptus elongation and mediate effects of interferon tau on the ovine uterine endometrium. Biol. Reprod. 84, 1119–1127. doi:10.1095/BIOL REPROD.110.089979 Dozier, B. L., Watanabe, K., and Duffy, D. M. (2008). Two pathways for prostaglandin F2 alpha synthesis by the primate peri-ovulatory follicle. Reproduction 136, 53–63. doi:10.1530/REP-07-0514 El-Sayed, A., Hoelker, M., Rings, F., Salilew, D., Jennen, D., Tholen, E., Sirard, M. A., Schellander, K., and Tesfaye, D. (2006). Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients. Physiol. Genomics 28(1), 84–96. doi:10.1152/PHYSIOLGENOMICS.00111.2006 Forde, N., Carter, F., Fair, T., Crowe, M. A., Evans, A. C., Spencer, T. E., Bazer, F.W.,McBride, R., Boland,M. P., O’Gaora, P., Lonergan, P., and Roche, J. F. (2009). Progesterone-regulated changes in endometrial gene expression contribute to advanced conceptus development in cattle.Biol. Reprod. 81, 784–794. doi:10.1095/BIOLREPROD.108.074336 Uterine prostanoid pathways at early dioestrus Reproduction, Fertility and Development K http://dx.doi.org/10.1071/RD14178 http://dx.doi.org/10.1095/BIOLREPROD67.1.161 http://dx.doi.org/10.1210/EN.2003-1607 http://dx.doi.org/10.1095/BIOLREPROD62.1.125 http://dx.doi.org/10.1095/BIOLREPROD.102.010660 http://dx.doi.org/10.1095/BIOLREPROD.102.010660 http://dx.doi.org/10.1530/REP.1.00996 http://dx.doi.org/10.1071/RD09302 http://dx.doi.org/10.1016/J.DOMANIEND.2013.10.003 http://dx.doi.org/10.1371/JOURNAL.PONE.0122874 http://dx.doi.org/10.1371/JOURNAL.PONE.0122874 http://dx.doi.org/10.1210/JC.2010-1589 http://dx.doi.org/10.1016/J.ANIREPROSCI.2012.06.013 http://dx.doi.org/10.1095/BIOLREPROD54.3.531 http://dx.doi.org/10.1071/RD07204 http://dx.doi.org/10.1530/REP.1.00861 http://dx.doi.org/10.1016/S0034-5288(02)00098-X http://dx.doi.org/10.1111/J.1439-0531.2008.01171.X http://dx.doi.org/10.1530/JRF.0.0590463 http://dx.doi.org/10.1071/RD11914 http://dx.doi.org/10.1095/BIOLREPROD.110.089979 http://dx.doi.org/10.1095/BIOLREPROD.110.089979 http://dx.doi.org/10.1530/REP-07-0514 http://dx.doi.org/10.1152/PHYSIOLGENOMICS.00111.2006 http://dx.doi.org/10.1095/BIOLREPROD.108.074336 Fortier, M. A., Krishnaswamy, K., Danyod, G., Boucher-Kovalik, S., and Chapdalaine, P. (2008). A postgenomic integrated view of prostaglan- dins in reproduction: implications for other body systems. J. Physiol. Pharmacol. 59(Suppl 1), 65–89. França, M. R., Mesquita, F. S., Lopes, E., Pugliesi, G., Van Hoeck, V., Chiaratti, M. R., Membrive, C. B., Papa, P. C., and Binelli, M. (2015). Modulation of peri-ovulatory endocrine profiles in beef cows: conse- quences for endometrial glucose transporters and uterine fluid glucose levels. Domest. Anim. Endocrinol. 50, 83–90. doi:10.1016/J.DOMA NIEND.2014.09.005 Garbarino, E. J., Hernandez, J. A., Shearer, J. K., Risco, C. A., and Thatcher, W. W. (2004). Effect of lameness on ovarian activity in postpartum hostein cows. J. Dairy Sci. 87, 4123–4131. doi:10.3168/jds.S0022-0302 (04)73555-9 Godkin, J. D., Roberts, M. P., Elgayyar, M., Guan, W., and Tithof, P. K. (2008). PhospholipaseA2 regulation of bovine endometrial (BEND) cell prostaglandin production. Reprod. Biol. Endocrinol. 6, 44. doi:10.1186/ 1477-7827-6-44 Hockett, M. E., Rohrbach, N. R., and Schrick, F. N. (2004). Alterations in embryo development in progestogen-supplemented cows administered prostaglandin F2alpha. Prostaglandins Other Lipid Mediat. 73, 227–236. doi:10.1016/J.PROSTAGLANDINS.2004.02.002 Ito, K., Utsunomiya, H., Suzuki, T., Saitou, S., Akahira, J., Okamura, K., Yaegashi, N., and Sasano, H. (2006). 17Beta-hydroxysteroid dehydro- genases in human endometrium and its disorders.Mol. Cell. Endocrinol. 248, 136–140. doi:10.1016/J.MCE.2005.11.038 Kankofer, M., and Wierciński, J. (1999). Prostaglandin E2 9-keto reductase from bovine term placenta. Prostaglandins Leukot. Essent. Fatty Acids 61(1), 29–32. doi:10.1054/PLEF.1999.0069 Kankofer, M., Wierciński, J., and Zerbe, H. (2002). Prostaglandin E(2) 9-keto reductase activity in bovine retained and not-retained placenta. Prostaglandins Leukot. Essent. Fatty Acids 66, 413–417. doi:10.1054/ PLEF.2002.0367 Kastelic, J. P., Pierson, R. A., and Ginther, O. J. (1990). Ultrasonic morphology of corpora lutea and central luteal cavities during the estrous cycle and early pregnancy in heifers. Theriogenology 34, 487–498. doi:10.1016/0093-691X(90)90006-F Lim, H., Paria, B. C., Das, S. K., Dinchuk, J. E., Langenbach, R., Trzaskos, J. M., and Dey, S. K. (1997). Multiple female reproductive failures in cyclo-oxygenase 2-deficient mice. Cell 91, 197–208. doi:10.1016/ S0092-8674(00)80402-X Lundström, S. L., Saluja, R., Adner, M., Haeggström, J. Z., Nilsson, G., and Wheelock, C. E. (2013). Lipid mediator metabolic profiling demon- strates differences in eicosanoid patterns in two phenotypically distinct mast cell populations. J. Lipid Res. 54, 116–126. doi:10.1194/JLR. M030171 Madore, E., Harvey, N., Parent, J., Chapdelaine, P., Arosh, J. A., and Fortier, M. A. (2003). An aldose reductase with 20 alpha-hydroxysteroid dehydrogenase activity is most likely the enzyme responsible for the production of prostaglandin F2 alpha in the bovine endometrium. J. Biol. Chem. 278, 11205–11212. doi:10.1074/JBC.M208318200 Mansouri-Attia, N., Aubert, J., Reinaud, P., Giraud-Delville, C., Taghouti, G., Galio, L., Everts, R. E., Degrelle, S., Richard, C., Hue, I., Yang, X., Tian, X. C., Lewin, H. A., Renard, J. P., and Sandra, O. (2009). Gene expression profiles of bovine caruncular and intercaruncular endometrium at implantation Physiol. Genomics 39, 14–27. doi:10.1152/PHYSIOLGE NOMICS.90404.2008 McNeill, R. E., Diskin, M. G., Sreenan, J. M., and Morris, D. G. (2006). Associations betweenmilk progesterone concentration on different days and with embryo survival during the early luteal phase in dairy cows. Theriogenology 65, 1435–1441. doi:10.1016/J.THERIOGENOLOGY. 2005.08.015 Mesquita, F. S., Pugliesi, G., Scolari, S. C., França, M. R., Ramos, R. S., Oliveira, M., Papa, P. C., Bressan, F. F., Meirelles, F. V., Silva, L. A., Nogueira, G. P., Membrive, C. M., and Binelli, M. (2014). Manipulation of the peri-ovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early dioestrus Nelore cows. Theriogenology 81, 861–869. doi:10.1016/J.THERIOGENOLOGY.2013.12.022 Mesquita, F. S., Ramos, R. S., Pugliesi, G., Andrade, S. C., Van Hoeck, V., Langbeen, A., Oliveira, M. L., Gonella-Diaza, A. M., Gasparin, G., Fukumasu, H., Pulz, L. H., Membrive, C. M., Coutinho, L. L., and Binelli, M. (2015). The receptive endometrial transcriptomic signature indicates an earlier shift from proliferation tometabolism at early dioestrus in the cow. Biol. Reprod. doi:10.1095/BIOLREPROD.115.129031 Mosher, A. A., Rainey, K. J., Giembycz, M. A., Wood, S., and Slater, D. M. (2012). Prostaglandin E2 represses interleukin 1 beta-induced inflam- matory mediator output from pregnant human myometrial cells through the EP2 and EP4 receptors. Biol. Reprod. 87(1), 7. doi:10.1095/BIOL REPROD.112.100099 Parent, J., Villeneuve, C., and Fortier, M. A. (2003). Evaluation of the contribution of cyclo-oxygenase 1 and cyclo-oxygenase 2 to the produc- tion of PGE2andPGF2alpha in epithelial cells frombovine endometrium. Reproduction 126, 539–547. doi:10.1530/REP.0.1260539 Pelletier, G., Luu-The, V., Têtu, B., and Labrie, F. (1999). Immunocyto- chemical localisation of Type 5 17beta-hydroxysteroid dehydrogenase in human reproductive tissues. J. Histochem. Cytochem. 47, 731–737. doi:10.1177/002215549904700602 Peres, R. F., Claro, I., Sá Filho, O.G., Nogueira, G. P., andVasconcelos, J. L. (2009). Strategies to improve fertility inBos indicus post-pubertal heifers and non-lactating cows submitted to fixed-time artificial insemination. Theriogenology 72, 681–689. doi:10.1016/J.THERIOGENOLOGY. 2009.04.026 Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45. doi:10.1093/NAR/29. 9.E45 Phillips, R. J., Al-Zamil, H., Hunt, L. P., Fortier,M. A., and López Bernal, A. (2011). Genes for prostaglandin synthesis, transport and inactivation are differentially expressed in human uterine tissues and the prostaglandin F synthase AKR1B1 is induced in myometrial cells by inflammatory cytokines.Mol. Hum.Reprod. 17, 1–13. doi:10.1093/MOLEHR/GAQ057 Pohler, K. G., Geary, T. W., Atkins, J. A., Perry, G. A., Jinks, E. M., and Smith,M. F. (2012). Follicular determinants of pregnancy establishment and maintenance. Cell Tissue Res. 349, 649–664. doi:10.1007/S00441- 012-1386-8 Pugliesi, G., Miagawa, B. T., Paiva, Y. N., França, M. R., Silva, L. A., and Binelli, M. (2014). Conceptus-induced changes in the gene expression of blood immune cells and the ultrasound-accessed luteal function in beef cattle: how early can we detect pregnancy? Biol. Reprod. 91, 95. doi:10.1095/BIOLREPROD.114.121525 Pugliesi, G., Santos, F. B., Lopes, E., Nogueira, É., Maio, J. R. G., and Binelli,M. (2015). Fertility response in suckled beef cows supplemented with long-acting progesterone after timed artificial insemination. Reprod. Fertil. Dev. 27, 98. doi:10.1071/RDV27N1AB11 Ramos, R. S., Mesquita, F. S., D’Alexandri, F. L., Gonella-Diaza, A. M., Papa Pe, C., and Binelli, M. (2014). Regulation of the polyamine metabolic pathway in the endometrium of cows during early dioestrus. Mol. Reprod. Dev. 81, 584–594. doi:10.1002/MRD.22323 Ramos, R. S., Oliveira, M. L., Izaguirry, A. P., Vargas, L. M., Soares, M. B., Mesquita, F. S., Santos, F.W., andBinelli,M. (2015). The peri-ovulatory endocrine milieu affects the uterine redox environment in beef cows. Reprod. Biol. Endocrinol. 13, 39. doi:10.1186/S12958-015-0036-X Robert, C. (2010). Microarray analysis of gene expression during early development: a cautionary overview. Reproduction 140, 787–801. doi:10.1530/REP-10-0191 L Reproduction, Fertility and Development M. L. Oliveira et al. http://dx.doi.org/10.1016/J.DOMANIEND.2014.09.005 http://dx.doi.org/10.1016/J.DOMANIEND.2014.09.005 http://dx.doi.org/10.3168/jds.S0022-0302(04)73555-9 http://dx.doi.org/10.3168/jds.S0022-0302(04)73555-9 http://dx.doi.org/10.1186/1477-7827-6-44 http://dx.doi.org/10.1186/1477-7827-6-44 http://dx.doi.org/10.1016/J.PROSTAGLANDINS.2004.02.002 http://dx.doi.org/10.1016/J.MCE.2005.11.038 http://dx.doi.org/10.1054/PLEF.1999.0069 http://dx.doi.org/10.1054/PLEF.2002.0367 http://dx.doi.org/10.1054/PLEF.2002.0367 http://dx.doi.org/10.1016/0093-691X(90)90006-F http://dx.doi.org/10.1016/S0092-8674(00)80402-X http://dx.doi.org/10.1016/S0092-8674(00)80402-X http://dx.doi.org/10.1194/JLR.M030171 http://dx.doi.org/10.1194/JLR.M030171 http://dx.doi.org/10.1074/JBC.M208318200 http://dx.doi.org/10.1152/PHYSIOLGENOMICS.90404.2008 http://dx.doi.org/10.1152/PHYSIOLGENOMICS.90404.2008 http://dx.doi.org/10.1016/J.THERIOGENOLOGY.2005.08.015 http://dx.doi.org/10.1016/J.THERIOGENOLOGY.2005.08.015 http://dx.doi.org/10.1016/J.THERIOGENOLOGY.2013.12.022 http://dx.doi.org/10.1095/BIOLREPROD.115.129031 http://dx.doi.org/10.1095/BIOLREPROD.112.100099 http://dx.doi.org/10.1095/BIOLREPROD.112.100099 http://dx.doi.org/10.1530/REP.0.1260539 http://dx.doi.org/10.1177/002215549904700602 http://dx.doi.org/10.1016/J.THERIOGENOLOGY.2009.04.026 http://dx.doi.org/10.1016/J.THERIOGENOLOGY.2009.04.026 http://dx.doi.org/10.1093/NAR/29.9.E45 http://dx.doi.org/10.1093/NAR/29.9.E45 http://dx.doi.org/10.1093/MOLEHR/GAQ057 http://dx.doi.org/10.1007/S00441-012-1386-8 http://dx.doi.org/10.1007/S00441-012-1386-8 http://dx.doi.org/10.1095/BIOLREPROD.114.121525 http://dx.doi.org/10.1071/RDV27N1AB11 http://dx.doi.org/10.1002/MRD.22323 http://dx.doi.org/10.1186/S12958-015-0036-X http://dx.doi.org/10.1530/REP-10-0191 Scenna, F. N., Edwards, J. L., Rohrbach, N. R., Hockett, M. E., Saxton, A. M., and Schrick, F. N. (2004). Detrimental effects of prostaglandin F2alpha on preimplantation bovine embryos. Prostaglandins Other Lipid Mediat. 73, 215–226. doi:10.1016/J.PROSTAGLANDINS.2004.02.001 Scenna, F. N., Hockett,M. E., Towns, T.M., Saxton, A.M., Rohrbach,N. R., Wehrman,M. E., and Schrick, F. N. (2005). Influence of a prostaglandin synthesis inhibitor administered at embryo transfer on pregnancy rates of recipient cows. Prostaglandins Other Lipid Mediat. 78, 38–45. doi:10.1016/J.PROSTAGLANDINS.2005.02.003 Schrick, F. N., Inskeep, E. K., and Butcher, R. L. (1993). Pregnancy rates for embryos transferred from early postpartum beef cows into recipients with normal oestrous cycles. Biol. Reprod. 49, 617–621. doi:10.1095/ BIOLREPROD49.3.617 Schuler, G., Teichmann, U., Kowalewski, M. P., Hoffmann, B., Madore, E., Fortier, M. A., and Klisch, K. (2006). Expression of cyclo-oxygenase-II (COX-II) and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD)/ prostaglandin F-synthase (PGFS) in bovine placentomes: implications for the initiation of parturition in cattle. Placenta 27, 1022–1029. doi:10.1016/J.PLACENTA.2005.11.001 Seals, R. C., Lemaster, J. W., Hopkins, F. M., and Schrick, F. N. (1998). Effects of elevated concentrations of prostaglandin F2 alpha on preg- nancy rates in progestogen-supplemented cattle. Prostaglandins Other Lipid Mediat. 56, 377–389. doi:10.1016/S0090-6980(98)00063-X Simmons, R.M., Satterfield,M. C.,Welsh, T. H., Bazer, F.W., and Spencer, T. E. (2010). HSD11B1, HSD11B2, PTGS2 and NR3C1 expression in the peri-implantation ovine uterus: effects of pregnancy, progesterone and interferon tau. Biol. Reprod. 82, 35–43. doi:10.1095/BIOLRE PROD.109.079608 Smith,W. L., DeWitt, D. L., and Garavito, R.M. (2000). Cyclo-oxygenases: structural, cellular and molecular biology. Annu. Rev. Biochem. 69, 145–182. doi:10.1146/ANNUREV.BIOCHEM.69.1.145 Song, B. S., Kim, J. S., Kim, C. H., Han, Y. M., Lee, D. S., Lee, K. K., and Koo, D. B. (2009). Prostacyclin stimulates embryonic development via regulation of the cAMP response element-binding protein-cyclo- oxygenase-2 signalling pathway in cattle. Reprod. Fertil. Dev. 21, 400–407. doi:10.1071/RD08180 Turzillo, A. M., and Fortune, J. E. (1990). Suppression of the secondary FSH surge with bovine follicular fluid is associated with delayed ovarian follicular development in heifers. J. Reprod. Fertil. 89, 643–653. doi:10.1530/JRF.0.0890643 Ulbrich, S. E., Schulke, K., Groebner, A. E., Reichenbach, H. D., Angioni, C., Geisslinger, G., and Meyer, H. H. (2009). Quantitative characterisation of prostaglandins in the uterus of early pregnant cattle. Reproduction 138, 371–382. doi:10.1530/REP-09-0081 Ulbrich, S. E., Wolf, E., and Bauersachs, S. (2013). Hosting the preimplan- tation embryo: potentials and limitations of different approaches for analysing embryo–endometrium interactions in cattle. Reprod. Fertil. Dev. 25, 62–70. doi:10.1071/RD12279 Vasconcelos, J. L., Sartori, R., Oliveira, H. N., Guenther, J. G., and Wiltbank, M. C. (2001). Reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate. Theriogenology 56, 307–314. doi:10.1016/S0093-691X(01)00565-9 Vilella, F., Ramirez, L., Berlanga, O., Martı́nez, S., Alamá, P., Meseguer, M., Pellicer, A., and Simón, C. (2013). PGE2 and PGF2a concentrations in human endometrial fluid as biomarkers for embryonic implantation. J. Clin. Endocrinol. Metab. 98, 4123–4132. doi:10.1210/JC.2013-2205 Walker, C. G., Littlejohn,M. D., Mitchell, M. D., Roche, J. R., andMeier, S. (2012). Endometrial gene expression during early pregnancy differs between fertile and subfertile dairy cow strains. Physiol. Genomics 44, 47–58. doi:10.1152/PHYSIOLGENOMICS.00254.2010 Walker, C. G., Littlejohn,M. D., Meier, S., Roche, J. R., andMitchell, M. D. (2013). DNA methylation is correlated with gene expression during early pregnancy in Bos taurus. Physiol. Genomics 45, 276–286. doi:10.1152/PHYSIOLGENOMICS.00145.2012 Weems, C. W., Weems, Y. S., and Randel, R. D. (2006). Prostaglandins and reproduction in female farm animals. Vet. J. 171, 206–228. doi:10.1016/ J.TVJL.2004.11.014 Wiltbank, M. C., and Ottobre, J. S. (2003). Regulation of intraluteal produc- tion of prostaglandins. Reprod. Biol. Endocrinol. 1, 91. doi:10.1186/ 1477-7827-1-91 Xiao, C. W., Liu, J. M., Sirois, J., and Goff, A. K. (1998). Regulation of cyclo-oxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-tau in bovine endometrial cells. Endocrinology 139, 2293–2299. www.publish.csiro.au/journals/rfd Uterine prostanoid pathways at early dioestrus Reproduction, Fertility and Development M View publication statsView publication stats http://dx.doi.org/10.1016/J.PROSTAGLANDINS.2004.02.001 http://dx.doi.org/10.1016/J.PROSTAGLANDINS.2005.02.003 http://dx.doi.org/10.1095/BIOLREPROD49.3.617 http://dx.doi.org/10.1095/BIOLREPROD49.3.617 http://dx.doi.org/10.1016/J.PLACENTA.2005.11.001 http://dx.doi.org/10.1016/S0090-6980(98)00063-X http://dx.doi.org/10.1095/BIOLREPROD.109.079608 http://dx.doi.org/10.1095/BIOLREPROD.109.079608 http://dx.doi.org/10.1146/ANNUREV.BIOCHEM.69.1.145 http://dx.doi.org/10.1071/RD08180 http://dx.doi.org/10.1530/JRF.0.0890643 http://dx.doi.org/10.1530/REP-09-0081 http://dx.doi.org/10.1071/RD12279 http://dx.doi.org/10.1016/S0093-691X(01)00565-9 http://dx.doi.org/10.1210/JC.2013-2205 http://dx.doi.org/10.1152/PHYSIOLGENOMICS.00254.2010 http://dx.doi.org/10.1152/PHYSIOLGENOMICS.00145.2012 http://dx.doi.org/10.1016/J.TVJL.2004.11.014 http://dx.doi.org/10.1016/J.TVJL.2004.11.014 http://dx.doi.org/10.1186/1477-7827-1-91 http://dx.doi.org/10.1186/1477-7827-1-91 https://www.researchgate.net/publication/282875300