Spatio-specific regulation of endocrine-responsive gene transcription by periovulatory endocrine profiles in the bovine reproductive tract Estela R. AraújoA, Mariana SponchiadoA, Guilherme PugliesiA, Veerle Van HoeckA, Fernando S. MesquitaB, Claudia M. B. MembriveCand Mario BinelliA,D ADepartment of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Avenida Duque de Caxias Norte, 225, Pirassununga, SP, 13635-900, Brazil. BSchool of Veterinary Medicine, Federal University of Pampa, Rodovia BR 472, 592, Uruguaiana, RS, 97508-000, Brazil. CCollege of Animal Science, University of São Paulo State ‘Júlio de Mesquita Filho’, DracenaRodovia Comandante João Ribeiro de Barros, km 651, Dracena, SP, 17900-000, Brazil. DCorresponding author. Email: binelli@usp.br Abstract. In cattle, pro-oestrous oestradiol and dioestrous progesterone concentrations modulate endometrial gene expression and fertility. The aimwas to compare the effects of different periovulatory endocrine profiles on the expression of progesterone receptor (PGR), oestrogen receptor 2 (ESR2), oxytocin receptor (OXTR), member C4 of aldo–keto reductase family 1 (AKR1C4), lipoprotein lipase (LPL), solute carrier family 2, member 1 (SLC2A1) and serpin peptidase inhibitor, clade A member 14 (SERPINA14): (1) between uterine horns ipsi- and contralateral to the corpus luteum (CL), (2) between regions of the ipsilateral horn and (3) in the vagina. Endometrium and vagina tissue samples were collected from cows that ovulated a larger (large follicle-large CL, LF-LCL; n¼ 6) or smaller follicle (small follicle-small CL, SF-SCL; n¼ 6) 7 days after oestrus. Cows in the LF-LCL group had a greater abundance of transcripts encoding ESR2, AKR1C4, LPL, SLC2A1 and SERPINA14, but a reduced expression of PGR and OXTR in the endometrium versus the SF-SCL group (P, 0.05). Expression of PGR and OXTR was greater in the contralateral compared with the ipsilateral horn (P, 0.05). Regardless of group, the anterior region of the ipsilateral horn had increased expression of PGR, ESR2, LPL, SLC2A1 and SERPINA14 (P, 0.05). Different periovulatory endocrine profiles, i.e. LF-LCL or SF-SCL, did not influence gene expression in the vagina and had no interaction with inter- or intra-uterine horn gene expression. In conclusion, inter- and intra-uterine horn variations in gene expression indicate that the expression of specific genes in the bovine reproductive tract is location dependent. However, spatial distribution of transcripts was not influenced by distinct periovulatory sex-steroid environments. Additional keywords: oestradiol, progesterone, transcript abundance, uterine horn, vagina. Received 29 May 2014, accepted 14 February 2015, published online 21 April 2015 Introduction Embryonic mortality (Diskin and Morris 2008) during the first weeks after ovulation is amajor cause of economic losses in beef cattle commercial operations (Ayres et al. 2008; Sales et al. 2011). Losses are associated with preimplantation embryo mortality that reaches 30 to 40% of inseminated cows (Diskin and Sreenan 1980). Between Day 4 and 5 after oestrus the embryo leaves the oviduct and enters the anterior region of the uterine horn relative to the ovary containing the corpus luteum (CL; ipsilateral horn), where it continues its further development supported by uterine secretions until implantation (Spencer and Bazer 2003). Volume and composition of uterine secretions are regulated by endometrial function, specifically, by the continuum of changes in the endometrial transcriptome and associated proteome that occur before implantation. Indeed, up to the time of maternal recognition of pregnancy during late dioestrus, progressive changes in the endometrial transcriptome occur independently of the presence of the con- ceptus (Bauersachs et al. 2005; Mitko et al. 2008; Clemente et al. 2009; Forde et al. 2011). This indicates that the uterine CSIRO PUBLISHING Reproduction, Fertility and Development, 2016, 28, 1533–1544 http://dx.doi.org/10.1071/RD14178 Journal compilation � CSIRO 2016 www.publish.csiro.au/journals/rfd tissues play a key role in providing an optimal environment for the early developing embryo. It may be proposed that the uterine environment is modulated by steroid hormones to reach a maternal receptive state, as supported by the positive association between the preovulatory follicle size and subsequent circulat- ing progesterone (P4) concentrations during the early luteal phase and the greater probability of embryo survival in beef cows (Baruselli et al. 2012). In this context, coordinated actions of oestradiol (E2) and P4 during pro-oestrus and dioestrus, respectively, stimulate uterine function towards embryo recep- tivity (Lopes et al. 2007; Bridges et al. 2012). Furthermore, there are positive associations between periovulatory concen- trations of E2 and P4, conceptus development and elongation (Garrett et al. 1988; Satterfield et al. 2006; Carter et al. 2008) and overall fertility (Demetrio et al. 2007; Meneghetti et al. 2009; Peres et al. 2009; Baruselli et al. 2012). Changes in the periovulatory endocrine milieu that stimulate embryodevelopment, i.e. pro-oestrus–oestrusE2andmetaoestrus– early dioestrus P4, affect endometrial gene expression. For example, Forde et al. (2009) increased P4 concentrations during the early luteal phase through insertion of a progestin-releasing device. They reported changes in expression of several genes in the endometrium 5, 7, 13 or 16 days after insemination and such changes were associated with increased elongation of concep- tuses recovered on Day 16. More recently, Mesquita et al. (2014) manipulated the growth of the preovulatory follicle (POF) to generate two groups of cows that presented distinctly different pro-oestrus concentrations of E2 and early dioestrus concentrations of P4. These different periovulatory endocrine environments significantly affected the expression of genes associated with endometrial function, such as steroid signalling (progesterone receptor, PGR; oestrogen receptor 2, ESR2), lipid metabolism (lipoprotein lipase, LPL) and eicosanoid synthesis (member C4 of aldo–keto reductase family 1, AKR1C4). More importantly, cows treated to ovulate larger POFs presented a 20% increase in pregnancy in a large AI fertility trial (G. Pugliesi and M. Binelli, unpubl. data). An important technical aspect ofmost reports on uterine gene expression is that either tissue from a single region within the uterine horn (i.e. medial region; Forde et al. 2009) or a pool of tissues from different regions (Mesquita et al. 2014) was used for treatment comparisons. However, regional gradients of gene expression levels, previously reported in the reproductive tract of cattle (Bauersachs et al. 2005), pigs (Buhi and Alvarez 2003) and sheep (Meikle et al. 1997, 2001), might be highly relevant for receptivity and pregnancy success. For example, early embryo transfer work determined that when two embryos were transferred bilaterally, the greatest chances of twinning were achieved when at least one embryo was transferred to the tip of the horn relative to the CL (Newcomb et al. 1980). In contrast, the lowest embryo survival was noted when embryos were transferred to the base of each horn. It is tempting to speculate that regional differences in receptivity within the uterus are due to molecular microenvironments specific for each region. Spe- cific microenvironments could be associated with different regional abundance of transcripts. Furthermore, differences in P4 concentrations between different regions of the uterine horn relative to the CL and between horns were reported earlier (Pope et al. 1982). This provides mechanistic support for a regional control of expression of P4-stimulated genes. In the present study we hypothesised that endocrine changes associated with growth, ovulation of follicles of different sizes and subsequent changes in dioestrous P4 concentrations modu- late the spatial expression patterns of specific steroid-responsive transcripts in the uterine and vaginal tissue of beef cows at a specific moment during the early luteal phase (Day 7 after induction of ovulation). Therefore, we aimed to compare peri- ovulatory endocrine influences on the expression levels ofPGR, ESR2, oxytocin receptor (OXTR), solute carrier family 2, mem- ber 1 (SLC2A1), AKR1C4, LPL and serpin peptidase inhibitor, clade A member 14 (SERPINA14) between the ipsi- and contralateral uterine horns, between different regions within the ipsilateral uterine horn and in the vagina. Materials and methods Animal model and reproductive management Animal procedures were approved by the Ethics and Animal Handling Committee of the School of Veterinary Medicine and Animal Science of theUniversity of São Paulo (Protocol number 6732280414). Forty-two non-lactating, multiparous Nelore (Bos indicus) cows containing no gross reproductive anomalies were kept in grazing conditions, supplemented with sugarcane and corn silage, concentrate and minerals and water ad libitum. As previously described by Mesquita et al. (2014), follicular growth was pharmacologically manipulated to generate two groups of animals, presenting a large (LF-LCL) or small (SF-SCL) preovulatory follicle and subsequent CL. Briefly, all cows were pre-synchronised by two intramuscular (i.m.) treat- ments of prostaglandin F2a (PGF2a) analogue (0.5mg of sodium clopostrenol, Sincrocio; Ouro Fino, Cravinhos, Brazil) 14 days apart. Cows that did not present oestrus after the second PGF2a treatment were excluded. To modulate the growth of the preovulatory follicle (POF), on Day �10 animals received a single i.m. treatment of PGF2a (LF-LCL) or not (SF-SCL) along with oestradiol benzoate (2mg; Sincrodiol, Ouro Fino). On the same day, all animals received an intravaginal P4-releasing device (1 g, Sincrogest; Ouro Fino) that was removed 42–60 h or 30–36 h before the gonadotrophin-releasing hormone (GnRH) treatment in the LF-LCL and SF-SCL groups, respectively. Ovulation was induced with buserelin acetate (GnRH, 10mg i.m., Sincroforte; Ouro Fino) on Day 0. Expectation was that POF growth would be reduced in the presence of greater circulating P4 concentrations in cows from the SF-SCL group because of P4 from both exogenous (device) and endogenous (CL) sources. All animals received an i.m. PGF2a treatment at P4 device removal and a second treatment 6 h later. Blood plasma samples were obtained for measurement of E2 concentrations from Day�2 to Day 0 and for P4measurements fromDay 0 to Day 7. Blood was collected by jugular venipuncture in heparinised tubes (BD, São Paulo, Brazil) and stored on ice. Plasma was obtained by centrifugation at 48C, 1500g for 30min and stored at �208C. Transrectal ultrasound examinations of the ovaries were performed on Days �10 and �6, daily from Day �2 to Day 0, every 12 h on Day 1 and Day 2 and daily from Day 3 to Day 7 to 1534 Reproduction, Fertility and Development E. R. Araújo et al. assess follicle growth, ovulation and CL development and vascularisation. The examinations were performed using a duplex B-mode (grey-scale) and pulsed-wave colour Doppler ultrasound instrument (MyLab30; Esaote Healthcare, São Paulo, Brazil) equipped with a multi-frequency linear transducer. From the 42 animals that started the synchronisation protocols, six animals per group were selected because they corresponded to the treatments as planned. Collection of reproductive tract samples Following slaughter on Day 7, the reproductive tract was removed, transported to the laboratory on ice within 10min and the uterus and the vagina were dissected. Each horn was opened from the antimesometrial side and intercaruncular endometrium dissected from the anterior, middle and posterior regions. Endometrium samples from each region of the uterine horn relative to the ovary containing the CL, from pooled regions of the contralateral horn and from vaginal tissue collected in the vaginal fornix were stored individually at�808C. Furthermore, endometrial fragments from the anterior and middle regions of the ipsilateral horn relative to the CL were collected from cows in the LF-LCL and SF-SCL groups, fixed in 4% buffered for- malin for 24 h and embedded in paraffin for microscopy. The CL was excised, measured and weighed. Volume (V) of the CL was calculated by applying measurements of height, width and length in the ellipsoid volume formula: V¼ 4/3p� r1� r2� r3, where p is the mathematical constant, r1 is height radius, r2 is width radius and r3 is length radius. Sample processing Radioimmunoassay for measuring P4 and E2 concentrations Plasma P4 concentrations were assayed with a solid-phase radioimmunoassay (RIA) kit containing antibody-coated tubes and 125I-labelled P4 (Coat-A-Count Progesterone; Diagnostic Products Corporation, Los Angeles, CA, USA) as reported previously (Garbarino et al. 2004). Plasma E2 concentrations were assayed using a commercial RIA kit (Double Antibody Oestradiol; Diagnostic Products Corporation) as reported (Siddiqui et al. 2009). The intra-assay CV and sensitivity for P4 and E2, respectively, were 6.2% and 0.07 ngmL�1 and 4.0% and 0.06 pgmL�1. Extraction of RNA and synthesis of cDNA RNA extraction was performed using an RNeasy mini columns kit (Qiagen Laboratories, São Paulo, Brazil), as per the manufacturer’s instructions. To maximise lysis and total RNA isolation, ,30mg of endometrial and vaginal tissue was submerged in liquid nitrogen and macerated using a stainless steel apparatus. The macerate was mixed with buffer RLT from the kit and the tissue suspension was passed at least 10 times through a 21-guage needle and centrifuged at 12 000g for 1min at 48C for removal of debris, before supernatant loading and processing in RNeasy columns. The samples were treated with DNAse I (Life Technologies, São Paulo, Brazil) for 15min at room temperature in 80-mL reactions during the RNA extraction protocol. Columns were eluted with 40 or 60 mL of RNase free water for endometrium and vagina, respectively. Concentration and purity of total RNA in extracts was estimated by a spectro- photometer (NanoDrop; Thermo Scientific, Waltham, MA, USA) by the absorbance at 260 nm and the 260/280 nm and 260/230 nm ratios, respectively. The RNA extracted samples were submitted to cDNA synthesis using the High-Capacity cDNAReverse Transcription Kit (Life Technologies), as per the manufacturer’s instructions. Samples were incubated at 258C for 10min, followed by incubation at 378C for 2 h and reverse-transcriptase inactivation at 858C for 5min and storage at �208C. Quantitative polymerase chain reaction (PCR) We selected seven genes participating in different pathways relevant to endometrium receptivity (Table 1). Gene expression of PGR, ESR2 and OXTR are upregulated by E2 and down- regulated by P4 (Okumu et al. 2010; Bishop 2013). SLC2A1, AKR1C4, LPL and SERPINA14 are genes related to histotroph composition and conceptus maintenance (Ulbrich et al. 2009a, 2009b; Forde et al. 2010). Peptidylprolyl isomerase A (PPIA), cyclophilin A, actin-b (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L15 (RPL15) were used as reference genes for normalisation of expression. Primer sequences were obtained from the literature or were designed based on Gen-Bank Ref Seq mRNA sequences using the National Center for Biotechnology Information tool, Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/) and PrimerQuest (http://www.idtdna.com/primerquest). Equivalent specificity searches were done using the software Basic Local Alignment Search Tool (BLAST; Altschul et al. 1990; http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Real-time reverse transcription polymerase chain reactions were carried out in 96-well plates in a Step One Plus apparatus (Life Technologies). The 20-mL reactions contained power SYBR green reagent (10 mL), cDNA samples (diluted 1 : 40) or diethylpyrocarbonate (DEPC) water in the negative control (4 mL) and a mixture of specific forward and reverse primers (6 mL). The thermal cycling parameters were set at 958C for 10min then 40 cycles of 15 s at 958C and 1min at 608C. For each gene the concentration of primers in the reaction mixture was tested (150, 300, 600 or 900 nM) and selected according to efficiency of amplification, lack of amplification in the negative control and melting (dissociation) curve analysis. Transcript abundance was determined using the relative standard curve method using serial dilutions of a cDNA pool (1 : 40 to 1 : 320). Primers were considered for further analysis when efficiency of amplification of the standard curve was between 85 and 110% and the coefficient of determination of the regression equation was .0.97. There was no linear amplification of the AKR1C4 gene in the vagina and this gene was not further analysed in this tissue. Cycle quantification (Cq) values were obtained from LinReg PCR software (http://www.hartfaalcentrum.nl/index. php?main=files&sub=LinRegPCR). The expression of each target gene was calculated following normalisation using GeNorm software (http://medgen.ugent.be/,jvdesomp/genorm/; Vandesompele et al. 2002). Initially four reference genes were used (PPIA, ACTB, GAPDH and RPL15). After selection performed by the software, PPIA and ACTB were chosen. Regional transcript abundance in the bovine uterus Reproduction, Fertility and Development 1535 http://www.ncbi.nlm.nih.gov/tools/primer-blast/ http://www.idtdna.com/primerquest http://blast.ncbi.nlm.nih.gov/Blast.cgi http://blast.ncbi.nlm.nih.gov/Blast.cgi http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR http://medgen.ugent.be/∼jvdesomp/genorm/ The geometric mean of the expression of these two genes was used for normalisation of the expression of the target genes. Identity of amplified PCR fragments was verified by sequencing and the resulting sequences were assessed by Chomas software (http://technelysium.com.au/) and tested for specificity using BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Immunohistochemistry Paraffin-embedded endometrium samples of the anterior and middle regions of the ipsilateral horn were cut into 4-mm sec- tions andmounted on an adhesive slide (StarFrost; Knittel Glass, Braunschweig, Germany). Sections were deparaffinised in xylene and rehydrated in a series of increasing dilutions of ethanol. Sections were incubated for 5min in 10 nM citrate buffer (pH 6) at room temperature and then incubated slides were processed for four cycles of 5min each at 750 W in a microwave oven for antigen retrieval. The container was refilled with distilled water as needed to replace evaporation of the solution and avoid section drying. Slides were cooled at room temperature for 20min and washed three times for 5min in phosphate-buffered saline (PBS) containing 0.3% Triton (PBS– Triton; pH 7.4). Endogenous peroxidase activity was blocked by incubation of the sections in 1.5% hydrogen peroxide in meth- anol for 30min at room temperature then sections were washed in PBS–Triton (3� 5min). To block nonspecific binding, pro- tein block was carried out using 2% non-fat dry milk for 15min at room temperature. Tissue sections were incubated in a humid chamber with a mouse monoclonal anti-progesterone receptor (PGR) primary antibody (Clone PR10A9, 1 : 75; Beckman Coulter, Villepinte, France) overnight at 48C. Negative-control reactions contained normal mouse IgG instead of primary antibody. Tissue sections were then washed (3� 5min) in PBS–Triton. The Biotinylated Link Universal Solution (Dako, Glostrup, Denmark) was used as secondary antibody and the incubation was conducted for 15min at room temperature in a humid chamber. Next, slides were washed in PBS–Triton buffer (3� 5min), incubated with a streptavidin–horseradish peroxi- dase complex (streptavidin–HRP; Dako) for 15min in a humid chamber and washed again in PBS–Triton. Sections were then incubated in diaminobenzidine (DAB; Dako) solution for 5min and washed in distilled water (3� 5min). Finally, sections were counterstained with haematoxylin, washed in running water for 10min, dehydrated in a series of increasing concentrations of ethanol, cleared in xylene andmounted on coverslips. The slides were photographed using light microscopy and staining inten- sity was subjectively scored in each of the following tissue compartments: luminal epithelium, stromal cells and glandular epithelium, by six independent evaluators. Intensity scores ranged from zero (i.e. absence of staining) to three (i.e. very strong staining). Statistical analyses All data were tested for normality of distribution of residues using Shapiro–Wilk’s test and homogeneity of variances using Welch’s method and transformed by natural or inverse loga- rithms if necessary. Discrete dependent variables were analysed by one-way ANOVA for the effect of group using the PROC MIXED procedure (SAS Software, Version 9.2; SAS Institute, Cary, NC, USA) with animal-within-group as a random vari- able. For gene expression, data was analysed by split-plot ANOVA. For measures repeated in space using PROCMIXED procedure (SAS software) two distinct models were used. The first model estimated the effects of group, side and their inter- action, whereas the secondmodel estimated the effects of group, Table 1. Symbols of genes, accession numbers, primer pair sequences, amplicon size and literature references used to generate primers Target gene GenBank number Sense and anti-sense primer sequences Size of amplicon (bp) References PGR NM_001205356.1 50-GCCGCAGGTCTACCAGCCCTA-30 199 Primer-Blast 30-GTTATGCTGTCCTTCCATTGCCCTT-50 ESR2 NM_174051.3 50-TCACGTCAGGCACGCCAGTAAC-30 155 Primer-Blast 30-CACCAGGTTGCGCTCAGACCC-50 OXTR NM_174134.2 50-AAGATGACCTTCATCGTCGTG-30 177 Krishnaswamy et al. 2009 30-CGTGAAGAGCATGTAGATCCAG-50 AKR1C4 NM_181027.2 50- TGAGCTATTACTGGGTGTTGGTCACCC-30 106 Primer-Blast 30-ACAGCCCTAGAAGCAAGATGTTCTGGT-50 SERPINA14 NM_174797.3 50-ATATCATCTTCTCCCCCATGG-30 126 Ulbrich et al. 2009a 30-GTGCACATCCAACAGTTTGG-50 LPL NM_001075120 50-CAGGTCGAAGTATCGGAATCCA-30 68 Forde et al. 2010 30-GAAAGTGCCTCCGTTAGGGTAAA-50 SLC2A1 NM_174602.2 50-ATCATCTTCACCGTGCTCCTGGTT-30 127 PrimerQuest 30-TGTCACTTTGACTTGCTCCTCCCT-50 GAPDH NM_001034034.2 50-GCCATCAATGACCCCTTCAT-30 70 Bettegowda et al. 2006 30-TGCCGTGGGTGGAATCA-50 ACTB NM_173979.3 50-GGATGAGGCTCAGAGCAAGAGA-30 78 Bettegowda et al. 2006 30-TCGTCCCAGTTGGTGACGAT-50 RPL15 NM_001077866.1 50-TGGAGAGTATTGCGCCTTCTC-30 65 Bettegowda et al. 2006 30-CACAAGTTCCACCACACTATTGG-50 PPIA NM_178320.2 50-GCCATGGAGCGCTTTGG-30 66 Bettegowda et al. 2006 30-CCACAGTCAGCAATGGTGATCT-50 1536 Reproduction, Fertility and Development E. R. Araújo et al. http://technelysium.com.au/ http://blast.ncbi.nlm.nih.gov/Blast.cgi region and their interaction using a compound symmetry (CS) covariance matrix structure with animal-within-group as a random variable. For target gene abundance in vaginal tissue, only the effect of group was considered. A probability of P# 0.05 indicated significant effects and a probability of 0.05,P# 0.1 indicated that significance was approached. Data are presented as the arithmetic mean � s.e.m. Results Animal model The animal model succeeded to generate two groups with larger or smaller follicle and subsequent CL and, consequently, greater or smaller plasma concentrations of E2 and P4 (Table 2). Briefly, follicle diameter on Day �2 and Day 0 and POF diameter were greater (P# 0.05) in the LF-LCL group than in the SF-SCL group. Concentrations of E2 were higher (P# 0.05) on Day �1 and Day 0 in the LF-LCL group (n¼ 3) compared with the SF-SCLgroup (n¼ 5). Althoughweight of CL onDay 7 was greater (P# 0.05) in the LF-LCL than in the SF-SCL group, CL volume did not differ (P. 0.1) between groups. Plasma P4 concentrations were also greater (P# 0.05) for several P4 end- points during early dioestrus in the LF-LCL group compared with the SF-SCL group. Endometrial transcript abundance Effect of side of the uterine horn relative to the to CL Abundance of transcripts for ESR2, AKR1C4, SLC2A1, LPL and SERPINA14 did not differ (P. 0.1) between the ipsilateral and contralateral horns to the CL. However, abun- dance of transcripts for PGR and OXTR was greater (P# 0.05) in the side contralateral to the CL (Fig. 1). For the proposed model to modulate the periovulatory endocrine profile, an effect of group (SF-SCL or LF-LCL) was detected for the expression of ESR2, AKR1C4, LPL, SLC2A1 and OXTR genes. Abundance of transcripts for ESR2, AKR1C4, LPL and SLC2A1 was greater (P# 0.05) and for SERPINA14 tended to be greater (P# 0.1) in the LF-LCL group than in SF-SCL group. In contrast, the transcript abundance was lesser (P# 0.05) or tended to be lesser (P# 0.1) in the LF-LCL group than in the SF-SCL group for OXTR and PGR, respec- tively. An interaction of group-by-side was observed only for the OXTR gene. Interpretation of the interaction revealed that while abundance of OXTR was increased in the contralateral side for the SF-SCL group, it was similar between sides in the LF-LCL group. Effect of region of the uterine horn relative to the CL An effect (P, 0.05) or an approached effect (P, 0.1) of region (anterior, medial or posterior) of the horn relative to the CL was detected for all transcripts studied, except for OXTR (Fig. 2). Generally, expression was greatest in the anterior region and lowest in the posterior region of the uterine horn. There was no significant group-by-region interaction. An effect of group (SF-SCL or LF-LCL) was also detected for all target genes studied and followed the same abundance direction observed in the above-mentioned side-by-group analysis. Therefore, transcript abundance was greater (P# 0.05) for ESR2, AKR1C4, SLC2A1, LPL and SERPINA14 Table 2. Follicle, CL and P4 measurements of cows ovulating within the first 48 h after GnRH Values are expressed as mean � s.e.m. Day 0 is the day of GnRH treatment to induce ovulation Parameter Group P value SF-SCL (n¼ 6) LF-LCL (n¼ 6) Follicle diameter (mm) D-2 6.92� 0.47 10.88� 0.76 0.002 D0 10.11� 0.41 13.35� 0.48 0.0004 Pre-ovulatory follicle 10.33� 0.33 13.88� 0.47 ,0.0001 Plasma E2 concentrations (pg mL�1)A Day �1 0.36� 0.23 2.30� 0.57 0.04 Day 0 0.93� 0.16 2.96� 0.36 0.006 CL Weight (g)B 1.64� 0.23 2.88� 0.27 0.005 Volume (cm3) 1.72� 0.31 2.31� 0.24 0.17 Plasma P4 concentrations (ngmL�1) Day 6 1.27� 0.25 3.52� 0.48 0.002 Day 3 to Day 6C 2.71� 0.59 7.83� 1.16 0.002 Difference between Day 6 and Day 3D 1.03� 0.19 3.03� 0.40 0.001 Interval (days) Day 0 to day of P4 .1 ngmL�1 E 6.17� 0.31 4.33� 0.33 0.003 AOestradiol concentrations were assayed in a subset of animals (n¼ 3–5 per group). BMeasured post mortem on Day 7. CSum of P4 concentration of Day 3 to Day 6. DSubtraction of P4 concentration on Day 3 from P4 concentration on Day 6. ENumber of days between Day 0 and the day that P4 was .1 ngmL�1. Regional transcript abundance in the bovine uterus Reproduction, Fertility and Development 1537 G: P � 0.06 G: P � 0.005 G: P � 0.008G: P � 0.005 S: P � 0.9 GS: P � 0.9 0.6 0.5 0.4 0.3 0.2 0.1 0 0.6 0.5 0.4 0.3 0.2 0.1 0 0.6 0.7 0.5 0.4 0.3 0.2 0.1 0 0.6 0.7 0.5 0.4 0.3 0.2 0.1 0 0.6 0.7 0.5 0.4 0.3 0.2 0.1 0 0.6 0.7 0.5 0.4 0.3 0.2 0.1 0 0.6 0.5 0.4 0.3 0.2 0.1 0 SERPINA14 R el at iv e ex pr es si on le ve ls (a rb itr ar y un its ) R el at iv e ex pr es si on le ve ls ( ar bi tr ar y un its ) R el at iv e ex pr es si on le ve ls ( ar bi tr ar y un its ) IPSI CONTRA LF-LCL SF-SCL IPSI CONTRA IPSI CONTRA IPSI CONTRA IPSI CONTRA IPSI CONTRAIPSI CONTRA PGR ESR2 OXTR AKR1C4 SLC2A1 LPL G: P � 0.09 S: P � 0.5 S: P � 0.4 G: P � 0.03 GS: P � 0.4 GS: P � 0.5 G: P � 0.05 GS: P � 0.5 S: P � 0.1 GS: P � 0.5 S: P � 0.03 S: P � 0.03 GS: P � 0.01 S: P � 0.6 GS: P � 0.4 ∗ ∗ Fig. 1. Mean � s.e.m. transcript abundance of target gene expression normalised to peptidylprolyl isomerase A (PPIA) and b-actin (ACTB) in the uterine horns ipsi (IPSI)- and contralateral (CONTRA) to the CL frombeef cows synchronised to ovulate a large (LF-LCL) or small follicle (SF-SCL). Effect of group (G), side (S) and interaction of group-by-side (GS) are indicated. IPSI, horn ipsilateral to the ovary bearing the CL;CONTRA, horn contralateral to the ovary bearing theCL.Genes: progesterone receptor (PGR), oestrogen receptor 2 (ESR2), oxytocin receptor (OXTR), aldo–keto reductase family 1, member C4 (AKR1C4), solute carrier family 2, member 1 (SLC2A1), lipoprotein lipase (LPL) and serpin peptidase inhibitor, clade A member 14 (SERPINA14, formerly known as UTMP). 1538 Reproduction, Fertility and Development E. R. Araújo et al. 0.6 0.5 0.4 0.3 0.2 0.1 0 0.6 0.7 0.5 0.4 0.3 0.2 0.1 0 0.6 0.8 0.4 0.2 0 0.6 0.5 0.4 0.3 0.2 0.1 0 0.6 0.8 0.4 0.2 0 0.6 0.7 0.5 0.4 0.3 0.2 0.1 0 0.6 0.5 0.4 0.3 0.2 0.1 0 G: P � 0.006LF-LCL SF-SCL R: P � 0.002 GR: P � 0.1 G: P � 0.02 R: P � 0.003 GR: P � 0.8 SERPINA14 PGR ESR2 OXTR AKR1C4 SLC2A1 LPL G: P � 0.01 R: P � 0.7 GR: P � 0.1 G: P � 0.008 R: P � 0.07 GR: P � 0.8 G: P � 0.005 R: P � 0.01 GR: P � 0.2 G: P � 0.05 R: P � 0.01 GR: P � 0.2 G: P � 0.05 R: P � 0.01 GR: P � 0.5 R el at iv e ex pr es si on le ve ls ( ar bi tr ar y un its ) R el at iv e ex pr es si on le ve ls (a rb itr ar y un its ) R el at iv e ex pr es si on le ve ls ( ar bi tr ar y un its )IPIMIA IPIMIA IPIMIAIPIMIA IPIMIA IPIMIA IPIMIA Fig. 2. Mean � s.e.m. transcript abundance of target gene expression normalised to peptidylprolyl isomerase A (PPIA) and b-actin (ACTB) in the regions of the horn ipsilateral to the CL from beef cows synchronised to ovulate a large (LF-LCL) or small follicle (SF-SCL). Effect of group (G), region (R) and interaction of group-by-region (GR) are indicated. IA, ipsilateral anterior; IM, ipsilateral medial and IP, ipsilateral posterior regions. Genes: progesterone receptor (PGR), oestrogen receptor 2 (ESR2), oxytocin receptor (OXTR), solute carrier family 2, member 1 (SLC2A1), aldo–keto reductase family 1, member C4 (AKR1C4), lipoprotein lipase (LPL), serpin peptidase inhibitor, clade A member 14 (SERPINA14, formerly known as UTMP). Regional transcript abundance in the bovine uterus Reproduction, Fertility and Development 1539 and lesser (P# 0.05) for PGR and OXTR in the LF-LCL group than in the SF-SCL group. Regarding intensity of immuno- staining for PGR, average staining intensity was similar between groups and regions (Fig. 3). Vaginal transcript abundance There was no difference (P. 0.01) between groups in transcript abundance for ESR2, OXTR, LPL, SLC2A1 and SERPINA14 genes in the vagina (Fig. 4). However, the transcript abundance SF-SCL group LF-LCL group Anterior Medial 200 μm Fig. 3. Localisation of PGR in the bovine uterus by immunohistochemistry. Representative images of PGR immunohistochemi- cal localisation in the anterior and medial regions of LF–LCL and SF–SCL groups at Day 7 of the oestrous cycle. Original magnification 200�. Scale bar¼ 200mm. Inset: negative control. PGR VAGINA ESR2 LF-LCL SF-SCL 0 0.2 0.4 0.6 0.8 ∗ G: P � 0.08 OXTR SLC2A1 LPL SERPINA14R el at iv e ex pr es si on le ve ls ( ar bi tr ar y un its ) Fig. 4. Mean � s.e.m. transcript abundance of target gene expression normalised to peptidylprolyl isomerase A (PPIA) and b-actin (ACTB) in the vagina of beef cows synchronised to ovulate a large (LF-LCL) or small follicle (SF-SCL). An approached effect (P, 0.01) of group is indicated by an asterisk between group bars within each gene. Genes: progesterone receptor (PGR), oestrogen receptor 2 (ESR2), oxytocin receptor (OXTR), solute carrier family 2, member 1 (SLC2A1), lipoprotein lipase (LPL), serpin peptidase inhibitor, clade A member 14 (SERPINA14, formerly known as UTMP). 1540 Reproduction, Fertility and Development E. R. Araújo et al. for PGR approached being greater (P# 0.1) in the SF-SCL group compared with the LF-LCL group (Fig. 4). Discussion Needed increases in reproductive efficiency depend on in-depth understanding of endocrine, cellular andmolecular mechanisms regulating fertility. Focussing on the maternal reproductive tract, the endometrium is a highly dynamic tissue that has the capacity to undergo transcriptional and physiological changes coordinated by ovarian hormones. Circulating E2 and P4 con- centrations orchestrate uterine events in a spatio–temporal manner. In the present study, we have for the first time evaluated whether specific periovulatory endocrine profiles can regionally influence gene transcription patterns in the different sections of the bovine uterus and in the vagina at a specific moment during early dioestrus (Day 7). Therefore, the gene expression patterns of genes encoding endocrine receptors, substrate provision pathways and receptivity markers were compared in the endo- metrium, between the uterine horns ipsi- and contralateral to the CL and in the vagina. In summary, we found that the transcript abundance of ESR2, AKR1C4, LPL, SLC2A1 and SERPINA14 was greater, and the abundance of PGR andOXTRwas lower, in the endometrium of cows from the LF-LCL group (higher pre- ovulatory E2 and postovulatory P4) when compared with the SF-SCL group. The expression patterns of the specific genes displayed inter- and intra-uterine horn variations atDay 7,which indicates that the transcriptome signature of the bovine female reproductive tract is modulated regionally. However, the absence of group-by-side and group-by-region interactions on the abundance of transcripts indicates that regional effects were not influenced by the endocrine changes associated with the treatment groups. This finding did not support our initial hypothesis. Finally, no effect of group was observed on gene transcript abundance in the vagina. Abundance of transcripts for PGR and OXTR was down- regulated in the endometrial tissue of cows with larger POF and CL, and thus higher circulating pro-oestrous E2 and early dioestrous P4 levels, compared with the cows with smaller POF and CL. In line with this finding, elevated concentrations of P4 during early dioestrus are known to downregulate P4 receptor (Okumu et al. 2010; Forde et al. 2011). Moreover, downregulated mRNA and protein expression of PGR in the luminal and glandular epithelium have been proposed as a prerequisite for proper embryo implantation (Spencer et al. 2008; Bazer et al. 2010). However, no difference in PGR staining intensity in the endometrium was observed between anterior andmedial regions or between the LF-LCL and SF-SCL groups. Similarly, the expression of OXTR is known to be downregulated during dioestrus; its transcript abundance is maximal around oestrus and decreases progressively during early to mid-dioestrus (Wijayagunawardane et al. 1998; Spencer and Bazer 2004; Spencer et al. 2004). The down- regulation of OXTR expression may be triggered by increased P4 levels via non-genomic actions (Bishop 2013). Interestingly, we observed a higher ESR2 expression in cows with greater circulating P4 at Day 7, which contrasts with the previous report of Okumu et al. (2010), who documented an inhibiting effect of P4 on ESR2 gene expression at mid-dioestrus. Bazer and Slayden (2008) suggested that the dioestrous inhibition of endocrine receptors might trigger the expression of genes encoding secretory proteins and transporter molecules that selectively transfer substrates into the uterine lumen in order to produce histotroph. In the present study, the favourable periovulatory endocrine profiles (higher E2 and P4) indeed resulted in an increased expression of the genes related to histotroph composition and conceptus maintenance (AKR1C4, SLC2A1, LPL, SERPINA14). The AKR1C4 gene encodes an enzyme related to PGF2a synthesis and P4 clearance (Gauvreau et al. 2010; Seo et al. 2011). Therefore, the greater expression of AKR1C4 in the LF-LCL group could be associated with the beneficial role of uterine PGF2a during early pregnancy, i.e. stimulating vascularisation of the endometrium to support blastocyst growth and embryo implantation (Charpigny et al. 1997; Reese et al. 1999; Ulbrich et al. 2009b). LPL and SLC2A1 may contribute to the composition of the histotroph, as LPL is involved in delivering triacylglycerol (TAG) to target tissues (Mead et al. 2002) and SLC2A1 is responsible for the facilitated transport of glucose across the plasma membrane by diffusion gradient (Wood and Trayhurn 2003). Actions of the latter molecules are important for energy source provision towards the embryo during early development in the uterus (Ferguson and Leese 2006). Interestingly, P4 sequentially enhances LPL expression on Day 7 and decreases it when the embryo starts to elongate (Day 13; Forde et al. 2010, 2011). Regarding SERPINA14, it is one of the most abundant proteins present in the pregnant ruminants uterus (Segerson and Bazer 1989). Its main functions are immunemodulation and transport of proteins such as immunoglobulins (Ulbrich et al. 2009a; Padua and Hansen 2010; Ledgard et al. 2011). A previous study (Bauersachs et al. 2005) reported that the expression of SERPINA14 is increased at late dioestrus compared with early dioestrus, indicating that the main function of SERPINA14 could be much later during pregnancy. However, the present results indicated a difference in SERPINA14 transcript abundance at Day 7 between the different periovulatory profiles, which may suggest favourable earlier modulation of SERPINA14 expression by higher preovulatory oestradiol and postovulatory P4 on embryo survival. In the present study, the maternal tissue samples were collected around early dioestrus (Day 7 after GnRH treatment to induce ovulation), which coincides with the presence of the embryo in the ipsilateral horn, i.e. located on the side of the ovary containing the CL. The embryo remains confined to that region until elongation, which starts 12 to 15 days after oestrus (Peippo et al. 2011). This implies that, during the first 10 days in the uterus, the embryo is free-floating in the endometrial tissue secretion (histotroph) at the anterior region of the ipsilateral horn, in which the first maternal–embryonic interactions occur. Around this timing, the expression of PGR and OXTR was less in the uterine horn relative to the CL compared with the contralateral horn, whereas expression of the remaining five genes was similar between the two horns. It is proposed that local P4 concentrations were different between horns as observed by Pope et al. (1982) and this affected horn expression of PGR and OXTR. It is possible that the differential expres- sion between horns may be needed to provide an optimal Regional transcript abundance in the bovine uterus Reproduction, Fertility and Development 1541 environment for the developing embryo in the lumen of the anterior region of the ipsilateral horn (Wallenhorst and Holtz 1999). This can be reinforced by the fact that embryos trans- ferred to the ipsilateral horn have greater chance of survival than embryos transferred to the contralateral horn (Tervit et al. 1977; Christie et al. 1979). When focusing on the regional transcript abundance patterns within the ipsilateral uterine horn, it is important to consider the importance of the specific regions, with particular focus on the anterior part, in providing the embryonic nutrition for its first 2 weeks of development (Bazer et al. 2012). The abundance of transcripts coding for PGR, ESR2, LPL, SERPINA14 and SLC2A1 was upregulated in the anterior region of the ipsilateral uterine horn. Greater expression of PGR and ESR2 in the anterior part was not expected, as greater P4 concentrations reported (Pope et al. 1982) in this portion may downregulate these receptors. However, similar results were previously reported by Meikle et al. (1997) in sheep treated with P4 or E2. In that study, higher levels of PGR and ESR2 were observed in the anterior section of the myometrium, endometrium and caruncles than in the medial. The analysis of vaginal gene expression was performed in an attempt to determine possible similarities between modulation of gene expression by the periovulatory endocrine environments in the uterus and in the vagina. Due to its easy access and to avoid invasive approaches such as endometrial biopsies, vaginal biopsiesmight provide hands-on information in order to forecast maternal receptivity without disturbing maternal–embryonic interactions (Bonnett et al. 1993; LeBlanc et al. 2002; Pugliesi et al. 2014). Although a trend was observed for an upregulated abundance of PGR in the vagina of SF-SCL cows comparedwith the LF-LCL cows, there was no effect of our endocrinemodel on the expression of any of the other five genes evaluated in vaginal tissue. The PGR gene expression in our study is in line with findings reported by Sağsöz et al. (2011), who reported that E2 and P4 receptors in the bovine cervix and vagina were higher during the follicular phase than the luteal phase. Such endocrine responsiveness of the vaginal transcriptome profile has been demonstrated previously in rats by Ohta et al. (1993), who showed that PGR levels were increased by E2 administration, but not by P4. From the results presented here, we concluded that periovulatory endocrine milieu-induced changes in the expres- sion of genes in the endometrium are not similar to changes in the vagina. In conclusion, the present study showed that the expression pattern of specific genes displays uterine inter- and intra-horn variations in response to periovulatory endocrine modifications, which indicates that the transcriptome signature of the bovine female uterus is endocrine- and location-dependent at Day 7 after oestrus, but that of the vagina is not. 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