Mar 30, 2012 - Preimplantation Phase of the Estrous Cycle in Cattle ... embryo development, preimplantation, uterus, bovine, global proteomics, estrous cycle.
Article pubs.acs.org/jpr
Proteomic Characterization of Histotroph during the Preimplantation Phase of the Estrous Cycle in Cattle Michael P. Mullen,*,†,‡ Giuliano Elia,§ Mark Hilliard,§ Mervyn H. Parr,†,‡ Michael G. Diskin,† Alex C. O. Evans,§,∥ and Mark A. Crowe‡,§ †
Animal and Bioscience Research Department, Animal & Grassland Research and Innovation Centre, Teagasc, Mellows Campus, Athenry, Co. Galway, Ireland ‡ UCD School of Veterinary Medicine, §Mass Spectrometry Resource, UCD Conway Institute of Biomolecular and Biomedical Research, and ∥UCD School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland S Supporting Information *
ABSTRACT: Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography−tandem mass spectrometry (LC−MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC−MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy. KEYWORDS: histotroph, embryo development, preimplantation, uterus, bovine, global proteomics, estrous cycle
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INTRODUCTION Embryo loss during the preimplantation period accounts for a large proportion of reproductive failure in mammals with embryo mortality rates in the order of approximately 30%.1−3 Embryo mortality rates in dairy cattle in particular have significantly increased over the past four decades to approximately 45%, largely associated with the intense genetic selection for milk yield.4 The developing conceptus during this preimplantation period is free floating in the uterine lumen and solely reliant on the composition of the surrounding histotroph for supply of nutrients to sustain rapid growth and development.5 A functional endometrium is a prerequisite for histotroph production, and successful pregnancy involves extensive changes in endometrial morphology, expression, and secretion patterns to support an implanting conceptus.6,7 Studies in sheep using a uterine gland knock out model (UGKO) have demonstrated the pivotal role of the histrotroph as the conceptus fails to elongate after hatching from the zona pellucida in uteri lacking glands.8,9 Synchrony between the © 2012 American Chemical Society
developing embryo and the uterine environment during early pregnancy is also crucial as maternal recognition of pregnancy (MRP), via inhibition of luteolytic pulses of prostaglandin F2α by the endometrium, is dependent on sufficient IFN-tau (IFNT) production by the conceptus trophectoderm5 and must occur by Day 16 in cattle.10,11 Comprehensive endometrial transcriptome analyses during the estrous cycle and early pregnancy in bovine have been reported.12−15 Microarray analysis on endometrial tissues from pregnant heifers at distinct stages of embryo/conceptus development on Days 5, 7, 13, and 16 identified that the greatest number of differentially expressed genes occurred between Day 7 and Day 13.13 Further studies comparing pregnant and non-pregnant cattle on the same days detected no differences in endometrial gene expression between groups until pregnancy recognition on Day 1614 with recent data Received: February 14, 2012 Published: March 30, 2012 3004
dx.doi.org/10.1021/pr300144q | J. Proteome Res. 2012, 11, 3004−3018
Journal of Proteome Research
Article
from the five heifers with an additional sample randomly selected per day to increase sample numbers to n = 6 per time point. All uterine flushings were carried out by using a commercial embryo flushing operative (BoviGenetics Ltd., Cavan, Ireland) as previously described.31
identifying differences on Day 15.16 These data suggest histotroph composition during the preimplantation period undergoes significant alterations and is independent of the presence of a conceptus before pregnancy recognition.14,17 However, conflicting evidence exists regarding the correlation between mRNA and protein abundance levels,18−27 emphasizing the relevance of a global proteomic approach to describe histotroph composition. Despite the importance of histotroph for embryo development and survival, only four studies have examined the global histotroph proteome in domestic animal species,17,27−29 with only one reported in the bovine. Ledgard et al.27 using 2D polyacrylamide gel electrophoresis (PAGE) examined uterine luminal fluid on Day 16 and Day 18 from confirmed pregnant and non-pregnant cattle. They identified decreased protein abundance for cystatin E/M, legumain (LGMN), retinolbinding protein (RPB4), and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) in pregnant compared with non-pregnant heifers. Koch et al.29 recently reported the use of a label-free LC−MS/MS analysis on histotroph from pregnant and non-pregnant ewes on Day 16 and identified 15 proteins more abundant in the pregnant state putatively involved in early embryonic development. However, no study has yet reported using a non-gel-based LC−MS/MS approach to characterize or examine any temporal effects on the histotroph proteome before implantation in bovine or any other domestic animal species. We hypothesized significant temporal modulation of histotroph composition would occur between Day 7 and Day 13 and propose that characterization of histotroph from highly fertile cyclic dairy cattle represents an environment supportive of successful early pregnancy. Tandem mass spectrometry incorporating a label-free shotgun approach was employed to achieve this goal. The specific aims were to (1) characterize the bovine histotroph proteome on Day 7 and Day 13 and (2) examine the temporal histotroph proteomic changes between Day 7 and Day 13 to identify specific proteins that were likely to contribute to embryo survival.
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Uterine Flushing Peptide Digestion
Proteins were extracted from uterine flushings using acetone precipitation as previously described.31 Albumin was subsequently depleted from extracted uterine flushes using a commercially available Seppro bovine albumin depletion column system, and protein concentrations were determined using the Bradford assay according to manufacturer’s instructions (Sigma-Aldrich, Dublin, Ireland). Depleted uterine samples were vacuum-dried and reduced by using 10 mM DTT in 100 mM ammonium bicarbonate for 1 h at 56 °C. The samples were alkylated with 50 mM iodoacetamide in 100 mM ammonium bicarbonate for 30 min at room temperature and digested with 100 ng of sequencing grade modified porcine trypsin (Sigma-Aldrich, Ireland) on a rotary shaker at 37 °C for 8 h. Samples were subsequently dried down and stored at −80 °C until further analysis. Chromatography
Peptides were resuspended in 1 mL of strong cation exchange (SCX) buffer A (10 mM K2PO4, pH 3.0 25% MeCN) and separated offline by strong cation exchange by using a Dionex/ LC Packings UltiMate. Samples were loaded onto a polysulfethyl A column (The Nest Group, Southboro, MA, USA) and eluted with an increasing linear gradient (0−40%) of SCX buffer B (10 mM K2PO4, pH 3.0 + 25% MeCN with 600 mM KCl) over 70 min at a flow rate of 200 μL/min. Seventy SCX fractions of approximately 200 μL were collected into 96well microtiter plates. Eluted peptide fractions were pooled into 10−12 fractions according to the UV activity (214 nm) and desalted using Silica C18 columns (The Nest Group, Southborough, MA, USA), dried under vacuum, and stored at −80 °C until further analysis. Proteomic analysis
Tryptic peptides from pooled SCX fractions were resuspended in 1% ACN and analyzed on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer (Thermo Fisher Scientific, Rockford, IL, USA) connected to an Exigent NANO LC.1DPLUS chromatography system with autosampler, as previously described.32 Each sample was loaded onto a Biobasic C18 Picofrit column (100 mm length, 75 mm i.d.) and was separated by an increasing ACN gradient, using a 120 min reverse phase gradient at a flow rate of 300 nL/min. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200 °C, a capillary voltage of 9 V, a tube lens voltage of 100 V, and with a potential of 1800 V applied to the frit.33 All data were acquired with the mass spectrometer operating in automatic data dependent switching mode. A highresolution MS scan (300−2000 Da) was performed using the Orbitrap to select the 5 most intense ions before MS/MS analysis using the Ion trap.
EXPERIMENTAL SECTION
Animals
All experimental procedures involving animals were licensed by the Department of Health and Children, Ireland. Protocols were in accord with the Cruelty to Animals Act (Ireland 1876) and the European Community Directive 86/609/EC. Animals used in this study were part of a larger cohort of Holstein Friesian (HF) heifers (n = 121) that underwent embryo survival rating classification based on four successive rounds of AI followed by transrectal ultrasound scanning at Day 30 as described by Parr et al.30 Heifers (n = 10) were randomly selected from the group of heifers (n = 30) displaying 100% embryo survival rating, i.e., embryos detected at Day 30 post estrus in all four AI rounds. Heifers were subjected to repeated prostaglandin F2α (Estrumate; Shering-Plough Animal Health, Herdfordshire, U.K.) induced synchronization and were checked for standing estrus; only those in estrus (Day 0) were used. Heifers were flushed on each of two cycles in an attempt to obtain samples on both Day 7 (n = 9) and Day 13 (n = 9). Uterine flush samples were successfully collected on both days from five heifers, and samples were collected on either day from an additional four heifers. One heifer failed to display estrus. Both Day 7 and Day 13 samples were analyzed
Database Search and Protein Identification
Protein identification was carried out using Flexomics (v 2.52, build 3316), an in-house data analysis platform encompassing the Mascot (v2.2) algorithm. Thermo.raw spectral data were converted to mzXML format using the ReAdw tool provided by the Trans-Proteomic pipeline34 with workflows automated by Flexomics. Mascot parameters: enzyme, trypsin; one missing 3005
dx.doi.org/10.1021/pr300144q | J. Proteome Res. 2012, 11, 3004−3018
Journal of Proteome Research
Article
cleavage allowed; parent tolerance ±10 ppm and ±0.6 Da for fragment ion masses; and oxidation of methionine as a variable and carboxymethylamidation as a static modification were specified. MS/MS spectra were searched against the UniprotSwissprot/TrEMBL Bos taurus v 7.6 database containing 35,297 sequences. A Mascot-integrated decoy database search calculated a false discovery of ≤4.62% when searching was performed on the concatenated mgf files with an ion score cutoff of 30 and a significance threshold of p < 0.01. In an effort to prevent over stringency and loss of potentially biologically pertinent data, all proteins with at least one unique peptide and only peptides containing an unbroken “b” or “y” ion series of a minimum of 4 amino acid residues were considered. More stringent criteria were subsequently applied to the data set.
1:200 (R&D Systems, Minneapolis, MN); anti-human TIMP-2 at 1:1000 (Sigma-Aldrich); anti-human Chromogranin A at 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human Superoxide dismutase (SOD) at 1:1000 (Santa Cruz Biotechnology) for 2 h at room temperature. Membranes were then washed in TBST before addition of 1:2500 dilution of streptavidin HRP (R&D systems) for 90 min at room temperature. Proteins were detected using Pierce ECL plus detection kits (Thermo Fisher Scientific) and imaged and quantified using a Flourochem CCD digital camera and AlphaEase FC densitometry software (AlphaInnotech/Cell Biosciences, Santa Clara, CA, USA). For relative quantification, the integrated density value (IDV; defined as Σ(each pixel value − background)) was determined for equal sized boxes (for each sample) drawn around bands, with background values taken below each band of interest to account for any lane to lane variation in background. All 12 uterine flush samples were randomized per blot and repeated in triplicate. Multiple exposures were carried out to ensure linearity with exposure times subsequently set to 2 min between comparisons.
Spectral Index
To assess differences in protein abundance on Day 7 compared with Day 13, an empiric test based on spectral counting termed Spectral Index (SpI) was used.35 In this study, where one group represented samples collected on Day 7 and the other samples collected on Day 13, the SpI is defined as
Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA)
D ⎞ ⎛ S̅ N̅ D ⎞ ⎛ SD7 N̅D7 ̅ D13 ⎟ ⎜ ⎟ SpI = ⎜ × D13 − × T T N̅D13 N̅D7 ̅ + SD13 ̅ ̅ + SD13 ̅ ⎠ ⎠ ⎝ SD7 ⎝ SD7
GO analysis was carried out using AMIGO40 (v1.8) and the DAVID (v6.7) bioinformatics resource,41,42 with Benjamini corrected and EASE score P values of
