Proteome of the Early Embryo–Maternal Dialogue in the Cattle Uterus

Article pubs.acs.org/jpr

Proteome of the Early Embryo−Maternal Dialogue in the Cattle Uterus Marta Muñoz,† Fernando J. Corrales,‡ José N. Caamaño,† Carmen Díez,† Beatriz Trigal,† María I. Mora,‡ David Martín,† Susana Carrocera,† and Enrique Gómez*,† †

Centro de Biotecnología Animal - SERIDA Camino de Rioseco, 1225 La Olla − Deva 33394 Gijón, Asturias, Spain Centro de Investigación Médica Aplicada (CIMA), Avda Pío XII, 55 31008, Pamplona, Navarra, Spain

‡

S Supporting Information *

ABSTRACT: We analyzed embryo−maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC−ESI−MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development. KEYWORDS: bovine, embryo, uterus, development, NFkB, TNF, Interleukin-1Beta

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INTRODUCTION Up to the blastocyst stage, mammalian embryonic development is relatively autonomous and independent of the maternal tract, the embryo itself being able to regulate cell division and differentiation.1 However, during the in vitro culture period, deprivation of factors mediating embryo−maternal communication may affect subsequent embryonic development and its viability2 and lead to pathologies in the offspring.3,4 Certainly, the passage through the genital tract confers to the embryos improved viability and survival to cryopreservation.5−7 However, within the genital tract, it is difficult to identify the respective contributing factors (i.e., ovary and oocyte; sperm; embryo; oviduct and uterus) that lead embryos to become healthy born individuals. Gaining insight into the embryo− maternal interactions would allow the design of novel strategies leading to reduced early embryonic losses8,9 and increase pregnancy rates following embryo transfer.10 Therefore, the development of validated models is necessary to analyze embryo maternal interactions. In the uterus, the early embryo does not float in a recognizable volume of maternal secretions,11 but probably © 2011 American Chemical Society

within a thin fluid layer stabilized by glycoproteins that protect the embryo against osmotic changes and fluctuations. Similar to the oviduct, this microenvironment would avoid dispersal of ions and essential compounds, particularly during cilia beating or muscular contraction.12 Progesterone and estradiol trigger peripheral changes leading to temporal modifications within the endometrium and the oviduct,12 which lead to an appropriate substrate for the ovum and the embryo throughout its development. The composition of UF would therefore be a reflection of general modifications dependent on oestrus cycle phases and interactions between the embryos and the genital tract. Analysis of local interactions between early embryos and genital tract in vivo still remains as a challenge because of intrinsic technical difficulties in exploring the uterine environment surrounding the embryo. At a comprehensive uterine level, nevertheless, we propose that the intensity of such modifications could be commensurate with the numbers of live embryos interacting with the endometrium at a specific time. Received: July 11, 2011 Published: December 9, 2011 751

dx.doi.org/10.1021/pr200969a | J. Proteome Res. 2012, 11, 751−766

Journal of Proteome Research

Article

Animals and Embryo Transfer (ET)

Thus, with multiple early stage embryos resident in the uterus, local changes could be detectable and measured by sensitive proteomic techniques analyzing the UF. Such analysis could provide crucial knowledge into the developmental programming and origins of health and disease.13,14 In this context, valuable data can be obtained using the bovine model for studies concerning developmental programming.15−18 In cattle, systemic uterine changes are better understood than local molecular signaling between endometrial cells and embryos, which are not sufficiently described at early stages, although they would surely lead to basic knowledge required to formulate media and develop in vitro culture systems. The first systematic embryo-maternal proteome analysis in bovine focused on the preimplantation period,19 and recently two studies analyzed endometrial gene expression at early embryonic stages (i.e., day 7) in cattle.20,21 Most published research in embryo−maternal interactions in bovine, as well as in other farm species, focus on pre- and implantation periods.22 At present, a global proteomic profiling of the UF covering the period of blastocyst development has not yet been performed in domestic species with two-dimensional difference gel electrophoresis (2D-DIGE) coupled to mass spectrometry (MS). In the study presented here, we used well-characterized pairs of UF protein samples from the same heifers transferred or not with IVP embryos at the time of their expected entry in the uterus (day 5). The use of the same animals allowed counteracting genetic variability. After embryo and UF recovery on day 8, and repeating such an approach through different estrous cycles, we compared proteomic profiles in UF containing live embryos vs sham transfers and validated our model with UF from artificially inseminated cows. Coupled to a panel of morphological and functional analysis, we obtained evidence of local embryo−maternal interactions in the early pregnant uterus. Our long-term objective was to understand the molecular mechanisms underlying embryo−endometrial interactions from the beginning of uterine development and to unveil conserved molecular pathways. Ultimately, such knowledge could be useful in improving in vitro embryo culture media and systems and shedding light on in uterus origins of health.

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Cyclic, cross-bred beef heifers, 15 to 20 months old (n = 16) were housed on an experimental farm. Food was administered by an automated concentrate dispenser, accompanied by oat straw and mixed vitamin-mineral blocks ad libitum. Body condition score was maintained on 3 ± 0.5 points over a 0−5 scale. Oestrus cycles were synchronized by using an intravaginal progestagen device (PRID ALPHA, Ceva, Barcelona, Spain) for 10 days combined with a prostaglandin analogue (Dynolitic, Pfizer, Madrid, Spain) injected 48 h before progestagen removal. Animals were observed at least 3 times per 30 min a day for oestrus detection, commencing 33 h after progestagen removal. Day 0 was considered a fixed time 48 h after progestagen removal, to coincide with the IVF onset in the laboratory. On day 0, ovaries were scanned by ultrasound and the preovulatory follicle was measured. IVP embryos were nonsurgically transferred on day 5 to the cranial third of the uterine horn ipsilateral to the formerly detected preovulatory follicle, under epidural anesthesia as previously described.24 Sham transfers were also performed with IMV (Instruments de Médecine Vétérinaire) embryo holding medium (Humeco, Huesca Spain). Progesterone was analyzed by ELISA (EIA1561, DRG Diagnostics, Germany) in blood plasma samples taken in EDTA-vacuum tubes from venycoccigeal puncture on day 0, just after ET time (day 5) and on day 8. Uterine Flushings

On day 8, prior to being flushed, recipients were monitored to verify the presence of a corpus luteum in the expected ovary. UF were performed using silicon coated, disposable Foley catheters (24 Fr) loaded to flush the two cranial thirds of the horn ipsilateral to the corpus luteum. All recipients were first flushed with 45 mL Fluid Recovery Medium (FRM), consisting of PBS + 10 μL/mL protease inhibitor (Protease Arrest; GE Healthcare, Madrid, Spain). The uterine horn was carefully filled with FRM for 2 min and gently moved without massage. Recovery of FRM was performed by aspirating with a 50 mL syringe (Becton-Dickinson, Zaragoza, Spain), only while a steady flow could be achieved. Those recipients transferred with embryos were next extensively flushed with PBS + 1 mg/mL PVP (mw 40000 P0930). Embryos were identified using a stereomicroscope and rapidly separated from FRM, which was centrifuged (2000× g) at 4 °C, aliquoted and stored at −145 °C. Embryos were processed for immunocytochemical analysis (see below) or separated for other experiments. Fresh UF samples were subjected to bacteriological culture and Gram staining in duplicates by incubation in Brain Heart Infusion (Pronadisa, Madrid, Spain) medium at 37 °C for 4 to 5 days.

EXPERIMENTAL SECTION

All experimental procedures involving animals were performed according to the European Community Directive 86/609/EC (Spanish Real Decreto 1201/2005), and were sanctioned by the Animal Research Ethics Committee of SERIDA. All reagents were purchased from Sigma-Aldrich (Madrid, Spain) unless otherwise stated.

Isolation of High Molecular Weight Factors from Uterine Fluid for in vitro Embryo Culture

In vitro Embryo Production

Uterine fluid factors were isolated from UFs by 3-kDa dialyzation using an Amicon Ultra-15 device (Millipore, Madrid, Spain). Briefly, frozen UF samples were thawed at room temperature and filtered through a low-protein binding 0.22 μM filter. Volumes of UF with equivalent protein amounts were loaded in the Amicon filter device and centrifuged for 90 min at 4000× g and 4 °C. The procedure was repeated twice by dialyzing against 4 mL SOF without additives, which led to the obtention of SOF + uterine factors (SOF-UF) as a retentate. Dialyzation allowed the discarding of protein inhibitor Protease Arrest, a low molecular weight compound as declared by the supplier. Retentates were recovered, measured in volume and adjusted to 380 μL with SOF (accounting for an estimated final

Embryos were produced in vitro from oocytes within slaughterhouse ovaries as previously reported.23 Briefly, cumulus-oocyte complexes (COCs) were matured in TCM199, NaHCO3 (2.2 g/L), fetal calf serum (FCS, F-4135) (10% v/v), pFSH-LH (Stimufol, ULg FMV France) and 17β-estradiol (1 μg/mL). In vitro fertilization was performed with frozen/thawed sperm by using a swim-up procedure. Presumptive zygotes were cultured in synthetic oviduct fluid (SOF) droplets with 6 g/L BSA at 38.7 °C, 5% CO2, 5% O2 and 90% N2. Embryos (morulae) were temporarily transferred to recipients on day 5 after in vitro fertilization (IVF: day 0) until day 8 (see below), or continued in in vitro culture up to day 8. 752



Article

and volume ratio normalization of different samples in the same gel. The Biological Variation Analysis (BVA) module was then used to match protein spots on different gels and to identify protein spots with substantial differences. Manual editing was performed in the BVA module to ensure that spots were correctly matched on different gels and to remove streaks and speckles. Differentially expressed spots were considered for mass spectrometry (MS) analysis if the corresponding t test p-value was 0.67). Protein concentration in UFs was dependent on numbers of viable embryos recovered (Figure 2A), both showing a significant negative correlation (r = −0.387; p = 0.005). However, numbers of viable embryos did not affect the rise in P4 in blood levels during the period at which the embryos were present in the uterus (Figure 2B). A subset of flushings selected for comparative proteomic analysis by 2-D DIGE-MS consisted of paired UFs from 8 heifers, with embryos from Bull-B and their respective sham transfers (n = 16 samples). Results of the selected samples for embryo recovery, P4 and protein are shown in Table 2, and are presented in comparison with AI values in UFs from the same animals. No significant differences were observed, although the recorded numbers followed the pattern observed in Table 1 in all animals analyzed. Bacterial cultures and Gram staining were negative in all UF samples analyzed.

Statistical Analysis

Data were analyzed in two steps. First, factors showing significant influence were identified by categorical data modeling (CATMOD) using SAS Version 8.2 software (SAS 1999). Second, significant factors were used to produce a linear model using the general linear models procedure (GLM; SAS software). The GLM was used to estimate the least-squares means (LSM) for each fixed effect having a significant F value. The Ryan-Einot-Gabriel-Welsch test was used to compare raw means calculated for the main effects. Embryo development data were transformed to frequency percentages, whereas blastocyst cell counts, progesterone and protein concentration were expressed as absolute values.

Table 1. Bull Effects on in vitro Embryo Development, Embryo Recovery Rates from Host-recipients, P4 Concentration Rise from Day 5 (embryo transfer) to Day 8 (flushing), Flushed Volume and Protein Contents in Flushes in vivo development in vitro development ET Bull-A Bull-B Sham

oocytes 1737 2002

R

blastocysts

9 8

20.8 ± 2.4 37.7 ± 2.4y

x

P < 0.01

transferred oocytes 1694 1421

recovered fluid

recovered embryos (%)

c

Na

morulaeb

viable

16 14 18

36.1 ± 4.4 46.4 ± 5.2

12.4 ± 2.2 18.2 ± 2.5e

9.8 ± 1.7 15.2 ± 2.2e

P < 0.03

P < 0.03

blastocysts d

d

P4 (ng/mL)

uterine protein

(day 8)(day 5)

% volume

μg/100 μL

total (μg)

16.1 ± 1.9 12.2 ± 1.7 15.2 ± 1.5 P = 0.16

67.0 ± 3.6 74.5 ± 3.8 73.0 ± 2.8 P > 0.18

15.1 ± 4.0 18.2 ± 4.1d 27.2 ± 3.1e P < 0.04

4560 ± 937d 5021 ± 961d 6869 ± 738e P < 0.04

d

a

N: 30 flushings from 6 replicates within 12 host-recipients. bMean of day 5 IVP morulae transferred per flushing. cAs a proportion of transferred Morulae. dData are LSM ± SEM. eEffects considered: cow, recovered live embryos; percentages of embryos recovered had no effect (p > 0.34). 755



Article

Within the ICM, UF protein supplementation yielded significantly higher cell counts than PVA. Figure 3 shows micrographs (40×) of all types of embryos used in these experiments, that is, day 5 morulae transferred (3A); day 8 embryos recovered (3B); day 8 embryos cultured in vitro with SOF-BSA (3C); and day 8 embryos cultured in vitro with SOF-UF (3D). Proteomics

By means of 2D-DIGE, we obtained the proteomic profile of eight day 8 paired samples between early pregnancy and estrous cycle in the cow species. Analysis of the spot (n = 3275 ± 182) profiles led to the detection of 50 differential protein spots (t test; p < 0.05; Figure 4) identified in all samples analyzed. Subsequent protein digestion and peptide identification by nano-LC−ESI−MS/MS followed by submission to the Phenyx search engine enabled us to identify 38 proteins with an average ratio of differential quantity greater than 1.18 (Table 4). To integrate our results into a more general model, the differentially expressed proteins were classified according to Gene Ontology (GO) and we then investigated the functional associations between altered proteins using the Ingenuity Pathway Analysis Network (IPA). The molecular and cellular functions represented were cell death (n = 21 molecules), cellular movement (n = 15), nucleic acid metabolism (n = 7), small molecule biochemistry (n = 23) and carbohydrate metabolism (n = 10). According to the physiological system development and function represented, proteins were distributed among tissue morphology (n = 9), hematopoiesis (n = 8), immune cell trafficking (n = 10), reproductive system (n = 4) and connective tissue (n = 6). The results are represented as a network (Figure 5) in which each connection between nodes is supported by data available in the literature. The network integrated 23 proteins with a highly significant score of 67 (threshold 12), and it suggests a central function of the NFkB complex mediating the effects associated with the presence of live embryos in the uterus on day 8.

Figure 2. Differences in recoverable protein concentration (A) in day 8 uterine flushings and (B) in blood P4 concentration rise from day 5 to day 8 by numbers of live embryos recovered. Superscripts express significant differences (p < 0.03).

The procedures used in these experiments did not prejudice the capacity of animals to become pregnant upon transfer of IVP embryos. Thus, out of 18 animals flushed several times and later subjected to ET, 11, 3, and 1 were made pregnant after 1, 2, and 3 ETs, respectively; nine of these animals have since calved healthy offspring. The embryotrophic effect of UFs from pregnant (i.e., embryo transferred) and cyclic (i.e., sham transferred) cows and blastocyst cell counts are shown in Table 3. UF from pregnant cows improved blastocyst development over UF from cyclic cows. Interestingly, within development rates during blastulation such differences were consistent with the numbers of viable embryos contained in the original UF samples used. Blastocyst cell counts and survival to vitrification and warming (not shown in tables), were not affected at all by the pregnant or cyclic origin of UFs, and individual effects (cow) were not observed either. The pregnant or cyclic origin of UFs had no effect at all, and individual effects (cow) were not observed either.

Validation of DIGE Results by Western Blot in Uterine Fluid from Pregnant and Cyclic Heifers

Of the 38 proteins that were identified and significantly altered in the ET profile or included in the proteome network, we studied in greater detail the involvement of 10 proteins in the early pregnancy. These proteins were selected because there was a significant difference in their amount between pregnant and cyclic UFs and/or because they were associated with a relevant biological function or pathway. For validation purposes, we selected 4 proteins represented within our proteome analysis (albumin, annexin A4, lactotransferrin and HSPA5). BSA is the

Table 2. Embryo Recovery Rates from Host-recipients, P4 Concentration Rise from Day 0 to Day 8 (flushing), and Protein Contents in Flushes within a Subset of Uterine Fluid Samples (UF) from Bull-B and Sham Transfers Selected for 2D-DiGE-MS Analysis, and UF Samples Obtained from the Same Cows after Artificial Insemination (AI) That Were Used for Validation Purposes

a

% viable embryosa

P4a,b (ng/mL)

flushed uterine proteina

Bull-B

N

recovered (n)

(day 8)−(day 0)

μg/100 μL

Total (μg)

ET (2D-DiGE-MS) AI Sham transfer

8 7 8

20 ± 2.8 (9.8) (1) (−)

15.6 ± 3.8 15.6 ± 4.8 20.6 ± 3.9 P = 0.60

15.6 ± 4.2 17.3 ± 5.2 27.6 ± 4.8 P = 0.07

5057 ± 1064 4364 ± 1311 6328 ± 1207 P = 0.55

Data are LSM ± SEM. bP4 analysis referred to day 0 and day 8 due to the inclusion of AI data. 756



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Table 3. In vitro Development and Differential Cell Counts (inner cell mass -ICM- and trophectoderm -TE-) within Blastocysts Derived of Day 5 IVP Bovine Morulae Cultured in mSOF with PVA or a Dyalizate (>3 kDa) of Fluid Recovered by Flushing on Day 8 from Uteri That Contained Live Embryos after Transfer on Day 5 (+) or Were Sham Transferred (−)a % blastocysts (day 8) cow

live embryos recovered

8351 8351 4899 4899 5708 5708

(+) (−) (+) (−) (+) (−) PVA Cumulative Live embryos Sham transfer PVA

b

N

1c

66 79 79 79 79 79 77 224 237 77

R

d

3 4 4 4 4 4 4

blastocysts 66.8 61.2 73.6 65.2 72.7 67.0 52.2

± ± ± ± ± ± ±

3.8a 3.2b 3.2a,b 3.2b 3.2a,b 3.2b 4.6c

71.1 ± 2.6x 64.5 ± 2.5y 52.2 ± 4.3z

cell counts

expanded

hatched

± ± ± ± ± ± ±

13.7 ± 4.6 8.6 ± 3.9 16.6 ± 3.9a 8.6 ± 3.9 18.9 ± 3.9a 15.7 ± 3.9a 2.6 ± 3.9b

13 15 16 16 17 16 14

36.8 38.7 32.9 32.5 34.8 31.4 24.7

16.5 ± 2.3* 11.0 ± 2.2 2.6 ± 2.2i

46 47 14

34.7 ± 1.9a 34.2 ± 1.8a 24.4 ± 3.8b

58.1 40.7 56.6 41.8 54.3 53.4 25.9

5.0a 4.2b 4.2a,b 4.2b 4.2a,b 4.2a,b 4.1c

56.1 ± 2.6x 45.3 ± 2.5y 25.9 ± 4.3z

N

2e

ICM ± ± ± ± ± ± ±

3.6 3.1 3.1 3.1 3.0 3.2 3.8

TE 124.6 124.5 137.4 145.0 147.1 136.9 131.2

± ± ± ± ± ± ±

8.8 7.7 7.6 7.7 7.4 8.0 9.5

137.8 ± 4.7 135.7 ± 4.6 131.1 ± 9.6

Data are LSM ± SE. Superscripts express significant differences: x,y: p < 0.02; a,b,c: p < 0.05; *i: p < 0.005. bFlushes from cows 8351, 4899, and 5708 contained 24, 9, and 6 live embryos recovered, respectively. cDay 5 cultured morulae. dReplicates. eBlastocysts counted.

a

while down-regulation will refer to higher abundance in cyclic UFs. All the selected proteins were validated in ET-pregnant and cyclic UF, and 3 of these proteins were also tested within AI-pregnant UF. All proteins validated by WB showed consistency with the DIGE data. Figure 6 shows representative WB (6A) and foldchange quantification (6B) of proteins used to validate the proteomics data. Lactotransferrin showed down-regulation (p < 0.05), while annexin-4 was up-regulated (p < 0.05). Of the proteins not identified in the proteomics profile but expected to be detected in UFs, insulin and the mature 17 kDa isoform of IL-1β were identified as down-regulated (p < 0.0005 and p < 0.05, respectively). The IL-1β 35 kDa precursor32 was also identified, although it showed a not-significant down-regulation (p > 0.45). PRDX1 was found as two molecular weight bands, one high molecular weight (HMW) band with a tend to up-regulation (p < 0.1; consistent with proteomics data), and a second 23−24 kDa not significantly down-regulated band (p > 0.45). NFkB-p65 was down-regulated (p < 0.05), while isoforms of its inducer TNF were detected in both types of UF samples. It was only possible to quantify the HMW bands, which probably correspond to the mature and the pro-TNF trimers (78 kDa and 51 kDa, respectively). A 78 kDa band tended to down-regulation (p < 0.1) whereas the 51 kDa band appeared in greater abundance in pregnant UF (p < 0.005). To validate our experimental model and the proteomics data, we analyzed three abundant proteins by WB, albumin, PNP and HSPA5 in UFs from pregnant, AI and cyclic cows (Figure 7). A representative WB is shown in Figure 7A, and significant differences for albumin (Sham vs ET and AI; p < 0.05), but not for PNP and HSPA5 are depicted in Figure 7B. Interestingly, in the three proteins analyzed, AI values were always in the range delimited by pregnant and cyclic values, suggesting that our experimental model did not introduce artifacts.

Figure 3. Micrographs (40×) of all types of embryos used in experiments. (A) Day 5 morulae transferred to cows. (B) Day 8 embryos recovered from cows (note the abundance of collapsed blastocysts, attributable to the protease inhibitor present in recovery medium and its composition that includes PVA and no protein supplements). (C) Day 8 embryos cultured in vitro with SOF-BSA. (D) Day 8 embryos cultured in vitro with SOF-UF (plastic filament residues derived from Amicon devices are observable).

most abundant protein compound in genital fluids, while Annexin A4 has anti-inflammatory properties. Lactotransferrin down-regulation relates to a lack of activation of the NFkB noncanonical pathway, and HSPA5 is widely expressed across species, protecting against the cytotoxic damage of some NFkB activators. The other 6 selected proteins were not identified in the IPA analysis, but were candidates to be involved in the observed effects, as inferred from the proteome network. Thus, we selected the subunit p65 of the NFkB complex and two of its major biological inducers (i.e., TNF-α and IL-1B), insulin, a NFkB inducer involved in embryonic and uterine metabolism, and purine nucleoside phosphorylase (PNP) and peroxiredoxin-1 (PRDX1), whose concentrations significantly increase in the cow uterus at implantation.31 For practical purposes, we will refer to up-regulation as higher abundance in pregnant UFs,

Immunocytochemical Analysis

Embryos were examined by confocal microscopy. IL-1B and TNFα were detected in all blastocysts examined, ET-recovered (n = 9 and n = 9, respectively), cultured in SOF-BSA (n = 10 and n = 8), and cultured in SOF- UF (n = 5 and n = 9). Figure 8 shows representative pictures of ET-recovered (A and D), cultured in SOF-UF (B and E) and cultured in SOFBSA (C and F) blastocysts, for IL-1B and TNFα, respectively. Cross sectioning showed IL-1B and TNFα localized to ICM 757



Article

Figure 4. Representative 2D-DiGE gel image of recovered uterine protein showing spots identified as up-regulated (blue circles) and down-regulated (red circles) by the presence of embryos.

(A. Fazeli, personal communication). We did not find regulated expression of TNFα, IL-1B or insulin in our proteomic analysis. Nevertheless, by targeted WB, pregnant UF showed downregulation of IL-1B, glycosylated TNFα, insulin and NFkB-p65, while nonglycosylated TNFα underwent up-regulation. Insulin might regulate glucose levels in uterus depending on the nutritional status of the mother, which could conduct decisions on the fate of developing embryos according to their sexual dimorphism.33 Glycosylation seems to be unnecessary for TNF bioactivity.43,44 In our work we found that TNFα and IL-1B exist in embryos from different origins (i.e., recovered from the uterus, exposed to UF during in vitro culture and entirely cultured in vitro). Expression of cytokine ligands and their receptors in embryos has been shown for the TNF family in human and mice,45−47 and for IL-1B in human, mice and pigs.48−51 Release of interleukins by the embryo to the culture medium suggests that they could work as uterine mediators.48,52 In the cow we have found an IL-1B release in media from single cultured embryos (unpublished data). TNF and their receptors TNFRI and TNFRII, are transcribed in the bovine uterus, while TNFRI and TNFRII proteins are weakly expressed in stromal cells, peaking on day 8 in the estrous cycle.53 TNFRII is strongly regulated and predominant in the immune systems,54 whose lymphoid cells usually infiltrate the bovine endometrium55 and are additional TNF sources. TNFRI is associated with apoptosis while TNFRII induces cell survival, growth promotion and differentiation.56,57 TNF production is regulated by TNF itself or by other cytokines in what is called a cytokine network that includes interleukins.58 In our work, the up-regulated proteins glyoxalase domain containing 4 and gelsolin might suppress TNF-induced NFkB activation.59,60 Cleavage of gelsolin by

and TE. The expression of both proteins was evenly distributed throughout the embryos. Control embryos in which the primary antibody was omitted did not show positive signals (Supplementary Figure 2, Supporting Information).

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DISCUSSION Development depends on a fine regulation of the maternal immune system, in such a way that the semiallograph embryo expressing paternal antigens will not be rejected. In this study we obtained the earliest evidence of involvement of the NFkB canonical pathway in the cattle uterus. The NFkB cascade can be induced by numerous factors, typically including cytokines such as TNFα and IL-1B, and reactive oxygen species (such as H2O2), end-products of the embryonic metabolism that may act as embryonic signals.33 Activation involves NFkB release from its cytoplasmic inhibitor IKBKE and translocation of NFkB dimers to the nucleus to activate transcription of responsive genes.34,35 Binding of toll-like receptors (TLRs) by bacterial lypopolysaccharide (LPS) also leads to NFkB activation in the bovine endometrium.36−38 Activation of TLRs is not exclusive to LPS, as lactotransferrin, an iron transporter, and fibrinogen, a blood-borne glycoprotein, bind to TLRs.39,40 Lactoferrin up-regulation exists in the ipsilateral bovine oviduct,41 while fibrinogen has been shown in the human uterus.42 Lactotransferrin and fibrinogen were downregulated in our analysis, and our bacteriological assays in UFs were negative, suggesting no evidence of NFkB activation via the TLR. Interestingly, with a gene expression profile similar to the proteome network we describe here, the pig uterus also shows alterations in endometrial transcription in response to embryos that potentially can lead to a decrease in NFkB expression and dampening of maternal innate immune responses 758



Article

Table 4. Identification and Description of 33 Proteins Found to Be Present in Different Amounts in Day 8 Uterine Fluids from Pregnant and Cyclic Cows

spot

t test

av ratio 1.44

ID

913

0.0088

1136

0.03

−2.14

1196

0.031

−1.35

1302

0.0098

−1.41

1380

0.044

−1.33

1559 1576 1736

0.0078 0.0056 0.03

−1.28 1.82 1.31

1796

0.032

1.34

1824

0.018

1.38

2049

0.018

1.19

2089

0.042

1.22

2146

0.038

1.20

2276

0.038

1.32

2472 2474 2654

0.014 0.034 0.006

1.55 1.25 1.35

2973 3003

0.039 0.0021

1.25 1.37

Glyoxalase domain containing 4 (mouse) Capping protein (actin filament) muscle Z-line, beta Capping protein (actin filament) muscle Z-line Rho GDP dissociation inhibitor (GDI) alpha Peroxiredoxin 2 Adenylate kinase isoenzyme 1 Phosphatidylethanolamine binding protein 1 PPIA PPIA

1467 1477

0.0087 0.019

1.32 1.28

Enolase 1/Enolase A Enolase 1/Enolase A

1186

0.0042

−2.02

3273

0.035

618

0.02

−1.52

625

0.0076

−1.92

630

0.0076

−1.65

797 815 1084 1336

0.047 0.023 0.011 0.025

−1.98 1.40 −1.77 −2.30

1362

0.0066

−2.45

1746

0.016

1.21

1338

0.048

−1.31

1.31

Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) Acyl-CoA dehydrogenase, very long chain T-complex protein 1 subunit delta 26S proteasome non-ATPase regulatory subunit 5 CNDP dipeptidase 2 (metallopeptidase M20 family) Argininosuccinate synthase 1 Keratin 19 SUMO1 activating enzyme subunit 1 Twinfilin, actin-binding protein, homologue 1 Glyceraldehyde-3-phosphate dehydrogenase

KPYM (rabbit) THIO

Phenyx protein scorea

peptide matches (unique pept)

% cov

Cytoplasm proteins Q0VCX2 HSPA5

220.2

29 (22)

P48818

36,0

AC

network symbol

ACADVL

Q2T9X2 Q0P5A6

PSMD5

Q3ZC84 P14568 P08728 A2VE14

KRT19

Q56JV6 P10096

GAPDH (includes EG:2597)

Q9CPV4

Mascot protein scoreb

pI

32

756 (>30)

5.09

72400

5 (5)

9

35 (>29)

7.26

66192

57.1

7 (7)

17

197 (>29)

7.83

58075

12.9

2 (2)

5

53 (>30)

5.22

55911

94.9

12 (12)

25

91(>30)

5.67

52655

28.6 193.6 60.2

4 (4) 32 (22) 8 (7)

8 51 24

50 (>29) 804 (>30) 211(>30)

7.64 4.96 5.22

46417 43885 38306

29.7

4 (4)

11

41 (>30)

6.64

40118

49.5

8 (7)

22

79 (>30)

8.71

35737

70.6

10 (9)

25

150 (>30)

5.35

32410

Mw

P79136

CAPZB

114.7

16 (14)

48

316 (>30)

6,24

33741

P79136

CAPZB

74.3

11(11)

36

208 (>30)

6,24

33741

P19803

ARHGDIA

96.0

25 (11)

41

514 (>28)

5.18

23421

Q9BGI3 P00570 P13696

PRDX2 AK1 PEBP1

125.7 71.5 51.1

60 (13) 12 (9) 7 (6)

49 45 32

599 (>30) 162 (>29) 225 (>30)

5.54 8.73 7.74

21946 21664 20986

46(11) 6(4)

54 33

606 (>29) 119 (>29)

8.68 8.68

17869 17869

28 (22) 131 (22)

49 51

693 (>30) 4750 (>30)

6.63 6.63

47195 47195

26 (17)

33

334 (>30)

8.21

57904

8 (3)

30

250 (>30)

5.16

11813

13

153 (>29)

7.88

82850

10

132 (>30)

7.88

82850

7

51 (>30)

8.72

56885

32 46 52 40

527 (>29) 588 (>30) 1375 (>30) 304 (>30)

8.73 5.61 5.65 5.55

78056 80731 66433 47652

39

326 (>28)

5.55

47652

38

167 (>30)

4.73

26604

14

108 (>30)

7.62

49264

P62935 99.3 P62935 34.6 Cytoplasm/Cell membrane Q9XSJ4 ENO1 202.4 Q9XSJ4 ENO1 220.2 Cytoplasm/Nucleus P11974 164.6 Cytoplasm/Nucleus/Secreted O97680 35.4 Extracellular space P81187 CFB 70.8

Complement factor B Bb 8 (8) fragment [CHAIN 0] Complement factor B Bb P81187 CFB 63.9 7 (7) fragment [CHAIN 0] Complement factor B Bb P81187 CFB 27.4 3 (3) fragment [CHAIN 2] Lactotransferrin P24627 LTF 204.6 24 (22) Gelsolin Q3SX14 GSN 251.7 56 (27) Albumin P02769 ALB 318.0 57 (33) Fibrinogen gamma-B chain P12799 FGG 137.7 18 (15) (Gamma’) [CHAIN 0] Fibrinogen gamma-B chain P12799 FGG 147.9 17 (15) (Gamma’) [CHAIN 0] Hepatoma-derived growth Q9XSK7 HDGF 71.4 14 (9) factor Prevacuolar compartment, peripheral and late endosome membranes Vacuolar protein sorting 4 Q0VD48 VPS4B 50.0 6 (6) homologue B 759



Article

Table 4. continued

spot

t test

av ratio

1079

0.02

1.33

1438

0.032

1.18

1674

0.028

1.35

620 1573 1677

0.021 0.013 0.031

−1.55 1.35 1.21

2030

0.02

1192

0.013

1.62 −2.51

ID Synaptotagmin binding, cytoplasmic RNA interacting protein (human) Sjogren syndrome antigen B (autoantigen La) 3′(2′), 5′-bisphosphate nucleotidase 1 Ezrin Ribosomal protein SA ARP2 actin-related protein 2 homologue (yeast) Annexin A4 FIB B- BOVIN

AC O60506

network symbol Nucleus SYNCRIP

Phenyx protein scorea

peptide matches (unique pept)

% cov

Mascot protein scoreb

9

61 (>29)

pI

43.7

5 (5)

93.8

10 (10)

25

344 (>29)

8.68

46534

Q3ZCK3

108.9

20 (14)

51

133 (>29)

5.47

33197

Plasma membrane P31976 EZR P26452 RPSA A7MB62 ACTR2

18.5 121.8 91.4

3 (3) 19 (12) 12 (11)

7 35 29

46 (>29) 665 (>30) 133 (>30)

6.17 4.85 6.49

68629 32884 44761

P13214

202.6

62 (21)

63

1571 (>30)

5.69

35889

189.7

29 (20)

44

770 (>30)

8.56

53340

Q0VCK0

101.8

14 (13)

28

180 (>29)

6.52

64483

Q5E956

125.4

19 (13)

42

378 (>29)

7.11

26558

P10881

ANXA4 Secreted

P02676

8,77

Mw 69471

Unknown 1142

0.029

−1.60

2352

0.047

1.22

Bifuntional purine biosynthesis protein Triosephosphate isomerase 1

a

Minimum significant protein score = 6. bNumbers in parentheses are the minimum ion score that indicates identity or extensive homology. Protein scores are derived from ion score.

Figure 5. Functional clustering of differential proteins in uterine fluid (UF) from pregnant (i.e., embryo transferred) and cyclic (i.e., sham transferred) cows using Ingenuity. Proteomic data were grouped according to Ingenuity interaction parameters. The best network with a score of 67 is represented. The selected proteins correspond to nodes, which are represented by different symbols (red and green are up- and down-regulated, respectively) according to their functional classification. The nodes are interconnected by specific connectors according to the type of interaction between the linked nodes (see a description in Supplementary Figure 3, Supporting Information). A cluster of NFkB target genes displayed differential expression pattern in pregnant UF, suggesting the impairment of this essential transcription factor. Down-regulation of NFkB complex was further deducted by the decrease of the subunit p65 as evidenced by Western blot analysis. 760



Article

Figure 7. (A) Representative analysis of PNP, HSPA5 and albumin by Western blot (WB) in bovine uterine fluid from cyclic (i.e., sham transferred, ST), artificially inseminated (AI) and pregnant (i.e., embryo-transferred, ET) heifers. (B) WB validation of proteomic data: Similar to proteomic data, albumin was found in lower abundance (a,b: p < 0.05) in uterine fluid from ET heifers (black bars) as compared with the uterine fluid from ST animals (white bars). Note that protein expression values from PNP, HSPA5 and Albumin within AI fluids (gray bars) do not exceed the range delimited by ET and ST cows.

Figure 6. (A) Representative validation of Lactotrasferrin, PRDX-1 and Annexin-4 by Western blot (WB) in bovine uterine fluid from cyclic (i.e., sham transferred, ST) and pregnant (i.e., embryotransferred, ET) heifers. NFkB, Insulin, TNFα and IL-1β expression was also detected by WB in the same experimental groups. (B) WB validation of proteomic data. Similar to proteomic data (lined bars) WB analysis (solid bars) demonstrate that PRDX-1 and Annexin 4 appear in higher abundance, while lactotrasferrin was found in lower abundance in the uterine fluid from ET heifers compared with the uterine fluid from ST animals. PRDX-1, NFkB, Insulin, glycosylated TNFα and IL-1β were found in lower abundance in the uterine fluid from ET heifers compared with the uterine fluid from sham transferred animals while nonglycosylated TNF expression was found in higher abundance.

which would contribute to explain the maternal immunological tolerance to an allograft early embryo. Purine nucleoside phosphorylase (PNP) is abundant in UF from pregnant cows at preimplantation.31 At this period, a general increase of amino acid concentrations in the uterine fluid exists,68 which may feed purine synthesis. In our study we could not clearly identify PNP up-regulation, which is in fact shown by other proteins involved in biosynthetic pathways (peptidylprolyl isomerase A −PPIA-, GAPDH and PEBP1). Collectively, these data reflect an intense metabolic activity induced in the uterus by early embryos. To the best of our knowledge, our report is the first describing decreased protein contents during early pregnancy in recoverable bovine UF. Such a reduction could reflect lower UF secretion accompanied by other changes that the embryos induce and affect major down-regulated proteins (i.e., albumin, fibrinogen). Interestingly, the protein shortage was observed with the two assayed bulls, which differed in their in vivo and in vitro development, being independent of blood progesterone levels and correlated with the amount of live embryos recovered from the uterus. A reduced fluid volume in uterus would help to keep a thinner layer of fluid,69 leading to tighter contact between embryos and endometrium, improving the efficiency of paracrine exchanges. Such a decreased volume secretion means that successfully developing embryos were able to counteract pro-inflammatory conditions. Albumin is a major component in neat uterine fluid,70 accounting for 18−25% total protein (approximately 50% of plasma albumin; Morris, D. personal communication). As most proteins we found were up-regulated (n = 32/50 spots), it is

caspase-3, an apoptotic effecter, inhibits the apoptotic response to TNF,61 while members of the gelsolin family can repress the pro-inflammatory caspase-1.62 Not only TNF activity, but that of IL-1B could be restricted by specific mechanisms. Thus, we found the Sjögren’s syndrome antigen B (autoantigen La) up-regulated, which is associated with increased levels of the interleukin inhibitor IL-1 receptor antagonist IL-1ra.63,64 Whether IL-1ra is present in the bovine uterus, such as has been observed to occur in cultured human endometrial cells,65,66 together with IL-1B and its receptor Il-1RI,65−67 increased activity in the interleukin 1 system could be counteracted by the autoup-regulation of IL-Ra, leading to reduced binding of IL-1B to its receptor. Therefore, on day 8, TNFα and IL1B released by embryos might locally depress their own endometrial expression. Decreased endometrial TNF and IL-1B levels would in turn reduce NFkB expression and its associated pro-inflammatory and/or pro-apoptotic responses, 761



Article

Figure 8. Representative single optical images of immunoreaction for IL-1B (A,B,C) and TNFα (D,E,F), and DAPI staining of blastocysts recovered after ET (A,D), in vitro cultured in SOF-UF (B,E) and in vitro cultured in SOF-BSA (C,F). The ICM area is highlighted within an ellipse.

belongs to the C3-convertase complex. As in the pregnant ewe uterus,22 we observed a decrease in CFB, which might indicate that the protein is being used by the embryos to generate iC3b. Other proteins up-regulated in our study could confer antioxidant protection to the embryos, promote cell proliferation and differentiation, and mediate intracellular signaling. By WB and MS, we respectively identified active phosphorilatedPRDX-179 and PRDX-2. Members of the PRDX family eliminate H2O2 that is produced during cell metabolism with reducing equivalents provided by thioredoxin.80 PRDX-1 and thioredoxin are up-regulated during the implantation window in the cow and mouse uterus,31,81 and thioredoxin improves blastocyst development of pig and cattle embryos in vitro.82,83 Interestingly, overexpression of PRDX2 inhibits H2O2-induced activation of NFKB,80 a response found to be antiapoptotic.84 We found HSPA5 up-regulated in pregnant UF. The endometrium of cow, mice, women and sheep express HSPA5.31,85−88 During the implantation window, HSPA5 seems to protect cells against the cytotoxic damage of TNFα.89,90 ARHGDIA is an antiapoptotic factor found to be up-regulated during the preattachment period in the cow19 (cited as RhoGDI) that mediates implantation in humans.91 Annexin A4 protein and mRNA are regulated by P4 in the human uterus.92 The protein shows anti-inflammatory properties,93,94 and its antiapoptotic and signal-transduction role occurs almost exclusively in epithelial cells. In cattle, the peri-implantation environment governed by interferon-tau (IFNT), whose secretion by the embryonic trophoblast results in maintenance of the corpus luteum and pregnancy,95,96 is remarkably different from the embryo-induced growth promoting profile we observed on day 8. Among such differences, we have identified down-regulation of ezrin, a membrane-cytoskeletal linking protein found in the human endometrium.97 Ezrin and the tissue inhibitor of metalloproteinases-2 (TIMP-2), a modulator of extracellular matrix integrity, are up regulated in the cow uterus at specific times during implantation31 by IFNT.98,99 Up regulation of the NFkB system during implantation exists in ungulates at transcriptional

likely albumin is used to compensate relative increases suffered by other proteins. The UF interphase is a complex mixture of uterine-secreted, immune-secreted and serum-borne proteins, immune resident cells, and shed endometrial cells and cell debris; such composition is consistent with the strong remodeling observed in the uterus of all mammalian species. In pregnant animals, the UF must also reflect interactions between embryos, endometrium and immune cells, which can also affect conceptus and endometrial growth by increasing the concentration of cytokines.71−73 Intracellular proteins present in UF may arise by natural degradation of immune and detached endometrial cells, a process that could be enhanced by flushings. However, the presence of intracellular proteins is normally reported in UF from humans, both directly recovered by aspiration74,75 and by uterine flushing,76 therefore being independent of the recovery method and indicating that such proteins can be considered normal constituents of the UF. Similar results have been reported in cows31 and ewes.22 We showed that recovered UF factors >3000 kDa conditioned by live embryos are embryotrophic in vitro, without altering ICM and TE cell counts or embryo survival to cryopreservation. Interestingly, embryotrophic effects occurred at a very much lower total protein concentration than that normally contained in raw uterine secretions (estimated at over 50−100 times). In addition, in vitro embryo development rates were consistent with the amount of embryos recovered alive within each of 3 UFs, which may represent a further proof of a uterine environment conditioned by embryos. In line with these effects, we identified up-regulated proteins that can favor or promote embryo development. Some of these proteins, such as twinfilin, HDGF and synaptotagmin-binding cytoplasmic RNA-interacting protein have not before been identified in the mammalian uterus. The mitogenic protein HDGF is regulated by NFkB1, playing a role in cell proliferation and differentiation.77 On the other hand, human embryos mediate the oviductal conversion of C3b to embryotrophic inactivated complement-3b (iC3b), which stimulates blastocyst development.78 The complement factor B (CFB) 762


Journal of Proteome Research and protein levels,22,100−103 which is contrast with the depressed NFkB expression in our work. Ultimately, IFNT did not trigger differences in recoverable protein between pregnant and cyclic cows.68 Collectively, we suggest that there is no involvement of IFNT in the effects we described during early development. All types of blastocysts we analyzed contained detectable levels of IL-1B and TNFα protein. In line with our findings, earlier recognition of pregnancy than at the implantation period has been suggested.104,105 When mothers are malnourished or stressed, development of embryos according to their sex could be in turn disrupted.33 As subfertility is more likely to be mediated by local events in the reproductive tract, 21 inappropriate signaling would contribute to very early embryo mortality.106,107 This may explain in part why starting from fertilization rates in heifers close to 90%, only 60% of inseminations result in births.8 We recorded increased paracrine interactions in the uterus leading to a response that improves embryo development without altering cell distribution or cryopreservation survival in blastocysts. The latter is consistent with the cryopreservation benefit that arises from the passage through the genital tract.7,108 Our biological model, with multiple embryos recovered from the uterus, should not be considered unphysiological, as superovulated cows can host tens of embryos in the uterus, which are capable of yielding viable, normal calves upon single transfer to recipients. In fact, several traits we identified are dependent on the amounts of live embryos recovered, perhaps something difficult to elicit with only one resident embryo in the uterus. The latter postulate is evidenced in a recent transcriptome analysis that did not show endometrial changes induced by one embryo on days 5, 7 and 13 but on day 16.20

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REFERENCES

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CONCLUSIONS The traits we found, obtained in the absence of unaltered progesterone values, consist of a decreased total protein contents, embryotrophic effects of UF, and validation against physiological UF obtained after AI. The pregnancy proteome profile on day 8 strongly contrasts with implantation events and is consistent with the proposed uterine local environment conditioned by embryos. We did not find evidence of new proteins induced by embryos in the uterus. Therefore, embryo signaling could not require the use of ad-hoc molecules strange to the uterus, but redundant uterine effectors able to depress NFkB expression that could act as decoys. Candidate agents could be TNFα, IL-1B, both signaling alone or in a collective dialogue with other proteins. The release of embryonic molecules that down-regulate NFkB in uterus might contribute to explain the immune privilege of the embryo at early stages. ASSOCIATED CONTENT

S Supporting Information *

Supplemental figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

Grant Support: M. Muñoz (Spanish Ministry of Science and Innovation − MICINN-; RYC08-03454). B. Trigal is a fellowship holder sponsored by Cajastur. Project: AGL200910059. This work has been made in the framework of the COST ACTION FA0702 (GEMINI). Dr Ana del Cerro by bacteriological culture. Dr Luis Mariá Sánchez by his valuable technical help and scientific comments. Dr Kevin P. Dalton by English review and comments. Proteomics were performed in the Proteome Core Facility of CIMA, a member of ProteoRed.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Telephone: 34 984502010 Fax: 34 984502012. 763



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