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Endocrinology, doi:10.1210/en.2008-0309
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Endocrinology Vol. 149, No. 7 3274-3278
Copyright © 2008 by The Endocrine Society

Imaging Mass Spectrometry Reveals Unique Protein Profiles during Embryo Implantation

Kristin E. Burnum, Susanne Tranguch, Deming Mi, Takiko Daikoku, S. K. Dey and Richard M. Caprioli

Department of Chemistry and Biochemistry (K.E.B., R.M.C.), Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37221; and Departments of Pediatrics (T.D., S.K.D.), Cell and Developmental Biology (S.T., S.K.D.), and Pharmacology (S.K.D.), Division of Reproductive and Developmental Biology, and Department of Biostatistics (D.M.), Vanderbilt University Medical Center, Nashville, Tennessee 37222

Address all correspondence and requests for reprints to: S. K. Dey, Vanderbilt University Medical Center, MCN D4100, 1161 21st Avenue South, Nashville, Tennessee 37232. E-mail: sk.dey{at}vanderbilt.edu; or Richard M. Caprioli, Mass Spectrometry Research Center, Vanderbilt University, 465 21st Avenue South, Suite 9160 MRB III, Nashville, Tennessee 37221. E-mail: r.caprioli{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A reciprocal interaction between the implantation-competent blastocyst and receptive uterus is an absolute requirement for implantation, a process crucial for pregnancy success. A comprehensive understanding of this interaction has yet to be realized. One major difficulty in clearly defining this discourse is the complexity of the implantation process involving heterogeneous cell types of both the uterus and blastocyst, each endowed with unique molecular signatures that show dynamic changes during the course of pregnancy. Whereas gene expression studies by in situ hybridization or immunohistochemistry have shown differential expression patterns of specific genes during implantation, there is no report how numerous signaling proteins are spatially displayed at specific times and stages of implantation in the context of blastocyst-uterine juxtaposition. Using in situ imaging (matrix assisted laser desorption/ionization) mass spectrometry directly on uterine sections, here we provide molecular composition, relative abundance, and spatial distribution of a large number of proteins during the periimplantation period. This approach has allowed us for the first time to generate in situ proteome profiles of implantation and interimplantation sites in mice in a region- and stage-specific manner with the progression of implantation. This application is reliable because patterns of expression of several proteins displayed by in situ imaging mass spectrometry correlate well with in situ hybridization results. More interestingly, the use of this approach has provided new insights regarding uterine biology of cytosolic phospholipase A2{alpha} null females that show implantation defects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HETEROGENEOUS CELL types of the uterus, each with unique functions, pose a challenge for studying events during early pregnancy. This complexity is increased because their actions change, depending on their proximity to the implanting embryo and with pregnancy progression. Therefore, studying the uterine molecular landscape using conventional approaches presents an arduous task.

Studies in mice have shown that uterine receptivity occurs for a limited period on d 4 (the day of implantation). The uterus becomes nonreceptive by late d 5 of pregnancy (1). Once implantation occurs, the uterine stromal cells surrounding the embryo undergo extensive remodeling, a process termed decidualization. One function of the deciduum is to provide nutritional support to the developing embryo before establishment of a functional placenta (2). It is expected that protein signatures differ between implantation and interimplantation sites and also in the cell types within.

Although expression studies by in situ hybridization and immunohistochemistry have provided profiles of specific genes and their gene products during implantation and decidualization (2, 3), the in situ spatiotemporal distribution of a large number of gene products in uteri during implantation remains poorly understood. Here we used in situ imaging [matrix-assisted laser desorption/ionization (MALDI)] mass spectrometry to generate proteomic profiles in the periimplantation mouse uterus. Whereas mass spectrometry is an effective technology for identifying proteins and their posttranslational modifications under various physiological conditions, imaging mass spectrometry is a powerful tool for in situ analysis of proteome profiles on tissue sections (4, 5). This technology allowed us for the first time to analyze hundreds of proteins involved in proliferation, differentiation, and apoptosis during the periimplantation period, providing unique and differential proteomic blueprints of implantation and interimplantation sites. The uterine proteomic profile in mice lacking Pla2g4a, the gene encoding for cytosolic phospholipase A2{alpha} (PLA2), was also compared with wild-type implantation sites, providing new insight regarding uterine biology in these null females that display implantation defects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Adult CD-1 mice were purchased from Charles River Laboratory (Raleigh, NC). Females were mated with fertile males of the same strain to induce pregnancy (d 1 = vaginal plug). The disruption of the Pla2g4a gene was originally achieved in J1 ES cells by homologous recombination as described (6). Genotyping was by PCR analysis of genomic DNA. Implantation sites on d 5 and 6 of pregnancy were visualized by the blue dye method, as previously described by us (1). All mice in the present investigation were housed and used in accordance with the National Institutes of Health and institutional guidelines on the care and use of laboratory animals.

Protein profiling and imaging experiments
Implantation sites (IS) and interimplantation sites (inter-IS) were dissected from the uterus, snap frozen, sectioned (11 µm), and thaw mounted onto MALDI mass spectrometry compatible glass slides and processed as described (supplemental text). Experimental designs are illustrated in Fig. 1Go, A–C.


Figure 1
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  		FIG. 1. A, Representative photographs of uteri collected on d 5, 6, and 8 of pregnancy. Arrowheads, ovaries; arrows, IS. Uterine sections of an IS (upper) and inter-IS (lower) are shown. Experimental design for profiling (top), imaging (bottom) (B) and identifying (C) proteins is shown. Bar, 700 µm. D, Profiling MS. Left panel, A representative photomicrograph of a section of IS and inter-IS on d 6 of pregnancy. Arrows show location of implanting blastocyst. Average mass spectra for ubiquitin, calcyclin, calgizzarin, and transthyretin (right panel). *, Peak, m/z protein of interest. Bar, 230 µm. LTQ, Linear trap quadrupole.

 
Statistical analysis
For statistical analysis, see supplemental text.

Protein identification experiments
Excised d 6 and 8 ISs were homogenized in tissue protein extraction reagent (Pierce, Rockford, IL) supplemented with protease inhibitors. Detailed methods are found (supplemental text) and illustrated in Fig. 1CGo.

In situ hybridization
cDNA clones for calgizzarin, calcyclin, transthyretin, and ubiquitin were generated by RT-PCR using specific primers. For in situ hybridization, sense or antisense 35S-labeled cRNA probes were generated by using appropriate polymerases as previously described (7). Sections hybridized with sense probes showed no positive signals and served as negative controls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein signatures differ within specific regions of implantation and interimplantation sites
In mice, the implantation process begins on the evening of d 4 of pregnancy. This attachment is accompanied by an increased endometrial vascular permeability at the sites of blastocysts, which can be visualized as distinct blue bands after an iv injection of a Chicago Blue dye solution (7). This blue dye method is a useful tool because implantation and interimplantation sites can be separately excised, and sections with and without the implanting embryo can be obtained for differential characterization (Fig. 1AGo). On d 8 of pregnancy, blue dye injection is not necessary, because implantation sites are visually distinguishable (Fig. 1AGo).

Implantation in mice occurs at the antimesometrial pole of the uterus. After attachment, proliferating stromal cells surrounding the implanting embryo begin to differentiate into decidual cells, forming the avascular primary decidual zone (PDZ). By d 6, the PDZ is well differentiated and formed, but a secondary decidual zone (SDZ) forms around the PDZ, coinciding with the cessation of proliferation in the PDZ with continuing proliferation in the SDZ. The PDZ then progressively degenerates through d 8 of pregnancy (3). The SDZ is surrounded by the myometrium comprising circular and longitudinal muscle layers. In Fig. 1DGo, the antimesometrial pole, mesometrial pole, PDZ, and SDZ are demarcated by faux matrix spots on a representative section from a d 6 implantation site. An interimplantation site, which contains all the major cellular regions except the PDZ and SDZ because they are formed in response to the implanting blastocyst, is also shown (Fig. 1DGo).

To characterize the proteome signatures between specific regions within ISs and inter-ISs, matrix spots were deposited on particular areas of interest in d 6 uteri (Fig. 1DGo). Profiling mass spectrometry generated spectra from those matrix spots, in which each mass-to-charge (m/z) value corresponds to a unique protein that can be identified. In this analysis, approximately 230 distinct peaks were found within the optimal mass range, which for MALDI-time-of-flight-mass spectrometry (MS) is 2–30 kDa. This is dictated by instrumental parameters. The instrument is operated under delayed extraction conditions; thus, enhanced sensitivity, resolution and mass accuracy are observed for ions under 50,000 Da. Microchannel plate detection is velocity dependent; thus, higher molecular mass ions (having lower velocities) have lower probabilities of being detected unless they are highly abundant. Matrix molecules below 2000 Da complicate the mass spectra and potentially mask expression of low-molecular-mass peptides. Statistical analyses of spectra from IS and inter-IS revealed 50 peaks as significantly changed due to the presence and proximity of the embryo. The average mass spectra from specific uterine regions for ubiquitin (m/z 8565), calgizzarin (m/z 10952), calcyclin (m/z 9962), and transthyretin (m/z 13641) are shown (Fig. 1DGo). These four proteins were selected and chosen for further analysis because of their unique expression patterns in the uterus and their reported involvement during pregnancy (8, 9, 10).

Statistical analysis shows that ubiquitin is up-regulated at IS by 3.4-fold, compared with inter-IS on d 6 of pregnancy. In contrast, calcyclin and calgizzarin are up-regulated by 5.2- and 2-fold, respectively, in the PDZ, compared with the SDZ, whereas transthyretin is up-regulated in the SDZ near the antimesometrial pole (5.1-fold higher in the SDZ, compared with the PDZ, and 2.9-fold higher in antimesometrial pole, compared with the mesometrial pole) (Fig. 1DGo).

To view these spatiotemporal differences in expression, IS and inter-IS were sectioned on d 5, 6, and 8 of pregnancy and prepared for imaging MS. We found that imaging analysis correlated well with profiling for these four selected proteins (Fig. 2Go, A–D, upper panel).


Figure 2
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  		FIG. 2. Imaging MS shows spatial localization for selected proteins of interest on d 5, 6, and 8 of pregnancy. Spatial localization of ubiquitin (A), calcyclin (B), calgizzarin (C), and transthyretin (D) by imaging MS (upper panel) and respective mRNAs by in situ hybridization (lower panel). Bar, 700 µm.

 
To determine whether protein expression patterns identified by imaging analysis correlates with their mRNA localizations, in situ hybridization of ubiquitin, calcyclin, calgizzarin, and transthyretin was performed (Fig. 2Go, A–D, lower panel). We found a fairly good correlation between protein and mRNA localizations with the exception of transthyretin whose transcripts were detected at low levels in the uterus with interesting patterns in d 6 and 8 embryos. It can be speculated that imaging did not detect transthyretin in d 6 and 8 embryos because it could be a different mass in these regions due to posttranslational modifications. In addition, imaging mass spectrometry showed distinct expression patterns of other proteins on d 4, 5, 6, and 8 of pregnancy (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org, and supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org). It is interesting to note that the SDZ shows relative increases in proteins involved in energy production and proliferation. Conversely, the PDZ shows increases in proteins influencing hormone signaling.

Imaging MS was also used to analyze serial sections of d 6 IS to explore the relative protein expression in the Z direction (Fig. 3AGo). Ubiquitin, calcyclin, and calgizzarin all show varying expression patterns, depending on their proximity to the implanting embryo, whereas expression of Purkinje cell protein 4 remains relatively unchanged (Fig. 3BGo).


Figure 3
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  		FIG. 3. Relative protein expression changes along the Z-axis of a d 6 IS. A, Experimental design: enlargement of a d 6 IS and resulting sections. Bar, 1 mm. B, Imaging MS shows differential protein localization depending on proximity to implanted embryo. Bright-field images of serial sections of an IS collected on d 6. Bar, 700 µm.

 
Differential proteome profiles in Pla2g4a null mice
The success of our application of imaging MS in wild-type (WT) periimplantation uteri prompted us to use this approach in Pla2g4a null females with known defects in implantation. Our previous study suggested that compromised pregnancy in Pla2g4a null females results from deferred on-time implantation, leading to subsequent retarded fetoplacental development and reduced litter sizes (11). This study advanced a new concept that a short delay in the initial attachment reaction propagates detrimental ripple effects later in pregnancy. This is now supported by a more recent study using Lpa3 null mice (12). This deferred implantation in Pla2g4a null females results in both on-time and deferred IS, morphologically distinguishable by their differing sizes on d 6 of pregnancy (Fig. 4AGo).


Figure 4
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  		FIG. 4. Proteome profiles differ between WT and Pla2g4a null uteri on d 6 of pregnancy, regardless of implantation timing. A, Representative photograph of Pla2g4a null uterus collected on d 6 of pregnancy. B, Optical images of a WT IS and inter-IS and Pla2g4a null (–/–)deferred and on-time IS (upper panel). Bar, 670 µm. Ion intensity maps are shown below respective bright-field images. C, Average mass spectra obtained from d 6 uterine sections. *, Peak, m/z protein of interest.

 
We used profiling and imaging mass spectrometry to see whether proteome signatures differ between on-time IS in WT and Pla2g4a null mice and between on-time and deferred IS in Pla2g4a null females. We expected proteome profiles of on-time IS in Pla2g4a null females to be similar to those of WT on d 6, and deferred IS in Pla2g4a null females to be similar to d 5 WT implantation sites. Surprisingly, we found that expression/localization of proteins showing remarkable changes were mostly attributed to the lack of Pla2g4a rather than to differences in implantation timing (supplemental Fig. 2).

The four proteins that were characterized in WT d 6 uteri were selected for further analysis in Pla2g4a null uteri. Whereas the expression of ubiquitin did not appreciably differ between WT and Pla2g4a null uteri, calgizzarin, calcyclin, and transthyretin showed pronounced differences. Specifically, calgizzarin expression was found to be reduced in Pla2g4a null uteri, but more interestingly, its localization shifted to the luminal epithelium in null uteri as opposed to its predominant expression in the antimesometrial decidua of WT uteri (Fig. 4Go, B and C). Calcyclin expression was evident in the PDZ of WT uteri and the implanting embryo in Pla2g4a null females, with lowest expression in deferred IS in Pla2g4a null uteri (Fig. 4Go, B and C). In contrast, transthyretin was higher in deferred IS, compared with on-time IS in Pla2g4a or WT females (Fig. 4Go, B and C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In situ localization and identification of a broad proteomic landscape in a given region of tissues remains difficult with current labeling procedures. Therefore, direct analysis of tissue sections by MALDI-MS holds great potential in providing region-specific detailed assessment of the complex protein patterns within a tissue. This approach also has the advantage of interrogating tissues for protein identification and localization without the limitation of selecting specific proteins for analysis.

In this study, four proteins of interest were selected for more detailed analysis because of their unique uterine expression patterns and previously implicated roles in pregnancy. For example, calcyclin is expressed at high levels in d 8 and 9 decidua (10) and influences placental lactogen secretion in mice (8). Although our imaging MS results show similar expression patterns of calcyclin protein on d 8 of pregnancy, its differential expression pattern in periimplantation uteri suggests that calcyclin is also important for early pregnancy events. In contrast, roles of calgizzarin in early pregnancy remain largely unexplored. However, one study identified this protein as a downstream target of neurokinin B, which is secreted at high levels from the placenta during preeclampsia and is known to suppress calgizzarin expression (13). These results suggest that calgizzarin has beneficial roles in pregnancy maintenance. This contention is supported by our present findings of reduced calgizzarin expression in Pla2g4a null females with implantation, decidualization, and placental defects (11).

Transthyretin, a protein involved in transporting T4 and retinol-binding protein, is aberrantly expressed in placental tissues of patients with pregnancy loss, suggesting its role in placentation (14, 15). Indeed, Ttr mRNA is detected in human placentas with protein expression specifically in the syncytiotrophoblast (9). Whereas our studies focused on implantation and early pregnancy events, it will be of great interest to use this proteomic approach to determine differential proteome profiles during later events of pregnancy including placentation.

Pla2g4a null females show deferral of on-time implantation leading to subsequent adverse ripple effects throughout the course of pregnancy and ultimately reduced litter size (11). We hypothesized that embryos implanting beyond the normal window of implantation undergo premature demise and those that implant on-time develop normally. Our proteomics results suggest that proteome signatures differ between WT and Pla2g4a null uteri, regardless of implantation timing. This finding is interesting and may help distinguish which proteins are critical for on-time implantation. In this respect, the power of imaging MS in generating proteome signatures within and between implantation sites is remarkable.

This study examining differential protein signatures between and within implantation sites and interimplantation sites on different days of pregnancy has opened up a new avenue of exploring protein profiles and their interactions during normal and defective implantation.


   Acknowledgments
 
We thank Pierre Chaurand and Shannon Cornett for their insight throughout this project.


   Footnotes
 
This work was supported by Grants GM58008-09 and DOD W81XWH-05-1-0179 (to R.M.C.) and HD12304, DA06668, and P01-CA-77839 (to S.K.D.). K.E.B. and S.T. are supported by a National Institute of Child Health and Human Development Training Grant 2T HD007043-31A2.

Disclosure Summary: The authors have nothing to disclose.

First Published Online April 10, 2008

Abbreviations: inter-IS, Interimplantation site; IS, implantation site; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; m/z, mass-to-charge; PDZ, primary decidual zone; PLA2, phospholipase A2{alpha}; SDZ, secondary decidual zone; WT, wild type.

Received March 4, 2008.

Accepted for publication March 31, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Paria BC, Huet-Hudson YM, Dey SK 1993 Blastocyst’s state of activity determines the "window" of implantation in the receptive mouse uterus. Proc Natl Acad Sci USA 90:10159–10162[Abstract/Free Full Text]
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  8. Farnsworth RL, Talamantes F 1998 Calcyclin in the mouse decidua: expression and effects on placental lactogen secretion. Biol Reprod 59:546–552[Abstract/Free Full Text]
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Mass Spectrometry Special Feature: Molecular imaging of proteins in tissues by mass spectrometry
PNAS, November 25, 2008; 105(47): 18126 - 18131.
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