Pulse of α-2-macroglobulin and lipocalin-1 in the pregnant uterus of European polecats (Mustela putorius) at the time of implantation

Uterine secretory proteins protect the uterus and conceptuses against infection, facilitate implantation, control cellular damage resulting from implantation, and supply embryos with nutrients. The early conceptus of the European polecat (Mustela putorius) grows and develops free in the uterus until implanting at about 12 days after mating. Using a proteomics approach we found that the proteins appearing in the uterus leading up to and including the time of implantation changed dramatically with time. Several of the proteins identified have been found in pregnant uteri of other placental mammals, such as α1-antitrypsin, serum albumin, lactoferrin, cathepsin L1, uteroferrin, and ectonucleotide pyrophosphatase. The broad-spectrum proteinase inhibitor α2-macroglobulin rose from relatively low abundance initially to dominate the protein profile by the time of implantation. Its functions may be to limit damage caused by the release of proteinases during implantation, and to control other processes around the site of implantation. Lipocalin-1 (also known as tear lipocalin) has not previously been recorded as a uterine secretion in pregnancy, and also increased substantially in concentration. If polecat lipocalin-1 has similar biochemical properties to the human form, then it may have a combined function in transporting or scavenging lipids, and antimicrobial activities. The changes in the uterine secretory proteome of Euroepan polecats may be similar in those species of mustelid that engage in embryonic diapause, but possibly only following reactivation of the embryo.


Introduction
A conceptus may implant soon after its arrival in the uterus (as in humans and muroid rodents), or may remain free in the uterus for several weeks before implanting (as in equids) [1]. Intermediate to these extremes are the conceptuses of many Carnivora and Artiodactyla which implant 10-20 days after mating. The delay is attributed to time required for mating-induced ovulation, travel through the fallopian tubes, and a variable period during which the blastocyst develops without close cellular contact with maternal tissues [1]. In some species of mustelid, ursids, phocids, and roe deer, implantation is postponed for several months, during which time the conceptus enters obligatory diapause at the blastocyst stage and must be maintained and protected until reactivation is permitted by the mother [2][3][4][5]. The European polecat, Mustela putorius [6], fits into the intermediate group, with an embryonic development period (without embryonic diapause) of approximately 12 days preceding implantation.
Proteomics of polecat uterine fluids in early pregnancy Page 3 Proteins secreted into the non-pregnant uteri of eutherian mammals have a range of presumptive functions such as maintenance of the mucocutaneous surface of the endometrium, antimicrobial protection, receptivity to sperm and the subsequent conceptus, and nutrition of an embryo. Distinct differences have been found among mammal groups in the proteins secreted into the uterus before placentation, although there are commonalities [7][8][9][10][11][12]. These proteins have been divided mainly into those involved in nutritional support for the conceptus, or in facilitating and controlling implantation events. Perhaps the most notable example of the nutritional function is the uterocalin/P19 protein in horses. This protein appears to deliver essential lipids such as retinol and polyunsaturated fatty acids across the glycoprotein capsule to the equine conceptus [13][14][15]. Lipids present a problem in their transfer from mother to conceptus because they tend to be insoluble, and in some cases susceptible to oxidation damage unless protected within a protein binding site. Uterocalin is also notable in that its primary structure is enriched in essential amino acids, which doubles its function as a nutrient source for the developing embryo [15]. Other examples of lipid carriers in uterine secretions include a modified form of plasma retinol binding protein, and serum albumin which binds a range of small molecules, fatty acids in particular [11,16,17]. Of those proteins considered to influence implantation events, the broad-spectrum proteinase inhibitor α-2-macroglobulin (α 2 M) is secreted into the pregnant uterus around the time of implantation in several species, and, among other roles, is thought to limit tissue damage during implantation and to control local inflammatory responses [7-12, 18, 19].
Overall, there is considerable diversity in the suite of proteins secreted into pre-placentation uteri by different clades of mammals, as exemplified by equids, artiodactyles and Carnivora, all of which exhibit distinctive repertoires [7][8][9][10]12]. We investigated the proteins found in uterine secretions of pregnant European polecats in order to explore the preparatory events that lead up to, and at, implantation. Analysis of chronological samples indicated that progressive and dramatic changes in the protein profile occur as the time at which implantation would occur approaches. Some of the proteins have been found in pre-implantation uterine flushes of other species (notable among which is α-2-macroglobulin), although not in the same combination or relative concentrations. At least one protein, lipocalin-1, which we found to peak in abundance at implantation, has previously not been reported as a uterine secretory protein. Not only does this study demonstrate that mustelids exhibit a different but overlapping repertoire of implantation-related uterine secretions from other species groups, but it may also provide a general framework for investigation of what happens during embryonic quiescence and subsequent reactivation in species that engage in embryonic diapause.
Proteomics of polecat uterine fluids in early pregnancy Page 4

Animals and sample collection
The subjects used in this study descended from a population of 70  The time of oestrus was estimated by the physical appearance of the vulva. Males used for mating were known to have been fertile the previous year, and matings were visually confirmed (a tie observed). Animals were randomly selected for sample collection on days 4, 6, 7, 9, 12 and 14 days after mating, and those sampled on days 4 and 14 were found not to have become pregnant and thereby acted as mated but non-pregnant controls. Each animal was anesthetized with medetomidine (Dorbene®; Laboratorios syva s.a., León, Spain) and ketamine (Ketalar®; Pfizer Oy, Helsinki, Finland), and the uterus removed under sterile surgical conditions. The animals were subsequently euthanized with T-61 (Intervet International B.V., Boxmeer, the Netherlands) while still under anesthesia. Once the uterus was removed, the tips of each horn were opened, and the cut ends cleared of blood. The cervix was closed with forceps, and the uterus flushed with 5 ml sterile physiological saline through one horn and out the other, with care to avoid blood contamination.
The flush fluid was filter-sterilised and frozen at -20 °C in approximately 2 ml aliquots. Pregnancy was confirmed by the presence of developing embryos in uterine flushes.
All of the work was approved by the National Animal Experiment Board (Eläinkoelautakunta, ELLA) of Finland.

Protein electrophoresis
1-dimensional vertical sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using the Invitrogen (Thermo Scientific, Paisley, UK) NuPAGE system using precast 4-12% gradient acrylamide gels, with β2-mercaptoethanol (25 μl added to 1 ml sample buffer) as reducing agent when required. Gels were stained for protein using colloidal Coomassie Blue (InstantBlue, Expedion, Harston, UK) and images of gels were recorded using a Kodak imager. Electronic images were modified only for adjustment of contrast and brightness. Pre-stained molecular mass/relative mobility (M r ) standard proteins were obtained from New England Biolabs, Ipswich, MA, USA (cat. number P7708S). Samples were run under non-reducing and reducing conditions using β-2 mercaptoethanol as reducing agent. Selected gel slices were taken from non-reduced, Coomassie Blue-stained gels and processed for application to fresh gels under reducing conditions. Samples were concentrated where required using a Vivaspin 3,000 MWCO PES (Sartorius, Epsom, Surrey, UK) centrifugal concentrator device operated according to the manufacturer's instructions.

Proteomics
Stained protein bands were excised from preparative 1-dimensional gels and analysed by liquid chromatography-mass spectrometry as previously described [20]. Protein identifications were assigned using the MASCOT search engine to interrogate protein and gene sequences in the NCBI databases and the Mustela putorius genome database [21] and linked resources, allowing a mass tolerance of 0.4 Da for both single and tandem mass spectrometry analyses. BLAST searches, or searches of genome databases of other Carnivora (e.g. dog and giant panda), were carried out to check the annotations. Prediction of secretory leader peptides and their cleavage sites were carried out using SignalP software (http://www.cbs.dtu.dk/services/SignalP/; [22]), and molecular masses calculated using ProtParam (http://web.expasy.org/protparam/).

Sequential changes in the protein profile of pre-implantation uterine secretions
The protein profiles of all the uterine flush samples collected from days 4 to 14 after mating are shown in the SDS-PAGE analysis shown in Supplementary Information Figure S1. The disparities between overall protein concentrations amongst the samples could be due to differences in efficiency of flushing, changes in the total tissue volume, differences between individual animals' secretion volumes, or changes in secretory activity. In order to improve comparability, selected samples were concentrated as described above and/or the volume of loaded sample adjusted to Proteomics of polecat uterine fluids in early pregnancy Page 6 approximately equalise the intensity of the band at ca. 65 kDa M r (band C; Figure 1), suspected (and subsequently confirmed by mass spectrometry), to be serum albumin from its size and slower migration under reduction.
Standardization of protein concentration to serum albumin revealed dramatic changes in the chronological protein profile of pregnant uterine flushes, in particular the proteins in bands A, B, D, E, G, U, and W ( Figure 1). The pregnant animals on days 6 and 7 showed few, if any, differences from the mated but non-pregnant animal at day 4, but by day 9 considerable differences were evident between pregnant and non-pregnant individuals.
In order to explore relationships between some of the proteins in the most intense bands, gel slices were excised from a non-reduced gel, and the proteins in them subjected to electrophoresis under reducing conditions. This showed that bands A and B reduced to a similar set of proteins, suggesting that they were related or identical ( Figure 2). The sizes of the resulting fragments were not commensurate with post-reduction cleavage products of large multi-chain immunoglobulins (IgM and IgA) which might be expected to appear in uterine secretions. Protein band C, which was used to standardize the loading protein concentrations of samples for Figure 1, migrated more slowly when reduced, which is typical of serum albumin. The change in its migration is usually attributed to cleavage of its intramolecular disulphide bond such that the unfolded protein is retarded in its migration. Similar behaviour was exhibited by band G protein, but the others separated as under non-reducing conditions. The reduction of band C protein uncovered a different protein that was subjected separately to proteomic analysis (band R).

Proteomics
Protein bands whose concentrations changed dramatically with time after mating, or whose concentrations remained constant, were selected for proteomic analysis from both reducing and non-reducing gels ( Figure 1). The separation between the bands in the SDS-PAGE gels was sufficient to allow proteomic identification of the proteins with high confidence, all directly from the M. putorius genome-derived protein sequence database. The deductions were unchanged upon checking by BLAST searching of genomic and other databases for other Carnivora. Table 1 lists the identifications and also the MASCOT scores and peptide matches of the proteins, and illustrates the high quality of the matches.
The two bands with the highest M r , bands A and B, were both identified as α-2-macroglobulin (α-2M), a was their reduced form in band J. These sizes are larger than expected from the protein's polypeptide mass with its predicted secretory signal peptide removed, and may be due to glycosylation and/or covalent linkage with other entities. The other proteins that appeared at high relative amounts around the time of implantation included lipocalin-1 (bands G, U; also known as tear lipocalin, and an enzyme of the ectonucleotide pyrophosphatase family, also represented in bands D, O), cathepsin L1 (bands E, W), uteroferrin (bands E, H, W), and zinc-α-2-glycoprotein (band W). Transferrin (bands F, P), α1-antitrypsin (bands C, R, X), and lactoferrin (bands F, P) remained at constant levels relative to serum albumin throughout the sampling period.

Discussion
The proteins secreted into the uteri of pregnant European polecats changed dramatically in abundance over the approximately 12 days between mating and the time of implantation by the embryo. The repertoire of proteins shows some similarities with that found in other groups of mammals, presumably reflecting a theme inherited from a common ancestor, or convergent evolution in order to satisfy similar functional requirements. In European polecats, however, the appearance of α 2 M was particularly pronounced. Our analysis also identified an abundance of lipocalin-1, which protein has not been reported as a uterine secretion before.

α 2 -macroglobulin
α 2 M was the most notable protein to appear in abundance shortly before the time of implantation.
It is closely related to pregnancy zone protein (PZP) and three members of the complement system [23]. It is the largest non-immunoglobulin protein in circulation in mammalian blood, comprises an approximately 160kDa polypeptide that is glycosylated, and usually occurs in circulation as a tetramer comprising a pair of non-covalently associated disulphide-linked dimers [23]. It is synthesised mainly in the liver, but also in the decidua and endometrium in mice and possibly also in humans, and is thought to be important for implantation [12,[24][25][26][27]. α 2 M is a minor acute-phase inflammation reactant in several species [28]. It operates via two mechanisms as a broad-spectrum proteinase inhibitor [23]. In one mechanism, an active proteinase cleaves a bait peptide that causes α 2 M to fold around and encapsulate the target enzyme. In the other, the target proteinase is covalently bound via a thioester bond. These mechanisms trap proteinases of all classes but do not necessarily inactivate them, instead removing them from access to intact proteins.
Proteomics of polecat uterine fluids in early pregnancy Page 8 In acute-phase and inflammatory responses, the proteinases targeted by α 2 M are thought to be those of pathogens, but, significantly, also those released by granulocytes and other immune cells in potentially damaging inflammatory reactions [23]. This function could be directly relevant to placentation, given that the sites of placentation in some mammals exhibit local inflammation-like maternal responses [29-31]. The inflammatory response may be amplified by localized apoptosis of epithelial cells, and proteinase inhibition by α 2 M may minimize damage caused by released enzymes [27,[32][33][34][35][36][37]. α 2 M also binds several hormones, growth factors and cytokines involved in inflammatory responses, and can modify the biological activity of these signalling molecules [19,23].
In pregnancy, α 2 M is found in uterine secretions of several species, and has been described to control trophoblast positioning and overgrowth in the mouse [7-12, 18, 19].
Several bands in the gels of polecat uterine flushes contained α 2 M, ranging in size from close to that of the monomeic glycoprotein (band J) to higher-order structures (dimer, tetramer; bands A and B).
Some that did not conform to simple monomers, dimers or tetramers of the protein may be covalent α 2 M:proteinase complexes that were not dissembled by the reducing agent ( Figure 2). The presence of α 2 M in the uterine lumen could be due to increased permeability of the uterine vessels to plasma proteins or tissue disruption. If plasma leakage were to have contributed to the presence of α 2 M, as would also be the case for blood contamination during sampling, then other major plasma proteins, such as immunoglobulins and complement C3, would be present in proportionately high amounts.
None of these were observed, although serum albumin was clearly present at all times. While this is the most abundant plasma protein in terms of relative molarity, it is found in uterine secretions of other species in the absence of other plasma proteins [8,12]. Moreover, α 2 M is known to be synthesised by uterine tissues in humans and mice (see above).

Lipocalin-1
The appearance of lipocalin-1 was unexpected, as it has not previously been described as a component of uterine secretions. Moreover, while other lipocalins have been described in the reproductive tissues of other species, only lipocalin-1 was found in this study of polecat uterine secretions. Also known as tear lipocalin and von Ebner's gland protein, lipocalin-1 is a member of a large family of proteins that operates predominately extracellularly. In addition to its eponymous presence in tears, lipocalin-1 has been found in a wide range of tissues [40], albeit not in uterine secretions.
Most lipocalins bind small hydrophobic ligands such as retinol, fatty acids, odorants, or pheromones, and some are enzymatic [40,41]. Others are involved in infection defence, such as α 1 -acid glycoprotein (orosomucoid), which is an acute-phase protein in humans that binds a range of small

Tissue-protective and embryo support proteins
In addition to α-2M and lipocalin-1, we identified several additional proteins that may protect the uterus against infection and tissue damage associated with implantation. These include the ironbinding proteins lactoferrin (lactotransferrin) and transferrin (serotransferrin), both of which were present at fairly constant levels throughout the sampling period. Lactoferrin in particular is associated with antimicrobial activity by sequestering iron in milk, it is also synthesised and stored in neutrophil granules [51], and it directly attacks bacterial membranes and has anti-viral activity [52][53][54][55][56]. α 1 -antitrypsin was also present throughout, and increased in concentration slightly with time. This is an inhibitor of serine proteinases, is a major acute-phase reactant in humans, and is increasingly recognized as an anti-inflammatory mediator [57]. Its primary function appears to be protection against excessive proteolysis of tissues (e.g. in the lower respiratory tract) by neutrophil elastase in inflammation [58,59]. It has been observed in pregnant uteri of other species, possibly to control proteinases released during tissue remodelling, trophoblast invasion, apoptosis, or inflammatory processes that may accompany placentation [60,61].
Other proteins increased with time. Cathepsin L1, a cysteinyl proteinase, is usually confined to lysosomes and is involved in MHC class II antigen processing. Its expression level in uterine tissues is progesterone-dependent, and its importance in placentation has been recognized [60,62,63]. Other progesterone-dependent proteins that appeared in the polecat uterine fluid included a member of the ectonucleotide pyrophosphatase family (a lysophospholipase that may be involved in the production of pharmacologically-active lipid mediators), and uteroferrin (pregnancy-associated acid phosphatase, which appears to function in transplacental iron transport and stimulation of erythropoeisis) [16,[64][65][66][67][68][69][70].
Curiously, we did not find any dedicated nutrient carrier protein similar to equine uterocalin that might act to support a developing conceptus during a pre-placentation period. Equine uterocalin may, however, be a special adaptation in equids to provide resources for a large, capsule-enclosed conceptus during a prolonged pre-placentation period. Nothing like it has been found in non-equids.
Mustelid blastocysts do have extended placentation periods, and they can even distend the uterus before they implant, but they have no glycoprotein capsule intervening between their surfaces and the endometrial surface [6]. Moreover, the surface to volume ratios of mustelid conceptuses are substantially smaller than in equids at maximum growth such that they may not need a specialized nutrient carrier. Equine uterocalin and lipocalin-1 bind a similar set of small lipids [15,40], but the former is atypically enriched in essential amino acids [15], a feature that is not true of lipocalin-1.
We did, however, find other lipid carriers that may supply the polecat conceptus with lipids, such as serum albumin and apolipoprotein A-I.

Conclusion
The protein secretory response of the pre-implantation phase of pregnancy that we observed in European polecats appears primarily to prevent infection, facilitate implantation, and protect against release of deleterious cellular components associated with the tissue trauma of implantation. Some of the lipid-binding proteins we found may also be involved in maternal:embryo communication by transporting insoluble signalling molecules such as progesterone, prostaglandins and leukotrienes, or their precursors (as postulated for equine uterocalin; [13,15]). It would now be interesting to establish whether species of mustelid that exhibit prolonged obligatory embryonic diapause (e.g. stoats, otters, badgers) show a similar protein secretory response when reactivation of blastocysts occurs and implantation is imminent. On the other hand, many of the proteins identified in this study could fulfil the need to protect and nourish a blastocyst that is free in the lumen and devoid of circulatory support and direct immune protection for a considerable time before it implants.

Constituent of milk.
e MASCOT (MOWSE) search score where scores greater than 38 are taken to be significant. The MASCOT score is the highest value obtained where the protein was identified in more than one band, as were the peptide match values.
f Number of peptides found to match with number of peptides unique to this identification in parentheses.
g Putative functions and comments are drawn from literature cited, or NCBI and UniProtKB/Swiss-Prot databases.  Figure S1 for SDS-PAGE of all of the samples collected and upon which the adjustments were based. Gel band codes indicated by letters and are referred to in the text and in Table 1. Mmarker/calibration proteins with relative mobilities (M r ) as indicated in kilodaltons (kDa).