Uterine flushing proteome of the tammar wallaby after reactivation from diapause

in Reproduction
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  • 1 School of BioSciences
  • 2 Bio21 Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Australia

The marsupial tammar wallaby has the longest period of embryonic diapause of any mammal, up to 11 months, during which there is no cell division or blastocyst growth. Since the blastocyst in diapause is surrounded by acellular coats, the signals that maintain or terminate diapause involve factors that reside in uterine secretions. The nature of such factors remains to be resolved. In this study, uterine flushings (UFs) were used to assess changes in uterine secretions of tammars using liquid chromatography–mass spectrometry (LC–MS/MS) during diapause (day 0 and 3) and reactivation days (d) 4, 5, 6, 8, 9, 11 and 24 after removal of pouch young (RPY), which initiates embryonic development. This study supports earlier suggestions that the presence of specific factors stimulate reactivation, early embryonic growth and cell proliferation. A mitogen, hepatoma-derived growth factor and soluble epidermal growth factor receptors were observed from d3 until at least d11 RPY when these secreted proteins constituted 21% of the UF proteome. Binding of these factors to specific cellular receptors or growth factors may directly stimulate DNA synthesis and division in endometrial gland cells. Proteins involved in the p53/CDKN1A (p21) cell cycle inhibition pathway were also observed in the diapause samples. Progesterone and most of the oestrogen-regulated proteins were present in the UF after d3, which is concomitant with the start of blastocyst mitoses at d4. We propose that once the p21 inhibition of the cell cycle is lost, growth factors including HDGF and EGFR are responsible for reactivation of the diapausing blastocyst via the uterine secretions.

Abstract

The marsupial tammar wallaby has the longest period of embryonic diapause of any mammal, up to 11 months, during which there is no cell division or blastocyst growth. Since the blastocyst in diapause is surrounded by acellular coats, the signals that maintain or terminate diapause involve factors that reside in uterine secretions. The nature of such factors remains to be resolved. In this study, uterine flushings (UFs) were used to assess changes in uterine secretions of tammars using liquid chromatography–mass spectrometry (LC–MS/MS) during diapause (day 0 and 3) and reactivation days (d) 4, 5, 6, 8, 9, 11 and 24 after removal of pouch young (RPY), which initiates embryonic development. This study supports earlier suggestions that the presence of specific factors stimulate reactivation, early embryonic growth and cell proliferation. A mitogen, hepatoma-derived growth factor and soluble epidermal growth factor receptors were observed from d3 until at least d11 RPY when these secreted proteins constituted 21% of the UF proteome. Binding of these factors to specific cellular receptors or growth factors may directly stimulate DNA synthesis and division in endometrial gland cells. Proteins involved in the p53/CDKN1A (p21) cell cycle inhibition pathway were also observed in the diapause samples. Progesterone and most of the oestrogen-regulated proteins were present in the UF after d3, which is concomitant with the start of blastocyst mitoses at d4. We propose that once the p21 inhibition of the cell cycle is lost, growth factors including HDGF and EGFR are responsible for reactivation of the diapausing blastocyst via the uterine secretions.

Introduction

The uterine environment provides a complex array of secreted factors under dynamic endocrine control (Clemetson et al. 1977, McRae 1988, Salleh et al. 2005). Several studies in the 1970s used uterine flushing (UF) to sample uterine fluid in order to characterise the changing composition of uterine secretions in humans and animal models (Urzua et al. 1970, Renfree 1972, 1973, Aitken 1974, Wolf & Mastroianni 1975, Ametzazurra et al. 2009, Boomsma et al. 2009), but the techniques of the time lacked sensitivity and specificity now possible with modern proteomic techniques. A major contribution to the uterine contents comes from secretions of the glandular epithelial cells (Salamonsen et al. 2009), including nutrients, proteases, hormones, cytokines and growth factors, associated with the regulation of uterine function and embryonic development (Kane et al. 1997, Hempstock et al. 2004, Dominguez et al. 2010, Hannan et al. 2011, Binder et al. 2014, Thouas et al. 2015). Human UFs are complex, with more than 800 proteins derived from epithelial cell secretions, transudates from blood, and from breakdown of cells distinct from the cellular cytoplasm (Casado-Vela et al. 2009, Scotchie et al. 2009, Hannan et al. 2010).

Reproduction in the tammar wallaby is characterised by embryonic diapause. Like all marsupials, tammars give birth to an immature young that undergoes extended development whilst attached to the teat (Tyndale-Biscoe & Renfree 1987). Within 1 h after birth, the female mates (Rudd 1994), and the resulting conceptus grows for only 7 days in the uterus until it reaches about 80 cells when further cell division is halted as a result of the sucking stimulus of the new pouch young (Renfree & Shaw 2000). This unilaminar blastocyst is surrounded by an acellular shell coat, a mucin layer and a zona pellucida. The diapausing embryo has no measurable metabolic activity, no cell division or cell growth (Moore 1978, Thornber et al. 1981, Shaw & Renfree 1986, Spindler et al. 1998, 1999), but removing the sucking pouch young (RPY) removes the neuroendocrine inhibition of the corpus luteum. Consequently, circulating progesterone rises after day 3 to a peak at day 5–6 after RPY (Hinds & Tyndale-Biscoe 1982), which stimulates increased endometrial secretory activity and reactivation of the diapausing blastocyst (Renfree 1973, Renfree & Tyndale-Biscoe 1973, Shaw & Renfree 1986, Fletcher et al. 1988, Shaw 1996, Renfree & Shaw 2000).

Reactivation commences after day 3 (Gordon et al. 1988, Hinds et al. 1989). By day 4 RPY, there is a dramatic increase in glucose oxidation and glutamine uptake and oxidation by the blastocyst and the first mitoses in the blastocyst are observed (Spindler et al. 1998, 1999, Fig. 1).

Figure 1
Figure 1

Schematic illustration of the sequential events from diapause to reactivation and birth at d26 RPY. Reactivation occurs after inhibition of the CL by the early morning pulse of prolactin which has been removed for three consecutive days. If a young is replaced onto the teat at any time up to 72 h after RPY, reactivation will not occur. On the fourth day of the removal of PY (d4 RPY), there is an increase in mitosis, protein synthesis and transport followed by an increase in RNA synthesis by d5 RPY. However, the first expansion of the blastocyst does not occur until d8 RPY. The sampled days are enclosed in boxes. (Adapted from Renfree & Shaw 2000).

Citation: Reproduction 152, 5; 10.1530/REP-16-0154

Tammars have two separate uteri, but, since they are monovular, only one becomes gravid; the contralateral uterus is non-gravid. Both uteri respond similarly because of local distribution of progesterone from the ipsilateral corpus luteum (Towers et al. 1986) until around day 15 when only the gravid uterus becomes larger and more secretory due to local embryonic signals (Renfree 1972, Renfree & Tyndale-Biscoe 1973, Renfree & Shaw 2000, Renfree 2015). Embryo transfer experiments (Tyndale-Biscoe 1963, 1970, Renfree 1972) confirm that the corpus luteum (CL) does not directly stimulate the quiescent blastocyst to resume development, but rather acts by inducing changes in the uterine secretions. Embryo transfer experiments in eutherian mammals have provided further evidence of the importance of the uterus during embryonic development in diapause and that the uterine signals regulating the embryo are conserved (Chang 1968, Ptak et al. 2012, Cha et al. 2013, Fenelon et al. 2014, Renfree & Shaw 2014).

Specific components of the uterine secretions undoubtedly play a role in regulating early embryo development. There is greater rate of protein synthesis in the gravid uterus of the tammar (Renfree 1972, 1973, Shaw & Renfree 1986) because progesterone is preferentially delivered to the gravid uterus (Towers et al. 1986). Furthermore, progesterone alone is sufficient to reactivate diapausing embryos (Renfree & Tyndale-Biscoe 1973). In some marsupial endometria, the region around the nucleus of each cell undergoes active protein synthesis; these secretions may include cell compartments that continue to flow into uterine lumen (Shorey & Hughes 1973, Walker & Hughes 1981) and these patterns are similar to that in the tammar (Tyndale-Biscoe & Renfree 1987). However, after day 15, differences between the two uteri emerge that are due to the presence of the developing embryo, and appear to be the result of stimulation by the placenta (Renfree 1972, 2015) perhaps mediated by placental hormones (Menzies et al. 2011), demonstrating that there is maternal recognition of pregnancy (Renfree & Shaw 2000, Renfree 2015). Thus, only the gravid uterus maintains secretory activity as pregnancy progresses (Renfree 1972, 2000, Renfree & Tyndale-Biscoe 1973, Tyndale-Biscoe & Renfree 1987).

In tammars, the embryo is separated from the uterine epithelium by an acellular shell coat during diapause and until after day 17 of the 26-day active gestation, preventing direct cellular interaction with the endometrium, so control of diapause and reactivation must be achieved through soluble factors in the uterine fluids (Renfree 1972, Renfree & Tyndale-Biscoe 1973, Shaw 1996). Components of UF are derived from secretions from the luminal epithelium and glands, proteins selectively transuded from blood, and include several uterine-specific pre-albumins during reactivation (Renfree 1973). Previous studies have not identified the specific uterine regulatory factor(s) that control diapause of the embryo. In this study, we have therefore used a non-biased global proteomics approach to characterise the proteome of the uterine fluid in diapause and reactivation. We profiled tammar proteins in UF from diapause until late gestation with the aim of identifying and evaluating if those proteins might potentially be implicated in the maintenance of diapause or embryonic reactivation, expansion and development.

Materials and methods

Animal maintenance

Tammars of Kangaroo Island, South Australia origin was kept in open grassy yards with shelters provided. Their diet was supplemented with fresh fruits, vegetables and lucerne cubes and water supplied ad libitum. Care and treatment of animals conformed to the National Health and Medical Research Council Australian Code for the Care and Use of Animals for Scientific Purposes 2013. Animal handling and experimentation were approved by the University of Melbourne Animal Experimentation Ethics Committees. In this study, adult females with a pouch young older than day 8 post-partum were presumed to be carrying a diapausing blastocyst (Tyndale-Biscoe & Renfree 1987). Reactivation was achieved during the seasonal breeding period (Jan–May) by removing the pouch young, and stages of pregnancy were determined relative to the day of the removal of the pouch young (designated day 0 after RPY).

Sample collection and preparation

Analysis of marsupial uterine fluids poses problems because of their small volumes, which preclude direct collection of the secretions, so we used uterine flushings. Females were killed humanely on days 0, 3, 4, 5, 6, 8, 9, 11 and 24 after removal of pouch young. Five animals at each stage with paired gravid and non-gravid UF samples except at days 3, 11 and 24, where three animals were used and at days 11 and 24 UF from only the gravid side, were collected. Immediately post-mortem, the reproductive tract was dissected out and gravid and non-gravid uteri were each flushed with 2 mL of 0.9% saline. The volume of uterine fluids is minute and effectively exists only as a moist surface, so the concentration of protein in the flush was taken as the amount of protein in the uterine fluids per 2 mL. The flushings were examined under a dissecting microscope and any blastocysts were retrieved. About 10 µL of protease inhibitors (Sigma Aldrich) was added to the collected flushings. It was then centrifuged at 16,000 g for 10 min at 4°C in a refrigerated microcentrifuge (Eppendorf, Hamburg, Germany) to remove cell debris. The supernatants were stored at −80°C until further use. This study characterises the protein composition of UF in tammar from diapause (day 0 and day 3), 4, 5, 6, 8 and 9, 11 and 24 RPY. Chemicals were purchased from Sigma Aldrich unless otherwise indicated.

The protein concentrations of gravid UF were determined using a BCA Protein Assay Kit−Reducing Agent Compatible (Thermo Fisher Scientific) following the manufacturer’s instructions.

An aliquot equivalent to 100 µg of protein was initially with reduced by the addition of 5 mM dithiothreitol (DTT) at 95°C for an hour and alkylation with 50 mM iodoacetamide (IAA) in the darkness. This was followed by overnight digestion with sequencing grade modified trypsin (Thermo Pierce) at 37°C on a shaker with the ratio of enzyme to protein, 1:50. The following day, the protein digestion was halted by adding formic acid to final 1% (v/v). The protein digest was then purified using an Oasis HLB (polymeric reverse phase) solid phase extraction (SPE) cartridge (Waters Corporation, Milford, MA, USA). Purified peptides were then lyophilised overnight in a freeze dryer (Virtis, PA, USA) and reconstituted in 0.1% formic acid before analysis using LC–MS/MS.

One microlitre of whole blood was collected from tammars and stored at room temperature for an hour. It was centrifuged for 10 min at 2000 g in a refrigerated centrifuge (Eppendorf, Hamburg, Germany) at 4°C for 10 min to remove the blood clot. The supernatant (serum) was carefully aspirated into a new tube and stored at −80°C until further use. An aliquot of 5 µL serum was made up to 100 µL with water and digested as described above.

LC–MS/MS analysis

LC–MS/MS was carried out on an LTQ Orbitrap Elite (Thermo Scientific) mass spectrometer with a nanoESI interface in conjunction with an Ultimate 3000 RSLCnano high-performance liquid chromatography (HPLC) (Dionex, CA, USA). The HPLC system was equipped with an Acclaim Pepmap nano-trap column (Dinoex-C18, 100 Å, 75 µm × 2 cm) and an Acclaim Pepmap RSLC analytical column (Dinoex-C18, 100 Å, 75 µm × 15 cm) (Dionex, CA, USA). The tryptic peptides were injected to the enrichment column at an isocratic flow of 5 µL/min of 3% (v/v) acetonitrile containing 0.1% v/v formic acid for 5 min applied before the enrichment column was switched in-line with the analytical column. The eluants were 0.1% v/v formic acid (solvent A) and 100% v/v acetonitrile in 0.l% v/v formic acid (solvent B). The flow gradient was (i) 0–5 min at 3% B; (ii) 5–6 min at 3–6% B; (iii) 6–18 min at 6–10% B; (iv) 18–38 min at 10–30% B; (v) 38–40 min at 30–45% B; (vi) 40–42 min at 45–80% B; (vii) 42–45 min at 80% B; (vii) 45–46 min at 80–3% B; and (viii) 46–53 min at 3% B. The LTQ Orbitrap Elite spectrometer was operated in the data-dependent mode with nano ESI spray voltage of 2.0 kV, capillary temperature of 250°C and S-lens RF value of 55%. All spectra were acquired in positive mode with full scan mass spectrometry (MS) spectra scanning from m/z 300–1650 in the FT mode at 240,000 resolutions after accumulating to a target value of 1.0e6. A lock mass of 445.120025 was used. The top 20 most intense precursors were subjected to collision-induced dissociation (CID) with normalized collision energy of 30 eV and activation q of 0.25. A dynamic exclusion duration of 45 s was applied for repeated precursors.

Data analysis

Protein concentrations in UF at the different stages in both gravid and non-gravid uteri were assessed using analysis of variance (ANOVA). Data are presented as mean ± s.d. unless otherwise indicated. All statistical analysis was carried out using R (R Core Team 2015).

The MS and MS/MS data were searched using Proteome Discoverer (Thermo Scientific Version 1.4) with the MASCOT search engine (Version 2.4.1) against a tammar proteome database generated in-house (15,344 protein sequences) from the tammar genome version 2.0 (Renfree et al. 2011). Search criteria used were trypsin digestion, variable modifications set as carbamidomethylation of cysteine (Cys) and oxidation of methionine (Met), allowance of up to two missed cleavages, precursor tolerance of 10 ppm and 0.6 Da on the fragment ions. A targeted false discovery threshold of <1% was applied for all peptides (marked ‘high’ in Supplementary Table 1, see section on supplementary data given at the end of this article). Proteins were inferred on a basis of at least two unique peptides in order to be confident of a match. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Vizcaíno et al. 2014, 2016) repository with the dataset identifier PXD004170.

Results

Protein concentrations in UF did not differ between the two sides at any stage (P > 0.05, Fig. 2). Concentrations rose progressively after d 3 from about 25 µg/mL at days 0 and 3 to about 150 µg/mL at d 9 (P < 0.0001, Fig. 2).

Figure 2
Figure 2

The protein concentrations (mean ± s.d.) measured by BCA assay (n = 5 for each stage and side) across the stages (d0–d9 RPY) in the gravid and non-gravid uteri. At no stage, there was a significant difference between the concentrations in the two sides (P > 0.05). Protein concentrations in the flushings from both uteri increased progressively from d3 to d9 (P < 0.0001). The significant differences with reference to d0 are denoted by *.

Citation: Reproduction 152, 5; 10.1530/REP-16-0154

We identified 994 proteins in uterine flushings based on MASCOT searches of the tammar genome (Supplementary Table 1). Six hundred and three proteins were filtered based on minimum of two unique peptides and the protein to be present in minimum of three biological replicates out of five (Supplementary Table 2). A classification based on the cellular location using TargetP 1.1 (Emanuelsson et al. 2000) (Supplementary Table 2 column M – location) predicted 128 secretory proteins, 24 mitochondrial proteins and 451 other proteins. To assess the contribution of serum proteins to UF, we conducted parallel proteomic analyses of blood serum of tammar. We identified 47 proteins in serum (Supplementary Table 3A); of these, all were also found in UF, with albumin being the most significant component. Although there is no tammar serum/plasma proteome database in the public domain, we were able to cross-reference to the human plasma proteome database (Nanjappa et al. 2014) and identified 342 proteins that were present both in UF and the human plasma proteome database (Supplementary Table 3B). Enzymes involved in glycolysis, gluconeogenesis, pentose phosphate pathway, glycogenolysis, fatty acid metabolism, arachidonic acid metabolism, tricarboxylic (TCA) cycle, galactose metabolism, creatinine and retinol metabolism were identified in samples from d0 RPY until d11 RPY (Table 1) after d11 proteins involved in adhesion, implantation and embryogenesis were being identified (Supplementary Table 2). Comparison of proteins in d0 and early stages of reactivation (d4–d6) showed that most of the proteins were present in all the stages except the cell cycle regulatory proteins that were present only in d0 and growth factors only in stages d4–d6 (Supplementary Table 4).

Table 1

Metabolic enzymes identified across the different stages in tammar wallaby with their corresponding pathways.

AccessionProtein named0d3d4d5d6d8d9d11d24Metabolic process
ENSMEUP00000006216Peroxiredoxin 1++++++++Detoxification of reactive oxygen species/selenium pathway
ENSMEUP00000007211Carbonyl reductase NADPH 1++++++++Arachidonic acid metabolism
ENSMEUP00000010739Glutathione S-transferase mu 3 brain+++++++++Glutathione metabolism
ENSMEUP00000012361Sepiapterin reductase+++++++Tetrahydrobiopterin biosynthesis/folate biosynthesis
ENSMEUP00000004359Phosphoglycerate kinase 1++++++++Glycolysis and gluconeogenesis
ENSMEUP00000000879Peroxiredoxin 5++++Thioredoxin/selenium pathway, detoxification of ROS
ENSMEUP00000001389Glutathione S-transferase P++++++++Glutathione metabolism/xenobiotic metabolism
ENSMEUP00000006370Triosephosphate isomerase 1+++++++++Gluconeogenesis
ENSMEUP00000003207Thioredoxin+++++++Detoxification of ROS
ENSMEUP00000005432Alpha enolase+++++++++Glycolysis
ENSMEUP00000001178Creatine kinase B chain++++++++Urea cycle/amino acid metabolism
ENSMEUP00000000833Carbonic anhydrase III muscle specific+Vitamin/coenzyme/sulphur compound metabolism
ENSMEUP00000007173Phosphoglycerate kinase 1+++++++Glycolysis and gluconeogenesis
ENSMEUP00000013868Dimethylarginine dimethylaminohydrolase 2+++Arginine metabolism/citrulline metabolism/nitric acid biosynthesis
ENSMEUP00000006724Alcohol dehydrogenase NADP++++++++Glycolysis/gluconeogenesis
ENSMEUP00000002721Creatine kinase B chain+++++++Vitamin/coenzyme/sulphur compound metabolism
ENSMEUP00000013946Glyceraldehyde 3-phosphate dehydrogenase Fragment EC 1.2.1.12+++++++++Glycolysis/gluconeogenesis
ENSMEUP00000012704Glutathione peroxidase 2 gastrointestinal++Lipid metabolism
ENSMEUP00000005468Peroxiredoxin 2++++++++Detoxification of reactive oxygen species/selenium pathway
ENSMEUP00000003077Glutathione S-transferase theta2B+++++++Glutathione metabolism
ENSMEUP00000004100Pyruvate kinase muscle+++++++++Glycolysis/gluconeogenesis
ENSMEUP00000004497Glutaredoxin thioltransferase+++++++Electron transport chain
ENSMEUP00000002725Cyclophilin B++Prolactin signalling pathway/collagen biosynthesis
ENSMEUP00000004406Lactate dehydrogenase B++++++Carbohydrate metabolism
ENSMEUP00000013345Catalase++++++++Electron transport chain
ENSMEUP00000005695Sulphotransferase 1A1+++Steroid metabolism
ENSMEUP00000000775Transaldolase 1++++++++Pentose phosphate pathway
ENSMEUP00000008411Fructosebisphosphate aldolase A++++++++Glycolysis/gluconeogenesis
ENSMEUP00000005681Dihydropyrimidinase-like 2++++++++BDNF signalling pathway
ENSMEUP00000001338Lactate dehydrogenase A+++++TCA cycle/pyruvate metabolism
ENSMEUP00000002583Cytosolic non-specific dipeptidase+++++++Nitrogen metabolism/glutathione biosynthesis
ENSMEUP00000001121Protein disulphide isomerase family A member 3+++++++Detoxification of ROS/cellular protein metabolism
ENSMEUP00000005305Esterase D++++++++Oestrogen/progesterone biosynthesis
ENSMEUP00000013368Protein phosphatase 2 regulatory subunit A alpha+++MAPK signalling, cell cycle and mitosis
ENSMEUP00000012631Glutathione peroxidase 3 plasma+Detoxification of ROS
ENSMEUP00000012662Gamma-glutamyl hydrolase conjugase folylpolygammaglutamyl hydrolase+Glutamine metabolism
ENSMEUP00000014632Glutathione peroxidase 1++lipid metabolism
ENSMEUP00000009000Isocitrate dehydrogenase 1 NADP+++TCA cycle/phospholipid biosynthesis
ENSMEUP00000012664Fructose bisphosphate aldolase C+Glucose metabolism/gluconeogenesis
ENSMEUP00000007804Glutathione S-transferase theta+++Glutathione metabolism
ENSMEUP00000011838Phosphoglycerate mutase 1++++++Glucose metabolism/gluconeogenesis
ENSMEUP00000005036Peroxiredoxin 4++++++++Detoxification of reactive oxygen species
ENSMEUP00000011992d-dopachrome decarboxylase++++++Melanin biosynthetic process
ENSMEUP00000001181Prostaglandin E synthase 3 cytosolic++++Arachidonic acid metabolism/prostaglandin biosynthesis
ENSMEUP00000008081Aldehyde dehydrogenase 1 family member A++++++++Retinol metabolism
ENSMEUP00000005444Acetyl-CoA acetyltransferase 2+++Mevolonate pathway
ENSMEUP00000013692Galactose mutarotase aldose 1-epimerase++Carbohydrate metabolism
ENSMEUP00000015288Prolyl 4-hydroxylase beta polypeptide++Redox homeostasis
ENSMEUP00000009171Cytidine monophosphate UMP-CMP kinase 1 cytosolic++Purine & pyrimidine metabolism
ENSMEUP00000009302Glucose-6-phosphate isomerase++++Glycolysis
ENSMEUP00000010438Glutamine–fructose-6-phosphate transaminase+++UDP-N-acetylglucosamine metabolism/protein metabolism
ENSMEUP00000014039Methylthioadenosine phosphorylase++++++Purine metabolism
ENSMEUP00000007229Enolase 3 beta muscle+++++++Glycolysis/gluconeogenesis
ENSMEUP00000001514Protein phosphatase 2 catalytic subunit alpha isozyme+++Meiotic/mitotic cell cycle/wnt signalling pathway
ENSMEUP00000013706Phosphoglucomutase 1+++Glucose/galactose/glycogen metabolism/pentose phosphate pathway
ENSMEUP00000003732Peroxiredoxin 6+++++Phenylalanine metabolism
ENSMEUP00000005004Puromycin-sensitive aminopeptidase+++++Hydrolysis of amino acids
ENSMEUP00000009792Acid phosphatase 1 soluble++++Phosphatase activity
ENSMEUP000000091286-Phosphogluconolactonase+++++Glucose metabolism/pentose phosphate pathway
ENSMEUP00000004425N-acetylneuraminate synthase+++Oligosaccharide/lipopolysaccharide biosynthesis
ENSMEUP00000001387Glutathione S-transferase theta-2+++Glutathione metabolism
ENSMEUP00000002646Quinoid dihydropteridine reductase++Phenylalanine and tyrosine metabolism/folate biosynthesis
ENSMEUP00000000713Aminoacyl-tRNA synthetase class II++tRNA modification/processing
ENSMEUP00000015196Carnosine synthase 1+Carnosine biosynthesis pathway
ENSMEUP00000008483Aldehyde dehydrogenase 1 family member L1++10-Formyltetrahydrofolate catabolic process/one carbon metabolic process
ENSMEUP000000065083-Hydroxybutyrate dehydrogenase type 2+Siderophore metabolism/beta fatty acid oxidation/ketone biosynthesis
ENSMEUP00000006320Alanine tRNA ligase+tRNA modification/processing
ENSMEUP00000002574Methionine adenosyltransferase II alpha++++S-adenosylmethionine biosynthetic process
ENSMEUP00000013099Protease serine 8+Positive regulation of sodium ion transport
ENSMEUP00000005092Aspartylglucosaminidase+++Protein deglycosylation and maturation
ENSMEUP00000005423UDP-glucose 6-dehydrogenase++Lipopolysaccharide biosynthesis
ENSMEUP00000012604Prostaglandin reductase-2++Prostaglandin metabolism
ENSMEUP00000013328GDP-mannose 46-dehydratase+++Nucleotide sugar biosynthesis
ENSMEUP00000013481Superoxide dismutase 1 soluble+++++Detoxification of oxygen species/activation of MAPK activity
ENSMEUP00000005428Dihydropyrimidinase-like 3++++Regulation of cell migration
ENSMEUP00000003945Peptidase D++Amino acid metabolism/collagen catabolism
ENSMEUP00000013977Quiescin Q6 sulfhydryl oxidase 1++++++Growth regulation
ENSMEUP00000010353Leukotriene A4 hydrolase++++Fatty acid metabolism
ENSMEUP00000005926Fumarylacetoacetate hydrolase fumarylacetoacetase++Arginine/phenylalanine/tyrosine metabolism
ENSMEUP00000004615UDP-GlcNAcbetaGal beta-13-N-Acetylglucosaminyltransferase 2+Carbohydrate/glycosaminoglycan metabolism
ENSMEUP00000004865Protein phosphatase 1 catalytic subunit alpha isozyme+Glycogen metabolism, triglyceride catabolism/cell division and cell cycle
ENSMEUP00000007839Methionine sulphoxide reductase A+Methionine metabolism
ENSMEUP00000000045ADP-ribosylhydrolase like 2+Cellular response to superoxide
ENSMEUP00000006354Ubiquitin specific peptidase 5 isopeptidase T+Ubiquitin-dependent protein catabolism
ENSMEUP00000003136Aldo-keto reductase family 1 member B1+++++Carbohydrate/doxorubicin/sorbitol metabolism
ENSMEUP00000001600Glycyl-tRNA synthetase+tRNA modification/processing
ENSMEUP00000011680Phosphopantothenoylcysteine synthetase+Coenzyme A/pantothenate/vitamin metabolism
ENSMEUP00000007002Protein disulphide isomerase family A member 4++++Detoxification of ROS/cellular protein metabolism
ENSMEUP00000004464Aldehyde dehydrogenase mitochondrial++++Carbohydrate/xenobiotic metabolism
ENSMEUP00000004313Prenylcysteine oxidase 1+Terpenoid synthesis
ENSMEUP00000015108Carbonic anhydrase II+++Vitamin/coenzyme/sulphur compound metabolism
ENSMEUP00000009844ATP citrate lyase++TCA cycle
ENSMEUP00000011277Phosphoglucomutase 2++++++Glycogen/carbohydrate metabolism
ENSMEUP00000001078Prolyl endopeptidase++Proteolysis
ENSMEUP00000013173Lysosomal alpha-mannosidase+Protein modification/mannose metabolism
ENSMEUP00000002183Phospholipase A2 group XV+Ceramide/glycerophospholipid/phosphatidylcholine metabolism, fatty acid catabolism
ENSMEUP00000010449Enolase-phosphatase 1+Methionine biosynthesis
ENSMEUP00000014768Malate dehydrogenase mitochondrial+Carbohydrate metabolism/TCA cycle
ENSMEUP00000013511Uroporphyrinogen decarboxylase+Uroporphyrinogen III metabolism/small molecule metabolism
ENSMEUP00000000258Aflatoxin B1 aldehyde reductase member 2+Carbohydrate metabolism/Xenobiotic metabolism
ENSMEUP00000005261Prostaglandin-E2 9-reductase++Prostaglandin biosynthesis
ENSMEUP00000007181Hexosaminidase B beta polypeptide+Carbohydrate metabolism/sphingolipid metabolism
ENSMEUP00000000995Arylacetamide deacetylase esterase 17+Triglyceride catabolism, xenobiotic metabolism
ENSMEUP00000009032Glutathione reductase+Glutathione metabolism/destruction of ROS
ENSMEUP00000000895Ectonucleotide pyrophosphatasephospho­diesterase 2+Phospholipid catabolism, cell migration
ENSMEUP00000007146Nuclear casein kinase and cyclin-dependent kinase substrate 1+Glucose homeostasis, insulin receptor signalling pathway
ENSMEUP00000006299Glutaminyl-tRNA synthetase+tRNA modification/processing
ENSMEUP00000002407Alcohol dehydrogenase 5 class III chi polypeptide+Destruction of ROS
ENSMEUP00000012523Carboxypeptidase N polypeptide 1+Bradykinin catabolism
ENSMEUP00000013478Aconitase 1 soluble++++TCA cycle
ENSMEUP00000004870Lecithin-cholesterol acyltransferase+Cholesterol/lipoprotein metabolism
ENSMEUP00000006047Phosphofructokinase liver+Carbohydrate metabolism
ENSMEUP00000001439Tryptase+Endopeptidase activity
ENSMEUP00000014818Galactosidase beta 1+Carbohydrate metabolism/sphingolipid metabolism
ENSMEUP00000002036Tryptophanyl-tRNA synthetase+Angiogenesis
ENSMEUP00000002335Aldehyde dehydrogenase 1 family member A3+++Retinol metabolism
ENSMEUP00000000288Phosphorylase glycogen brain+Carbohydrate metabolism
ENSMEUP00000006031Protease serine 35+Serine protease
ENSMEUP00000011456X-prolyl aminopeptidase aminopeptidase P 1 soluble+Bradykinin catabolism
ENSMEUP00000000536Serine hydroxymethyltransferase 1 soluble+Carnitine biosynthesis, vitamin/nitrogen/folic acid metabolism
ENSMEUP00000008533Leucine carboxyl methyltransferase 1+Protein modification/regulation of mitotic cell cycle spindle assembly
ENSMEUP00000001467Peptidylprolyl isomerase F+++++Negative regulation of oxidative phosphorylation
ENSMEUP00000007250Aspartyl aminopeptidase+Peptide metabolism
ENSMEUP00000003548Glutamyl-prolyl-tRNA synthetase+Protein biosynthesis
ENSMEUP00000004150Deoxyribose-phosphate aldolase putative+Carbohydrate/deoxyribosenucleotide catabolism
ENSMEUP00000004805N-acylaminoacyl-peptide hydrolase+Endopeptidase activity
ENSMEUP00000011308Dihydropyrimidine dehydrogenase+Beta-alanine biosynthesis, purine-pyrimidine catabolism
ENSMEUP00000001344Protein disulphide isomerase family A member 6++Detoxification of ROS/cellular protein metabolism
ENSMEUP00000015002Methylenetetrahydrofolate dehydrogenase+One carbon/tetrahydrofolate metabolism
ENSMEUP00000003224Glutamate-cysteine ligase catalytic subunit+Xenobiotic/glutathione/sulphur amino acid/cysteine/glutamate metabolism
ENSMEUP00000008144UDP-N-acteylglucosamine pyrophosphorylase 1+Uridylyltransferase activity
ENSMEUP00000003820Mitogen-activated protein kinase 1+Activation of MAPK activity/negative regulator of cell differentiation/apoptosis/cell cycle
ENSMEUP00000015019Histidyl-tRNA synthetase+Protein biosynthesis
ENSMEUP00000009382Aldehyde dehydrogenase 9 family member A1++++Carnitine biosynthesis/nitrogen metabolism
ENSMEUP00000010778Tyrosyl-tRNA synthetase+Protein biosynthesis
ENSMEUP00000010557Protein disulphide isomerase+Detoxification of ROS
ENSMEUP00000011501Ubiquitin specific peptidase 14 tRNA-guanine transglycosylase+Ubiquitin-dependent protein catabolism
ENSMEUP00000010927Mannosidase alpha class 1A member 1+Protein modification
ENSMEUP00000007934Lysyl-tRNA synthetase+Protein biosynthesis
ENSMEUP00000005076Alpha-lactalbumin+Carbohydrate metabolism

Apart from the other types of proteins, by d11 RPY, there were more proteins associated with cell adhesion (Stavréus-Evers et al. 2002, Wang & Dey 2006). It is likely that proteins involved in adhesion present on the luminal epithelial surface are important for the maintenance of integrity between adjacent epithelial cells. Hepatoma-derived growth factor, Transforming growth factor (TGFβ) 68 kDa protein and epidermal growth factor receptor (EGFR) were identified at different stages of reactivated UFs and in d3 UFs but not in the d0 samples. UFs contain significant number of growth factors, which were found to be secreted into the lumen from the day of reactivation (d4 RPY) and these increased up to late gestation (Table 2). Proteins involved in the p53/p21 cell cycle inhibition pathway (Fig. 3A) including Septin 2 (SEPT2), Septin 7 (SEPT7), mitogen-activated protein kinase 1 (MAPK1), proliferation-associated protein 2G4 (PA2G4), glia maturation factor beta (GMFB), neural precursor cell expressed, developmentally downregulated 8 (NEDD8) protein, cullin-associated NEDD8-dissociated protein 1(CAND1) and RNA binding protein fused in sarcoma (FUS) were only seen in d0–d3 samples (Supplementary Table 2). Lectin, galactoside-binding soluble 1 (LGALS1) and proliferating cell nuclear antigen (PCNA), which regulates cell proliferation and differentiation, were observed only in the reactivation samples (Supplementary Table 2).

Table 2

Growth factors and associated binding proteins that may be involved in regulating diapause and later pregnancy that have been identified from d3 RPY to d24 RPY uterine flushings of the tammar wallaby.

Growth factorsStage present (after RPY)References
Hepatoma-derived growth factord3, d4–d5, d6–d9, d11Gómez et al. (2012, 2014), Muñoz et al. (2014)
Granulind8, d9Gerton et al. (2000), Bateman et al. (2003)
Insulin-like growth factor binding proteins 1,2,3,4,5,6,7d24Simmen et al. (1995), Bagnell et al. (1997), Seidel et al. (1998), Costello et al. (2014)
Transforming growth factor beta-2d24Li et al. (2014, 2015)
MMP-2d24Howe et al. (1999), Aslan et al. (2007)
Growth differentiation factor 15d24Trovik et al. (2014),Chudecka-Głaz et al. (2015)
sEGFRd3–d4, d8, d11Perez-Torres et al. (2008), Adamczyk et al. (2011), Maramotti et al. (2012)
Insulin-like growth factor 2d24Geisert et al. (2001), Costello et al. (2014)
Figure 3
Figure 3

(A) A protein interaction map of some of the factors identified in UF. p53, PCNA and CDKN1A are the main players in this regulatory pathway. CDKN1A induction by p53 leads to cdk2 inhibition, thereby inhibiting cellular and blocking cell cycle progression. STIP1, HSPs and CDC37 act as molecular chaperones promoting their binding and stabilisation of protein complexes. NEDD8 and CAND1 also play important roles in cell cycle control by degradation of cyclins and other regulatory proteins. PTGES3 in a hormone-dependent manner disrupts receptor-mediated transcription. PCNA interaction with DNA polymerases is sensitive to changes in CDKN1A levels. (B) A kinase-dependent p53/CDKN1A regulatory pathway controls the cell cycle.

Citation: Reproduction 152, 5; 10.1530/REP-16-0154

Discussion

Earlier studies of the tammar UF using approaches such as gradient gel electrophoresis (Renfree 1973) and radioactive tracer studies (Shaw & Renfree 1986) demonstrated that there are stage-specific changes in the proteins present in the uterine secretions, but this is the first study using modern proteomics techniques and provides new data on the presence of individual proteins identified by MASCOT search (Perkins et al. 1999) of the recently developed tammar genome. This approach allowed us to study the qualitative changes in protein composition in UF taking into consideration whether a protein was consistently found in at least three biological replicates during diapause, reactivation and up to late gestation.

Studies in a range of eutherian and marsupial species show that UF contains selectively transudated serum proteins as well as proteins unique to the uterus (Junge & Blandau 1958, Stevens et al. 1964, Renfree 1973, Beier 1974). The protein repertoire of UF in tammar using gradient acrylamide gel electrophoresis identified numerous small uterine-specific pre-albumin proteins (Renfree 1973) and similarly in human secretions using two-dimensional polyacrylamide gel electrophoresis (Maclaughlin et al. 1986, Hannan et al. 2010). However, the studies in tammar did not reveal the identities of several UF proteins. Our study demonstrated the presence of many serum proteins in the UF (Supplementary Table 3A). These may be passing though the endometrial epithelial barrier by selective entry or apocrine secretions. There is also evidence to demonstrate localisation of some of these serum proteins in the endometrium (DeSouza et al. 2005, Fowler et al. 2007). In marsupials and primates, uterine-specific pre-albumins may pass into the blastocyst and exert some effect on its growth (Renfree 1973, 1978, Peplow et al. 1974, Hearn & Renfree 1975), but this idea has not been tested directly. The predictions by TargetP 1.1 revealed that 21% of the proteins in UF are classed as secretory proteins.

Exosomes and microvesicles derived from endometrial epithelium or trophectoderm have been identified in human (Kshirsagar et al. 2012, Ng et al. 2013, Tannetta et al. 2014) and ovine UF (Racicot et al. 2012, Burns et al. 2014), which provide an alternative mode of maternal embryo communication. Exosomes that contain lipids, proteins or micro-(mi)RNA are acquired by the endometrial epithelial cells or the blastocyst to promote implantation (Valadi et al. 2007, Simpson et al. 2008). Exosomal marker proteins include CD9, CD63 and CD81 along with heat-shock proteins HSP70 and HSP90 (Mathivanan et al. 2010). Among the exosomal proteins reported in earlier studies, glyceraldehyde phosphate dehydrogenase (GAPDH), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ), HSP70 and high mobility group B1 (HMGB1) have been identified in our study. However, given that the tammar blastocyst is enclosed in an acellular shell coat for the first 18 days of pregnancy, it has yet to be determined whether microsome- or exosome-mediated transfer from the uterus to the blastocyst is possible in this species.

Pregnancy is also regulated by cytoskeleton-associated proteins involving cytoskeletal rearrangements, apoptosis and constant remodelling (Lee et al. 1998, Miehe et al. 2005), which may in turn regulate the developing embryo (Jensen et al. 2013). Keratins are usually considered as a source of contamination during proteomic procedures, but 51 specific keratins were detected in pre-implantation mouse embryos, and several other studies confirm the presence of keratins and cytokeratins in the early mammalian embryonic development (Lehtonen et al. 1983, Magin et al. 2000). The changes in the keratin components in UF in our study possibly reflect changes resulting from the cellular differentiation of the endometrium. Adhesion protein Vanin 3 was specifically found in d11RPY UF samples in the tammar previously known to be present on the surface of equine endometrium (Hayes et al. 2012). Furthermore, synthesis of several classes of proteins, including adhesion factors, cytokines and growth factors, increases immediately after the oestrogen pulse that induces mouse implantation (Dey et al. 2004) as well as at reactivation from diapause in the tammar (Cha et al. 2013). Several ECM components associated with apocrine secretion from the glandular epithelium (Demir et al. 2002) are up-regulated in the pre-implantation endometrium in mice, including fibronectin, laminin and collagen type IV (Armant et al. 1986, Carson et al. 1988). These ECM proteins were observed in tammar UFs during diapause and most of the reactivation stages and not limited to any stage.

HMGB1 acts in an autocrine/paracrine fashion, and immunohistochemical analysis of human and rat endometrial tissue revealed the higher levels of HMGB1 in the nuclei of glandular epithelial, stromal and luminal epithelial compartments during the pre-receptive phase compared with their counterparts in the receptive phase in human (Cui et al. 2008, Bhutada et al. 2014). HMGB1/p53 complex are known to regulate autophagy and apoptosis (Livesey et al. 2012). In the tammar, HMGB1 was present from d0 until d11RPY, and high mobility group B2 (HMGB2) was present in d0–d3 and d11 of pregnancy.

UF contains many enzymes involved in important metabolic pathways that have been studied in the uterus and embryo (Murdoch & White 1968, Kirchner et al. 1971, Denker & Petzoldt 1977, Peplow 1982, Zavy et al. 1984). During diapause, the blastocyst remains viable but has a low metabolic rate that must provide sufficient energy for the maintenance and homeostasis of the embryo during the long period of arrest (Spindler et al. 1995, 1997, 1999). The first significant increase in blastocyst metabolism is at day 4 RPY when the first mitoses are seen in the blastocyst, subsequent to activation of the CL and progesterone stimulation of the uterus (Spindler et al. 1995, 1997). To maintain viability during diapause, it is important for the blastocyst to avoid damage from reactive oxygen species (ROS). Within the uterus, this may be achieved by local action of enzymes in the uterine fluid. This reducing environment is preserved by enzymes that maintain the reduction–oxidation (REDOX) state through a constant input of metabolism-derived energy (Gilbert & Colton 1999). Cells have several mechanisms to protect against reactive oxygen species (ROS). Prolonged, experimentally induced ROS production severely inhibits embryo development (Johnson & Nasresfahani 1994, Guerin et al. 2001), and its regulation is necessary for optimal embryo growth (Burdon 1996). The apocrine secretion of enzymes identified by our study (Table 1), which catalyse the destruction of ROS, are transcribed in pre-implantation embryos (Harvey et al. 1995, Takahashi 2012, Ramos et al. 2015). REDOX enzymes also known to support cell cycle progression (Yamauchi & Bloom 1997). Glutathione peroxidase, identified in our study, is an important REDOX regulator and is found in reproductive tract fluid (Gardiner et al. 1998) and decreases in concentration as early cleavage proceeds (Gardiner & Reed 1995). A family of peroxiredoxins identified in our study is known to be secreted by endometrial epithelium into the uterine lumen in mice and play crucial roles as antioxidants in the development of pre-implantation mouse embryos (Wang et al. 2010, Bhutada et al. 2013). Another autocrine secretory factor cyclophilin A, commonly seen during oxidative stress (Jin et al. 2000), was observed in all UFs especially more in days d0–d3 and early reactivation stages.

Progesterone and oestradiol induce changes in the uterine environment and the production and release of some cytokines and growth factors from the uterus that can have both autocrine and paracrine actions to regulate the pre-implantation embryo and prepare the endometrium for implantation (Harvey et al. 1995, Sharkey 1998, Hannan et al. 2011, Binder et al. 2014. Nineteen proteins previously shown to be regulated by progesterone and oestradiol in human endometrial and myometrial cells (Tamm et al. 2009, Soloff et al. 2011) were identified in tammar UF (Supplementary Table 5).

Several growth factors play important roles during pre-implantation mammalian development. Leukaemia inhibitory factor (LIF), insulin-like growth factors (IGFs), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), platelet activating factor (PAF), vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β) are present in the uterus and influence the development and growth of the pre-implantation embryo in several species including mouse, rat, cow and sheep and tammar (Thouas et al. 2015). Roles of growth factors during early development have been demonstrated by the addition of purified growth factors to culture medium or by molecular genetic techniques that interfere with gene expression and necessary for successful development of the blastocyst. A few of the previously identified proteins like progestagen-associated endometrial protein (PAEP), LIF and uteroglobin found in UF (Renfree 1973, Li et al. 1993) were not identified by our study likely due to them below the detection limit of this technique. Epidermal growth factor receptor identified in our study is important in intricate signalling and transcriptional networks (Large et al. 2014), which regulate diverse cellular functions, promoting cell proliferation, differentiation, migration, cell growth, and survival. These receptors bind epidermal growth factors (EGFs), a mitogen known to terminate diapause in ovariectomized rats in the absence of the oestrogen pulse (Johnson & Chatterjee 1993). Oestrogen stimulates the expression of the EGF family, and EGF is a potent mitogen expressed in the uterus during implantation in the mouse (Dey et al. 2004). EGF receptors are present in truncated forms on porcine endometrium during d9–d11 of pregnancy (Kliem et al. 1998) and in dormant carnivore embryos (Paria et al. 1994). Upregulation of epidermal growth factor receptor (EGFR) signalling is often observed in carcinomas and promotes uncontrolled cell proliferation and metastasis. The soluble forms (sEGFR) are diagnostic and/or prognostic cancer biomarkers (Perez-Torres et al. 2008, Adamczyk et al. 2011, Maramotti et al. 2012). The soluble secretory forms of EGFR were present in the tammar UF from d3 onwards correlating with the start of cell proliferation.

Hepatoma-derived growth factor (HDGF), a component of p53/p21 cell cycle control pathway, mediates cell proliferation, is activated by mitotic phosphorylation (Everett 2011) and secretion of HDGF requires processing of the N-terminus (Thakar et al. 2010). HDGF was absent at d0 but was identified from day 3 RPY onwards. Thus, it is the earliest specific secretory change identified, and may be associated with the start of cell proliferation (Fig. 3A). HDGF in bovine UF promotes embryonic development and cell proliferation and is synthesized by the endometrium and embryo (Gomez et al. 2014), therefore performing a dual role by receptor-mediated action or directly by DNA binding.

Fibroblast growth factors activate mink embryos from diapause and there is a gradual increase from day 3 after activation (Desmarais et al. 2004). Most of the growth factors identified have roles in malignancy due to their involvement in proliferation and metastasis (Witsch et al. 2010). The absence, or low concentrations, of specific growth factors and the presence of cell cycle inhibitors during lactation and up to d3 RPY in the tammar may be a potent reason the blastocyst remains dormant (Fig. 4).

Figure 4
Figure 4

Summary of the uterine embryo interactions that occur during reactivation from diapause in the tammar. The blastocyst is surrounded by an acellular coat, so uterine control of the blastocyst must be mediated by soluble factors that can pass through this barrier. Uterine flushings contain significant quantities of enzymes, products of cellular differentiation and growth factors from the day of reactivation (d4 RPY), which increase up to late gestation. The absence or low concentrations of growth factors during diapause and presence of cell cycle regulatory proteins that causes cell cycle arrest at G0/G1 phase may maintain diapause, whilst the surge of progesterone from the CL at reactivation induces the release of specific growth regulators from the endometrium that lead to reactivation of the blastocyst.

Citation: Reproduction 152, 5; 10.1530/REP-16-0154

Reduction or cessation of mitotic activity in the embryo is controlled by cell cycle regulatory mechanisms (Lopes et al. 2004). The cell cycle arrest markers may cause activation of mitogen-activated protein (MAP) kinase signal pathway (Moscatello et al. 1998), downregula­tion of cyclins or cyclin-dependent kinases (cdks) (Kim et al. 1999, Wang et al. 2013), which leads to upregulation of p21 and cell cycle arrest. A p53/p21 cell cycle inhibition pathway was evident in all diapause samples (d0 and d3) by the presence of p21-associated proteins that mediate cell cycle arrest at G0/G1 phase. This cell cycle arrest pathway was illustrated by STRING 10 using equivalent proteins from human (Szklarczyk et al. 2010) (Fig. 3A). Different domains of p21 interact with Cdks and PCNA, and both of these domains can independently inhibit DNA replication when present in cells (Fig. 3B) (Cayrol et al. 1998). The presence of LGALS1 at d3–d4 and PCNA at d6 by autocrine secretions indicates their roles in DNA replication and cell cycle control of the endometrium during reactivation. These findings are consistent with those identified in the mouse embryo in which dormancy is associated with the decrease in DNA replication genes (Hamatani et al. 2004).

Future studies of the validation of these growth factors and other molecules of interest in the UF could be carried out using MALDI (Matrix Absorption Laser Desorption Ionisation Time of Flight) Imaging Mass Spectrometry (MALDI IMS) (Caprioli et al. 1997) coupled to LC–MS/MS experiments. IMS is advantageous over other techniques since it does not require target-specific labelling reagents such as antibodies or tissue homogenization and utilizes intact tissue, which enables to correlate molecular information with histological details. This technique can help to localise specific protein signatures within different tissue compartments, thus preserving the spatial localisation of the molecules of interest (Balluff et al. 2011, Fehniger et al. 2014). Comparison of the peak list from MALDI IMS for identification of proteins using a parallel liquid chromatography (LC)–MS approach enables the identification of hundreds of proteins (Schober et al. 2012, Cole et al. 2013, Franck et al. 2013).

Conclusion

The proteomics approach using multiple biological replicates to characterise the UF proteome from d0 until late gestation coupled with LC–MS/MS analysis provided a global profile of proteins in the UFs and provides evidence for autocrine, paracrine and apocrine pathways. The data from our study collectively suggest that p21 may be responsible for the inhibition of the cell cycle in the uterine epithelium, thus preventing growth factor synthesis, but once diapause is terminated, numerous growth factors including HBGF and EGFR may have a role in reactivation of the diapausing blastocyst.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-16-0154.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research was funded by the Australian Research Council (ARC Discovery project grant # DP110101727, awarded to G S and D K G).

Acknowledgements

The authors thank Scott Brownlees, Helen Clark and the entire wallaby research group for assistance with the wallabies. They thank their collaborators Associate Professor Andrew Pask, Professor Asao Fujiyama (National Institute of Informatics, Japan), Professor Rachel O’Neill and Mr Tom Heider (University of Connecticut) for their contributions to the generation of an updated tammar genome that was used to derive the proteome database used in this study. They are also grateful to David Perkins at the proteomics facility Bio21 institute for setting up the tammar protein database and enabling automated BLAST searches.

The mass spectrometry proteomics data can be publicly accessed at the ProteomeXchange Consortium via the PRIDE database.

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    Schematic illustration of the sequential events from diapause to reactivation and birth at d26 RPY. Reactivation occurs after inhibition of the CL by the early morning pulse of prolactin which has been removed for three consecutive days. If a young is replaced onto the teat at any time up to 72 h after RPY, reactivation will not occur. On the fourth day of the removal of PY (d4 RPY), there is an increase in mitosis, protein synthesis and transport followed by an increase in RNA synthesis by d5 RPY. However, the first expansion of the blastocyst does not occur until d8 RPY. The sampled days are enclosed in boxes. (Adapted from Renfree & Shaw 2000).

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    The protein concentrations (mean ± s.d.) measured by BCA assay (n = 5 for each stage and side) across the stages (d0–d9 RPY) in the gravid and non-gravid uteri. At no stage, there was a significant difference between the concentrations in the two sides (P > 0.05). Protein concentrations in the flushings from both uteri increased progressively from d3 to d9 (P < 0.0001). The significant differences with reference to d0 are denoted by *.

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    (A) A protein interaction map of some of the factors identified in UF. p53, PCNA and CDKN1A are the main players in this regulatory pathway. CDKN1A induction by p53 leads to cdk2 inhibition, thereby inhibiting cellular and blocking cell cycle progression. STIP1, HSPs and CDC37 act as molecular chaperones promoting their binding and stabilisation of protein complexes. NEDD8 and CAND1 also play important roles in cell cycle control by degradation of cyclins and other regulatory proteins. PTGES3 in a hormone-dependent manner disrupts receptor-mediated transcription. PCNA interaction with DNA polymerases is sensitive to changes in CDKN1A levels. (B) A kinase-dependent p53/CDKN1A regulatory pathway controls the cell cycle.

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    Summary of the uterine embryo interactions that occur during reactivation from diapause in the tammar. The blastocyst is surrounded by an acellular coat, so uterine control of the blastocyst must be mediated by soluble factors that can pass through this barrier. Uterine flushings contain significant quantities of enzymes, products of cellular differentiation and growth factors from the day of reactivation (d4 RPY), which increase up to late gestation. The absence or low concentrations of growth factors during diapause and presence of cell cycle regulatory proteins that causes cell cycle arrest at G0/G1 phase may maintain diapause, whilst the surge of progesterone from the CL at reactivation induces the release of specific growth regulators from the endometrium that lead to reactivation of the blastocyst.