From Peptide Masses to Pregnancy Maintenance: A Comprehensive Proteomic Analysis of The Early Equine Embryo Secretome, Blastocoel Fluid, and Capsule

Swegen, Aleona; Grupen, Christopher G.; Gibb, Zamira; Baker, Mark A.; Ruijter-Villani, Marta de; Smith, Nathan D.; Stout, T.A.E.; Aitken, R. John

doi:10.1002/pmic.201600433

Cited by 37 publications

(55 citation statements)

References 94 publications

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“…Similarly, Demetriou et al, [52] suggested a prospective crucial role for this gene in the maintenance of pregnancy in humans. Recently, the importance of the role of FKBP4 protein was showed also in 10-day old horse embryo as it was found within the blastocoel fluid [53]. However, this study could not confirm that this protein was actively secreted into the uterine lumen, or was capable of interaction with the mare's endometrium [53].…”

Section: Discussionmentioning

confidence: 74%

“…Recently, the importance of the role of FKBP4 protein was showed also in 10-day old horse embryo as it was found within the blastocoel fluid [53]. However, this study could not confirm that this protein was actively secreted into the uterine lumen, or was capable of interaction with the mare's endometrium [53]. Although our results showed an up regulation of FKBP4 protein in cyclic heifers, further studies are needed to determine its functions in bovine pregnancy and early embryo development in the oviduct.…”

Section: Discussionmentioning

confidence: 98%

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Spatial and Pregnancy-Related Changes in the Protein, Amino Acid, and Carbohydrate Composition of Bovine Oviduct Fluid

Rodríguez‐Alonso

Maillo

Acuña

et al. 2020

IJMS

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Knowledge of how the biochemical composition of the bovine oviduct is altered due to the oviduct anatomy or the presence of an embryo is lacking. Thus, the aim of this study was to assess the effect of (I) oviduct anatomy and (II) embryo presence on oviductal fluid (OF) protein, amino acid, and carbohydrate composition. Cross-bred beef heifers (n = 19) were synchronized and those in standing estrus were randomly allocated to a cyclic (non-bred) or pregnant (artificially inseminated) group. All heifers were slaughtered on Day 3 after estrus. The oviducts ipsilateral to the corpus luteum from each animal were isolated, straightened and cut, separating ampulla and isthmus. Each portion was flushed with 500 µl of PBS enabling recovery of the oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (8-cell embryos; pregnant group) were located in the isthmus of the oviduct. Samples of flushing medium from the isthmus and ampulla were used for proteomic (n = 2 per group), amino acid (n = 5), and carbohydrate (n = 5) analysis. For proteomic analysis, total protein from cyclic and pregnant samples were labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3-10, 17 cm, Protean ® IEF cell system, Bio Rad). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). Images were obtained with a Typhoon 9410 scanner and analyzed with Progenesis SameSpots software v 4.0. Amino acid content in the OF was determined by high performance liquid chromatography (HPLC). Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. For the proteomic assessment, the results of the image analysis were compared by ANOVA. For both amino acid and carbohydrate analyses, statistical analysis was carried out by 2-way ANOVA with the Holm-Sidak nonparametric post hoc analysis. On Day 3 post-estrus, OF composition varied based on (I) anatomical region, where isthmic metabolites were present in lower (i.e., lactate, glycine, and alanine) or higher (i.e., arginine) concentrations compared to the ampulla; and (II) embryo presence, which was correlated with greater, arginine, phosphoglycerate kinase 1, serum albumin, α-1-antiproteinase and IGL@ Int.The oviduct is a tubular seromuscular organ connecting the ovary to the uterus providing a physiological environment for gamete interaction, fertilization, and early embryo development. During the first four days of embryo development, which occurs within the oviduct [1], embryonic nutritional requirements are presumed to be fulfilled largely by oviduct fluid (OF)-a complex mixture of proteins, amino acids, carbohydrates, ions, hormones, and extracellular vesicles, amongst other constituents [2]. OF composition is regulated by (I) selective transudate from the vasculature, and (II) the secretory cells within the oviduct epithelial monolayer [3]. Moreover, OF composition is influenced spatially by the anatomical...

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Section: Discussionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 98%

Spatial and Pregnancy-Related Changes in the Protein, Amino Acid, and Carbohydrate Composition of Bovine Oviduct Fluid

Rodríguez‐Alonso

Maillo

Acuña

et al. 2020

IJMS

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“…Other processes, such as "lipid biosynthetic processes" and "fatty acid metabolic process" were also up-regulated during conceptus development [19]. Comparative proteomics similarly highlighted changes in functional categories of ULF proteins including developmental processes, "binding", response to a stimulus and lipid and cholesterol activity [21,22].…”

Section: Introductionmentioning

confidence: 93%

“…ligand-receptor associations) between the endometrial and conceptus transcriptomes on day 16 [20]. The proteomic profile of day 9 and 10 blastocyst secretions and blastocoele fluid [21], and day 13 uterine luminal (ULF; cyclic and pregnant mares) and yolk sac (blastocoele) fluid have also been reported [22]. The latter studies identified differences in the ULF induced by the presence of a conceptus, and identified proteins secreted by the endometrium or the conceptus into the ULF or yolk sac fluid.…”

Section: Introductionmentioning

confidence: 99%

Asynchronous Embryo Transfer Followed by Comparative Transcriptomic Analysis of Conceptus Membranes and Endometrium Identifies Processes Important to the Establishment of Equine Pregnancy

Gibson¹,

Ruijter-Villani²,

Bauersachs³

et al. 2020

Preprint

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Pre-implantation horse conceptuses require nutrients and signals from histotroph, the composition of which is regulated by luteal progesterone and conceptus-secreted factors. To distinguish progesterone and conceptus effects we shortened the period of endometrial progesterone-priming by asynchronous embryo transfer. Day 8 embryos were transferred to synchronous (day 8) or asynchronous (day 3) recipients, and RNA sequencing was performed on endometrium and conceptuses recovered 6 and 11 days later (embryo days 14 and 19). Asynchrony resulted in many more differentially expressed genes (DEGs) in conceptus membranes (3473) than endometrium (715). Gene ontology analysis identified upregulation in biological processes related to organogenesis and preventing apoptosis in synchronous conceptuses on day 14, and in cell adhesion and migration on day 19. Asynchrony also resulted in large numbers of DEGs related to 'extracellular exosome'. In endometrium, genes involved in immunity, the inflammatory response, and apoptosis regulation were upregulated during synchronous pregnancy and, again, many genes related to extracellular exosome were differentially expressed. Interestingly, only 14 genes were differentially expressed in endometrium recovered 6 days after synchronous versus 11 days after asynchronous transfer (day 14 recipient in both). Among these, KNG1 and IGFBP3were consistently up-regulated in synchronous endometrium. Furthermore bradykinin, an active peptide cleaved from KNG1, stimulated prostaglandin release by cultured trophectoderm cells. The horse conceptus thus responds to a negatively asynchronous uterus by extensively adjusting its transcriptome, whereas the endometrial transcriptome is modified only subtly by a more advanced conceptus. the donor (synchronous; n=10), or 5 days after the donor mare (asynchronous; n=10). Conceptuses were then recovered on day 14 or day 19 of development (n=5 per group). Day 14 conceptuses were recovered 6 days after ET by uterine lavage via a sterile endotracheal tube, and day 19 conceptuses were recovered 11 days after ET using an endoscopically-guided net, after puncture of the membranes and aspiration of the yolk-sac fluid using a sharpened PTFE catheter [41]. Following conceptus recovery, an endometrial biopsy was collected, using alligator forceps (141965; Jørgen Kruuse A/S, Langeskov, Denmark), from the base of one or other uterine horn for day 14 conceptuses (mobile phase), or from the site of conceptus apposition for day 19 conceptuses (fixed location; n=5 per group) under videoendoscopic guidance. For conceptus membrane culture, pregnancy status was monitored by transrectal ultrasonography from day 14 after ovulation, and conceptuses were recovered on day 19 by uterine lavage via a sterile endotracheal tube (n=5 conceptuses).Conceptuses and endometrial biopsies were washed with sterile 0.9% NaCl. Under a stereomicroscope, the blastocyst capsule was removed from the conceptuses and the embryonic disc region, or embryo proper, were dissected from the conceptus membranes...

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“…A further study looked for possible relationships (e.g., ligand-receptor associations) between the endometrial and conceptus transcriptomes on day 16 [20]. The proteomic profile of day 9 and 10 blastocyst secretions and blastocoele fluid [21], and day 13 uterine luminal (ULF; cyclic and pregnant mares) and yolk sac (blastocoele) fluid have also been reported [22]. The latter studies identified differences in the ULF induced by the presence of a conceptus, and identified proteins secreted by the endometrium or the conceptus into the ULF or yolk sac fluid.…”

mentioning

confidence: 99%

Asynchronous Embryo Transfer Followed by Comparative Transcriptomic Analysis of Conceptus Membranes and Endometrium Identifies Processes Important to the Establishment of Equine Pregnancy

Gibson

Ruijter-Villani

Bauersachs

et al. 2020

IJMS

View full text Add to dashboard Cite

Preimplantation horse conceptuses require nutrients and signals from histotroph, the composition of which is regulated by luteal progesterone and conceptus-secreted factors. To distinguish progesterone and conceptus effects we shortened the period of endometrial progesterone-priming by asynchronous embryo transfer. Day 8 embryos were transferred to synchronous (day 8) or asynchronous (day 3) recipients, and RNA sequencing was performed on endometrium and conceptuses recovered 6 and 11 days later (embryo days 14 and 19). Asynchrony resulted in many more differentially expressed genes (DEGs) in conceptus membranes (3473) than endometrium (715). Gene ontology analysis identified upregulation in biological processes related to organogenesis and preventing apoptosis in synchronous conceptuses on day 14, and in cell adhesion and migration on day 19. Asynchrony also resulted in large numbers of DEGs related to 'extracellular exosome'. In endometrium, genes involved in immunity, the inflammatory response, and apoptosis regulation were upregulated during synchronous pregnancy and, again, many genes related to extracellular exosome were differentially expressed. Interestingly, only 14 genes were differentially expressed in endometrium recovered 6 days after synchronous versus 11 days after asynchronous transfer (day 14 recipient in both). Among these, KNG1 and IGFBP3 were consistently upregulated in synchronous endometrium. Furthermore bradykinin, an active peptide cleaved from KNG1, stimulated prostaglandin release by cultured trophectoderm cells. The horse conceptus thus responds to a negatively asynchronous uterus by extensively adjusting its transcriptome, whereas the endometrial transcriptome is modified only subtly by a more advanced conceptus.Int. J. Mol. Sci. 2020, 21, 2562 2 of 25 to be essential for MRP, since restricting migration to a single uterine horn results in failure to avert luteolysis [7]. In the absence of pregnancy, luteolysis occurs between approximately days 13 and 16 as a result of pulses of prostaglandin F2α (PGF2α) released by the endometrium following the establishment of an oxytocin-PGF2α positive feedback loop [8,9]. To prevent luteal regression, the embryo's MRP signal must prevent the cyclical increase in oxytocin sensitivity that starts at approximately day 10 after ovulation [10,11]. Although the identity of the equine embryonic MRP signal(s) remains unknown [12], it is increasingly clear that MRP involves the transient suppression of PGF2α release by a combination of inhibiting prostaglandin-endoperoxidase-synthase 2 expression (PTGS2; the rate-limiting enzyme in PGF2α synthesis) and delaying the upregulation of the endometrial oxytocin receptors critical to the oxytocin-PGF2α loop needed to generate luteolytic pulses of PGF2α [8,10,13].Recent transcriptomic and proteomic analyses have improved our understanding of embryo-maternal communication during the conceptus migration and MRP phase. Studies have compared gene expression in endometrium between cycling and pregnant mares on days 8 an...

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From Peptide Masses to Pregnancy Maintenance: A Comprehensive Proteomic Analysis of The Early Equine Embryo Secretome, Blastocoel Fluid, and Capsule

Cited by 37 publications

References 94 publications

Spatial and Pregnancy-Related Changes in the Protein, Amino Acid, and Carbohydrate Composition of Bovine Oviduct Fluid

Spatial and Pregnancy-Related Changes in the Protein, Amino Acid, and Carbohydrate Composition of Bovine Oviduct Fluid

Asynchronous Embryo Transfer Followed by Comparative Transcriptomic Analysis of Conceptus Membranes and Endometrium Identifies Processes Important to the Establishment of Equine Pregnancy

Asynchronous Embryo Transfer Followed by Comparative Transcriptomic Analysis of Conceptus Membranes and Endometrium Identifies Processes Important to the Establishment of Equine Pregnancy

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