Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle

Abstract

•Evaluation of UF-EV proteome changes at day 0, 7, and 16 of the simulated bovine estrous cycle with LC-MS/MS.•UF-EV proteome changes in key mechanisms needed for endometrial development.•Five putative human receptivity markers (ALPL, ANXA1, B2M, OLFM4, PPIA) were significantly enriched at day 16 of the estrous cycle.•Appropriate concentration of UF-EVs in embryo coculture is established.•Supplementing UF-EVs to embryo culture improved blastocyst rates significantly when cocultured with follicular phase UF-EVs. Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signaling and communications. Interestingly, the changes in UF-EV proteome during the bovine estrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics–based mass spectrometry was employed to compare UF-EV proteomes at day 0, 7, and 16 of the estrous cycle to understand the estrous cycle–dependent dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. One hundred fifty-nine UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle-dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development, and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion, and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased in vitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (p ≤ 0.05). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the estrous cycle. Interestingly, comparison of hormone-synchronized EV proteomes to natural cycle UF-EVs indicated shift of signaling. Finally, UF-EVs can be used to improve embryo production in vitro. Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signaling and communications. Interestingly, the changes in UF-EV proteome during the bovine estrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics–based mass spectrometry was employed to compare UF-EV proteomes at day 0, 7, and 16 of the estrous cycle to understand the estrous cycle–dependent dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. One hundred fifty-nine UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle-dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development, and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion, and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased in vitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (p ≤ 0.05). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the estrous cycle. Interestingly, comparison of hormone-synchronized EV proteomes to natural cycle UF-EVs indicated shift of signaling. Finally, UF-EVs can be used to improve embryo production in vitro. Bovine (Bos taurus) estrous cycle is a dynamic process under ovarian hormone regulation that controls cows’ endometrial physiology and subsequent pregnancy status. The duration of estrous cycle in cattle is 18 to 24 days, which can be divided to two discrete periods: luteal (14–18 days) and follicular phases (4–6 days). The luteal phase starts after the ovulation when the corpus luteum (CL) is formed, while follicular phase begins from the regression of functional CL and ends at ovulation (1Forde N. Beltman M.E. Lonergan P. Diskin M. Roche J.F. Crowe M.A. Oestrous cycles in Bos taurus cattle.Anim. Reprod. Sci. 2011; 124: 163-169Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Throughout this process, spatiotemporal changes transpire in the uterus regulated by the two key steroid hormones, progesterone and estrogen (2Faulkner S. Elia G. O’Boyle P. Dunn M. Morris D. Composition of the bovine uterine proteome is associated with stage of cycle and concentration of systemic progesterone.Proteomics. 2013; 13: 3333-3353Crossref PubMed Scopus (42) Google Scholar, 3Martins T. Pugliesi G. Sponchiado M. Gonella-Diaza A.M. Ojeda-Rojas O.A. Rodriguez F.D. et al.Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle.J. Anim. Sci. Biotechnol. 2018; 9: 70Crossref PubMed Scopus (19) Google Scholar). Uterine fluid (UF) is a mixture of growth factors, hormones, enzymes, lipids, glucose, transport proteins, and other molecules, which is typically secreted by uterine glands, epithelial cells, vasculature, and the composition of UF is known to change along with cycle dynamics (3Martins T. Pugliesi G. Sponchiado M. Gonella-Diaza A.M. Ojeda-Rojas O.A. Rodriguez F.D. et al.Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle.J. Anim. Sci. Biotechnol. 2018; 9: 70Crossref PubMed Scopus (19) Google Scholar, 4Moran E.T. Nutrition of the developing embryo and hatchling.Poult. Sci. 2007; 86: 1043-1049Crossref PubMed Scopus (271) Google Scholar). Uterine environment is rich in intercellular and intracellular communications owing to the complex physiology. Recent studies have discovered that these communications, especially the intercellular communications, are not only mediated by different secretory factors in UF (5Idelevich A. Vilella F. Mother and embryo cross-communication.Genes. 2020; 11: 376Crossref PubMed Scopus (26) Google Scholar) but also by the extracellular vesicles (EVs). EVs are lipid bilayer–bound nanoparticles, which contain wide range of biomolecules (e.g., proteins, lipids, miRNAs, DNA) (6Capra E. Lange-Consiglio A. The biological function of extracellular vesicles during fertilization, early embryo—maternal and their in and 2020; PubMed Scopus Google Scholar, D. N. Vilella F. Extracellular vesicles in human in and 2018; PubMed Scopus Google Scholar). and EVs from different of such as uterine M. A. et vesicles from and uterine supplementation in in vitro culture bovine embryo Anim. Sci. Biotechnol. 13: PubMed Scopus Google Scholar, F. M. et improve of cell 2018; PubMed Scopus Google M. A. et vesicles from and uterine supplementation in in vitro culture bovine embryo Anim. Sci. Biotechnol. 13: PubMed Scopus Google Scholar, M. A. P. et of bovine extracellular vesicles on embryo development and in PubMed Scopus Google and follicular fluid et extracellular vesicles from bovine ovarian follicular fluid the to the 2020; PubMed Scopus Google Scholar). on the cargo EVs normal and D. N. Vilella F. Extracellular vesicles in human in and 2018; PubMed Scopus Google Scholar). Recent studies have that UF-EVs such as endometrial development receptivity A. S. et extracellular vesicles proteome of endometrial cells the phase from that of endometrial cells the 13: PubMed Scopus Google Scholar, G. A. A. A. A. of bovine uterine fluid extracellular vesicles at follicular and luteal phases of the PubMed Scopus Google Scholar, M. M. M. M. et from endometrial fluid Reprod. PubMed Scopus Google during to and and pregnancy in PubMed Scopus Google regulation of embryo A. M. of in extracellular vesicles on regulation during the in Scopus Google and of embryo S. A. A. in the uterine a for at 2013; Scholar, T. are for bovine 2018; PubMed Scopus Google Scholar). the of EVs and composition which these biological processes are yet to be discovered. One of the important components of EVs are proteins, which can be used as various of the cells at time in A. S. M. et composition of extracellular vesicles by time of PubMed Scopus Google Scholar). the changes in UF-EV cargo in the uterus for studies have to the of changes for human UF-EVs during different time points of cycle A. M. S. et of human uterine extracellular vesicles dynamic regulation of key of embryo and during Scopus Google Scholar) and for embryo M. A. A. A. of extracellular vesicles secreted by human epithelial endometrial cells key proteins to embryo PubMed Scopus Google but bovine UF-EV dynamic changes during the estrous cycle and endometrial development are these human studies that dynamic changes in proteins are needed for endometrial development and embryo A. M. S. et of human uterine extracellular vesicles dynamic regulation of key of embryo and during Scopus Google Scholar, M. A. A. A. of extracellular vesicles secreted by human epithelial endometrial cells key proteins to embryo PubMed Scopus Google for proteins to cell extracellular and M. A. A. A. of extracellular vesicles secreted by human epithelial endometrial cells key proteins to embryo PubMed Scopus Google antioxidant A. M. S. et of human uterine extracellular vesicles dynamic regulation of key of embryo and during Scopus Google cell and M. A. A. A. of extracellular vesicles secreted by human epithelial endometrial cells key proteins to embryo PubMed Scopus Google Scholar). the the bovine embryo development is not on the uterine but on a complex fluid secreted by the (3Martins T. Pugliesi G. Sponchiado M. Gonella-Diaza A.M. Ojeda-Rojas O.A. Rodriguez F.D. et al.Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle.J. Anim. Sci. Biotechnol. 2018; 9: 70Crossref PubMed Scopus (19) Google Scholar). bovine not to the stage due to early in cattle is from which day 16 after (3Martins T. Pugliesi G. Sponchiado M. Gonella-Diaza A.M. Ojeda-Rojas O.A. Rodriguez F.D. et al.Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle.J. Anim. Sci. Biotechnol. 2018; 9: 70Crossref PubMed Scopus (19) Google Scholar, M. Morris D. and early in cattle and other Anim. PubMed Scopus Google Scholar). of the during the can be to functional of the uterine to the and the embryo to the (3Martins T. Pugliesi G. Sponchiado M. Gonella-Diaza A.M. Ojeda-Rojas O.A. Rodriguez F.D. et al.Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle.J. Anim. Sci. Biotechnol. 2018; 9: 70Crossref PubMed Scopus (19) Google Scholar). of the uterine for embryo development to to early embryo in embryo N. embryo and uterine environment of 16 in PubMed Scopus Google Scholar, The of human of Reprod. PubMed Scopus Google Scholar, D. A. M. the culture environment and embryo to embryo Full Text Full Text PDF PubMed Scopus Google Scholar). and signaling endometrial cells during is in embryo and endometrial which is needed for are of the of EVs from in in establishment of pregnancy and development of the embryo the M. M. M. M. et from endometrial fluid Reprod. PubMed Scopus Google Scholar, The of human of Reprod. 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PubMed Scopus Google Scholar) only of (ALPL, ANXA1, B2M, OLFM4, PPIA) were significantly enriched at day 16 of the estrous cycle to day and not other the which was significantly at day 16 day of the estrous cycle, The to the change in UF-EVs during the estrous cycle indicating the from is In the embryo culture the of different of bovine UF-EVs on blastocyst was The blastocyst ± from was in the supplemented with UF-EVs concentration of The of to in the supplemented with UF-EV at was significantly the EVs were supplemented (p and the of UF-EVs were supplemented (p and on the to on the of the the UF-EVs supplementation was with UF-EVs at a concentration of from follicular and luteal In this the phase of the estrous cycle to embryo development was cultures supplemented with luteal UF-EVs significantly (p improved blastocyst rates ± from to the ± the follicular phase also increased blastocyst rates ± to was not (p The of to was (p only luteal phase EVs supplemented and The dynamic regulation of changes in UF and its for endometrial and embryo development, which is for the normal physiology and for a pregnancy (3Martins T. Pugliesi G. Sponchiado M. Gonella-Diaza A.M. Ojeda-Rojas O.A. Rodriguez F.D. et al.Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle.J. Anim. Sci. Biotechnol. 2018; 9: 70Crossref PubMed Scopus (19) Google Scholar, M. A. et vesicles from and uterine supplementation in in vitro culture bovine embryo Anim. Sci. Biotechnol. 13: PubMed Scopus Google Scholar, F. M. et improve of cell 2018; PubMed Scopus Google Scholar). in many human and the of UF-EVs in the normal endometrial and pregnancy establishment are in this study, to the UF-EV changes in cattle at different time points of the estrous cycle and its in endometrial physiology and bovine in vitro embryo development. UF different which be with especially in the EV of these be which are with UF-EV is EV which the EVs from other of of proteins in to which of EVs from The of EVs be the to the EV in the EV D. N. Vilella F. 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Full Text Full Text PDF Scopus Google Scholar). in the and of uterine for be to understand its mechanisms to improve the bovine of the in the comparison of two have by the different used in the two of in the bovine estrous cycle, with ovulation and natural cycle, and also have the this of natural and simulated changes of the bovine EV proteomes are the to understand the on uterine with with to the be to the mass spectrometry M. et in Scopus Google Scholar). The development of endometrial cells receptivity involves which is on different et the and dynamics of the human in and in PubMed Scopus Google Scholar). In the study, many pathways and their changes in different of the estrous cycle that are key mechanisms needed for endometrial development. pathways were to cell and epithelial cell and and these pathways in normal be in even the are throughout the estrous cycle. studies have that abnormal of can to of signaling which to M. et of uterine and signaling and progesterone in the of with 11: Google Scholar). In study, a dynamic of proteins to for cell and from day to day 16 of the estrous cycle. One of the differentially enriched proteins was which is in endometrial The of abnormal endometrial which is to be with to A. et to the cell PubMed Scopus Google Scholar). The and its and is for the development of studies are needed to understand the signaling of UF-EV proteome components in for different processes of endometrial development, which to for abnormal endometrial development to uterine The of pregnancy in cattle to of the pregnancy on day 16 (3Martins T. Pugliesi G. Sponchiado M. Gonella-Diaza A.M. Ojeda-Rojas O.A. Rodriguez F.D. et al.Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle.J. Anim. Sci. Biotechnol. 2018; 9: 70Crossref PubMed Scopus (19) Google Scholar, M. Morris D. and early in cattle and other Anim. PubMed Scopus Google Scholar). this changes in the uterine are in the UF-EVs (5Idelevich A. Vilella F. Mother and embryo cross-communication.Genes. 2020; 11: 376Crossref PubMed Scopus (26) Google Scholar). changes to of a that is for the establishment of In human many have to endometrial receptivity markers even EV S. A. A. in the uterine a for at 2013; Scholar, S. et of extracellular vesicles in endometrial receptivity and their in and PubMed Scopus Google the endometrial receptivity markers for cattle are yet to be S. et changes in bovine and markers of uterine PubMed Scopus Google Scholar, M. T. Sponchiado M. et receptivity in the Reprod. PubMed Google Scholar). In study, with the of identified bovine UF-EV proteins that were to be putative receptivity markers for A. M. S. et of human uterine extracellular vesicles dynamic regulation of key of embryo and during Scopus Google Scholar, M. A. A. A. of extracellular vesicles secreted by human epithelial endometrial cells key proteins to embryo PubMed Scopus Google Scholar, P. P. A.

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