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© 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ ; From a broken bone to a major earthquake, the fracture of a material usually means trouble. However, in some practical applications engineers have learned how to harness the mechanisms of fracture. This is illustrated by the well-known process of hydraulic fracturing, or "fracking," in which injection of pressurized fluid in shale rocks opens cracks to extract oil or gas (1). On page 465 of this issue, Dumortier et al. (2) show that the developing mouse embryo uses this same principle to transiently disrupt its shape and sculpt a more complex one. Fracking is the key mechanism that enables the early embryo to develop its first symmetry axis, a key stage in fetal morphogenesis. ; The authors are supported by the European Research Council (CoG-616480 to XT, CoG-681434 to MA), European Union (H2020-FETPROACT-01-2016-731957), Generalitat de Catalunya and CERCA program, "ICREA Academia" award (MA), the Spanish Ministry for Science and Innovation/FEDER, Obra Social "La Caixa (XT)", and Severo Ochoa Award (XT). ; Peer Reviewed ; Postprint (author's final draft)

This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/stem.2067 ; During mammalian pre-implantation development, the cells of the blastocyst's inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived extra-embryonic endoderm (XEN) cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and extra-embryonic endoderm differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the re-organization of membrane trafficking machinery and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes. ; This work was supported by the European Union 7th Framework Program (PRIME-XS project grant number 262067 to K.S.L., L.G and C.M.M), the Biotechnology and Biological Sciences Research Council (BBSRC grant number BB/L002817/1 to K.S.L and L.G.), as well as a HFSP ...

Prologue -- 1 Collection of Gametes in Laboratory Animals and Preparation of Sperm for in Vitro Fertilization -- Collection of Ova for in Vitro Fertilization -- Methods Presently Used for Sperm Collection -- Differences in Sperm as a Function of Collection Method -- Preparation of Ejaculated Sperm for Use in in Vitro Fertilization -- Methods Used to Induce Capacitation of Ejaculated Sperm -- References -- 2 Ovum Collection and Induced Luteal Dysfunction in Primates -- Ovum Collection -- Evaluation of Corpus Luteum function -- Induced Folliculogenesis -- Discussion and Conclusions -- References -- 3 Analysis of Culture Media for in Vitro Fertilization and Criteria for Success -- Culture Media -- Criteria for Success -- Conclusions -- References -- 4 In Vitro Culture of the Zygote and Embryo -- General Observations on Fertilization and Early Development in Vitro -- Metabolic Requirements for Embryonic Development in Vitro -- Complex Media and Biological Fluids for Embryo Culture -- Conclusions -- References -- 5 Mechanisms of Fertilization in Mammals -- Epididymal Maturation and Capacitation of Spermatozoa -- Acrosome and Acrosome Reaction -- Hyperactivation of Spermatozoa -- Interaction of Spermatozoa with the Cumulus Oophorus -- Interaction of Spermatozoa with the Zona Pellucida -- Sperm-Egg Fusion -- Decondensation of the Sperm Nucleus and Development of Sperm and Egg Pronuclei -- Conclusions -- References -- 6 The Mammalian Egg's Block to Polyspermy -- Zona Reaction -- Zona Reaction Mechanisms -- Egg Plasma Membrane Block -- Block to Polyspermy Mechanisms -- Conclusions -- References -- 7 Gamete Interaction in the Sea Urchin A Model for Understanding the Molecular Details of Animal Fertilization -- Fertilization in the Sea Urchin -- The Plasma Membrane of Sea Urchin Sperm -- Sperm-Specific Surface Antigenicity Common to Seven Animal Phyla -- Isolation of Acrosome Granules and Identification of Bindin as the Major Component Involved in Sperm Adhesion -- Identification of a Bindin Receptor Glycoprotein from the Egg Vitelline Layer -- Conclusions -- References -- 8 Awakening of the Invertebrate Egg at Fertilization -- Sperm-Egg Fusion and the Rapid Block to Polyspermy -- Insertion and Localization of Sperm Components in the Egg -- The Cortical Reaction and Extracellular Peroxidative Reactions -- Activation of Egg Metabolism -- Conclusions -- References -- 9 Chromosome Aberrations and Mammalian Reproduction -- The Newborn -- Postimplantation Embryos -- Preimplantation Embryos -- Germ Cells -- Conclusions -- References -- 10 The Effects of Chromosomal Aneuploidy on Early Development Experimental Approaches -- Products of Aneuploid Mouse Embryos -- The Consequences of Monosomy -- Identical Twin Embryos -- Conclusions -- References -- 11 Blastocyst Fluid Formation -- Na+/ K+ ATPase and Blastocyst Fluid Accumulation -- Oxygen Consumption and Active Transport -- Developmental Aspects of Solute Transport in Blastocysts -- Conclusions -- References -- 12 Water and Electrolyte Transport by Pig Chorioallantois -- Porcine Allantoic Fluid Volume and Composition -- Effect of Lactogenic Hormones on Transport Properties of the Porcine Chorioallantoic Membrane -- Effect of Bromocryptine on Allantoic Fluid Volume and Electrolyte Composition at Day 30 of Gestation -- Discussion and Conclusions -- References -- 13 Critical Review of Embryo Transfer Procedures with Cattle -- Normalcy of Superovulated Ova -- A Note on Experimental Design -- Morphological Evaluation of Embryos -- Morphological Normalcy of Superovulated Ova with Time -- In Vitro Culture of Bovine Embryos -- Stage of the Estrous Cycle to Initiate Superovulation -- Regimens for Inseminating Superovulated Cows -- Side of Transfer -- Stage of Embryonic Development and Surgical Transfer -- Donor-Recipient Estrous Cycle Synchrony -- Factors Affecting Pregnancy Rates after Nonsurgical Transfer -- Summary and Conclusions -- References -- Epilogue.

Embryonic stem cell research holds great promise for biomedical research, but involves the destruction of human embryos. Katrien Devolder explores the tension between the view that embryos should never be deliberately harmed, and the view that such research must go forward. She provides an in-depth analysis of major attempts to resolve the problem.

Embryonic implantation is a key step in the establishment of pregnancy. In the present work, we have carried out an in-depth proteomic analysis of the secretome (extracellular vesicles and soluble proteins) of two bovine blastocysts embryonic trophectoderm primary cultures (BBT), confirming different epithelial–mesenchymal transition stages in these cells. BBT-secretomes contain early pregnancy-related proteins and angiogenic proteins both as cargo in EVs and the soluble fraction. We have demonstrated the functional transfer of protein-containing secretome between embryonic trophectoderm and maternal MSC in vitro using two BBT primary cultures eight endometrial MSC (eMSC) and five peripheral blood MSC (pbMSC) lines. We observed that eMSC and pbMSC chemotax to both the soluble fraction and EVs of the BBT secretome. In addition, in a complementary direction, we found that the pattern of expression of implantation proteins in BBT-EVs changes depending on: (i) their epithelial–mesenchymal phenotype; (ii) as a result of the uptake of eMSC-or pbMSC-EV previously stimulated or not with embryonic signals (IFN-τ); (iii) because of the stimulation with the endometrial cytokines present in the uterine fluid in the peri-implantation period. ; Ministerio de Economía Industria y Competitividad to Ramírez M. A. (AGL2015-70140-R), Spanish Ministerio de Ciencia e Innovación to Ramírez M. A. (PID2019-107145RB-I00) and to Yáñez-Mo M. (BIO2017-86500-R), Spanish Ministerio para la Transición Ecológica y el Reto Demográfico, through Fundación Biodiversidad to Ramírez M. A. (PRCV00820) and European Union's Horizon 2020

19 págs, ; Embryonic implantation is a key step in the establishment of pregnancy. In the present work, we have carried out an in-depth proteomic analysis of the secretome (extracellular vesicles and soluble proteins) of two bovine blastocysts embryonic trophectoderm primary cultures (BBT), confirming different epithelial–mesenchymal transition stages in these cells. BBT-secretomes contain early pregnancy-related proteins and angiogenic proteins both as cargo in EVs and the soluble fraction. We have demonstrated the functional transfer of protein-containing secretome between embryonic trophectoderm and maternal MSC in vitro using two BBT primary cultures eight endometrial MSC (eMSC) and five peripheral blood MSC (pbMSC) lines. We observed that eMSC and pbMSC chemotax to both the soluble fraction and EVs of the BBT secretome. In addition, in a complementary direction, we found that the pattern of expression of implantation proteins in BBT-EVs changes depending on: (i) their epithelial–mesenchymal phenotype; (ii) as a result of the uptake of eMSC- or pbMSC-EV previously stimulated or not with embryonic signals (IFN-τ); (iii) because of the stimulation with the endometrial cytokines present in the uterine fluid in the peri-implantation period ; This study was supported by grants from the Spanish Ministerio de Economía Industria y Competitividad to Ramírez M. A. (AGL2015-70140-R), Spanish Ministerio de Ciencia e Innovación to Ramírez M. A. (PID2019-107145RB-I00) and to Yáñez-Mo M. (BIO2017-86500-R), Spanish Ministerio para la Transición Ecológica y el Reto Demográfico, through Fundación Biodiversidad to Ramírez M. A. (PRCV00820) and European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 731014 (Ramírez M. A) ; Peer reviewed