Blastocyst Development

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Human Blastocyst (day 5)[1]

(Greek, blastos = sprout + cystos = cavity) or blastula, the term used to describe the hollow cellular mass that forms in early development.

The blastocyst consists of cells forming an outer trophoblast layer, an inner cell mass and a fluid-filled cavity. The blastocyst inner cell mass is the source of true embryonic stem cells capable of forming all cell types within the embryo. In humans, this stage occurs in the first and second weeks after the zygote forms a solid cellular mass morula stage) and before implantation.

Links: Fertilization | Week 1 | Morula | Blastocyst Development | Implantation
Mouse blastocyst development
Mouse blastocyst development[2]

Some Recent Findings

  • Four simple rules that are sufficient to generate the mammalian blastocyst[2] "We implemented experimentally reported mechanisms for polarity, cell-cell signaling, adhesion, and apoptosis as a set of developmental rules in an agent-based in silico model of physically interacting cells. We find that this model quantitatively reproduces specific mutant phenotypes and provides an explanation for the emergence of heterogeneity without requiring any initial transcriptional variation. It also suggests that a fixed time point for the cells' competence of fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) sets an embryonic clock that enables certain scaling phenomena, a concept that we evaluate quantitatively by manipulating embryos in vitro. Based on these observations, we conclude that the minimal set of rules enables the embryo to experiment with stochastic gene expression and could provide the robustness necessary for the evolutionary diversification of the preimplantation gene regulatory network."
  • Asymmetric division of contractile domains couples cell positioning and fate specification[3] "During pre-implantation development, the mammalian embryo self-organizes into the blastocyst, which consists of an epithelial layer encapsulating the inner-cell mass (ICM) giving rise to all embryonic tissues. In mice, oriented cell division, apicobasal polarity and actomyosin contractility are thought to contribute to the formation of the ICM. However, how these processes work together remains unclear. Here we show that asymmetric segregation of the apical domain generates blastomeres with different contractilities, which triggers their sorting into inner and outer positions."
  • Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst[4] "The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established."
More recent papers  
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This table shows an automated computer PubMed search using the listed sub-heading term.

  • Therefore the list of references do not reflect any editorial selection of material based on content or relevance.
  • References appear in this list based upon the date of the actual page viewing.

References listed on the rest of the content page and the associated discussion page (listed under the publication year sub-headings) do include some editorial selection based upon both relevance and availability.

Links: References | Discussion Page | Pubmed Most Recent | Journal Searches

Search term: Blastocyst Development

Dong-Hui Wang, Hong-Xia Zhou, Shu-Jun Liu, Cheng-Jie Zhou, Xiang-Wei Kong, Zhe Han, Cheng-Guang Liang Glial cell line-derived neurotrophic factor supplementation promotes bovine in vitro oocyte maturation and early embryo development. Theriogenology: 2018, 113;92-101 PubMed 29477014

Gabriella S Antoniotti, Melinda Coughlan, Lois A Salamonsen, Jemma Evans Obesity associated advanced glycation end products within the human uterine cavity adversely impact endometrial function and embryo implantation competence. Hum. Reprod.: 2018; PubMed 29471449

M Vera-Rodriguez, A Diez-Juan, J Jimenez-Almazan, S Martinez, R Navarro, V Peinado, A Mercader, M Meseguer, D Blesa, I Moreno, D Valbuena, C Rubio, C Simon Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Hum. Reprod.: 2018; PubMed 29471395

Xiaomin Su, Chenglei Wu, Xiaoying Ye, Ming Zeng, Zhujun Zhang, Yongzhe Che, Yuan Zhang, Lin Liu, Yushuang Lin, Rongcun Yang Embryonic lethality in mice lacking Trim59 due to impaired gastrulation development. Cell Death Dis: 2018, 9(3);302 PubMed 29467473

Zhiren Zhang, Yanhui Zhai, Xiaoling Ma, Sheng Zhang, Xinglan An, Hao Yu, Ziyi Li Down-Regulation of H3K4me3 by MM-102 Facilitates Epigenetic Reprogramming of Porcine Somatic Cell Nuclear Transfer Embryos. Cell. Physiol. Biochem.: 2018, 45(4);1529-1540 PubMed 29466785

Older papers  
  • Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage[5] "We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA)."
  • Blastocyst gene expression correlates with implantation potential[6] "Compared with blastocysts that resulted in healthy fetal development, blastocysts that failed to implant (negative) showed decreased B3gnt5 and Eomes gene expression, while blastocysts that resulted in spontaneous pregnancy loss (absorption) displayed decreased Wnt3a and Eomes gene expression."
  • FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst[7] "Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. ... In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner."


Human Blastocyst

Human Blastocyst (day 3 to 6)
 ‎‎Day 3 to 6
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Human blastocyst day 5-6.jpg
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Human blastocyst hatching movie icon.jpg
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Model Development

Model embryo to 32 cell stage icon.jpg
 ‎‎Morula Model
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Model embryo to 128 cell stage icon.jpg
 ‎‎Blastocyst Model
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Human Blastocyst Formation

The table below shows human blastocyst in vitro development changes during week 1.[8]

Human blastocyst formation-in vitro.jpg

Labeled Blastocyst

Human embryo day 5 label.jpg Human embryo day 5 label2.jpg

Blastocyst Hatching

Mouse Blastocyst hatching[9]
Mouse-hatching blastocyst.jpg Human carnegie stage 3 label.jpg
Blastocyst hatching from zona pellucida (mouse) Blastocyst hatching from zona pellucida (human)

Model Human Blastocyst Development

The following figure is from a recent study[5] using video and genetic analysis of in vitro human development during week 1 following fertilization.

Model human blastocyst development.jpg

  • EGA - embryonic genome activation
  • ESSP - embryonic stage–specific pattern, four unique embryonic stage–specific patterns (1-4)
Links: Figure with legend

Mouse Blastocyst Gene Expression

Mouse- preimplantation gene expression.jpg

General gene expression patterns are indicated from genomic profiling.[10]

  • red - loss of maternal mRNAs
  • green - activation of embryonic genome (EGA)
  • purple - maternal gene activation (MGA)
  • orange - continuous expression

Inner Cell Mass

Human Blastocyst (day 5)[1]

This outer layer of cells is also called the "embryoblast", a cluster of cells located and attached on one wall of the outer trophoblast layer.

Trophoblast Layer

This outer layer of cells is also called the "trophectoderm" (TE) epithelium. A key function is for the transport of sodium (Na+) and chloride (Cl-) ions through this layer into the blastocoel.

Differentiation of this layer has been shown to be regulated by the transcription factors Tead4[11] and then Caudal-related homeobox 2 (Cdx2).

Links: Trophoblast | OMIM -Tead4 | OMIM - Cdx2

Blastocoel Formation

Mouse - blastocoel formation[10]
  • trophectoderm transports of Na+ and Cl- ions through this layer into the blastocoel
  • generates an osmotic gradient driving fluid across this epithelium
  • distinct apical and basolateral membrane domains specific for transport
  • facilitates transepithelial Na+ and fluid transport for blastocoel formation
  • transport is driven by Na, K-adenosine triphosphatase (ATPase) in basolateral membranes of the trophectoderm [12]

Blastocyst Metabolism

Mouse blastocyst GLUT8 expression.[13]

At the blastocyst stage, mammalian development metabolism switches on anaerobic glycolysis metabolism to satisfy metabolic demands of growing blastocyst and formation of the blastocoel. This is thought to be driven by the integral membrane protein family of facilitative glucose transporters (GLUT or SLC2A).

  • aerobic - oxidation of lactate and pyruvate via the citric acid cycle (Krebs cycle) and oxidative phosphorylation
  • glycolysis- converts glucose into pyruvate
  • GLUT - GLUcose Transporter (divided into 3 classes I-III)
  • SLC2 - Solute Carrier Family 2

Glucose Transporter Expression

  • GLUT1 - from zygote to blastocyst. (all mammalian tissues, basal glucose uptake)
  • GLUT2 and GLUT3 - from late eight cell stage to blastocyst. (GLUT2, liver and pancreatic beta cells; GLUT3, all mammalian tissues, basal glucose uptake)
  • GLUT4 - not expressed. (muscle and adipose tissue)
  • GLUT8 - up-regulated at blastocyst stage. (central nervous system and heart)
(Data mainly from mouse development, adult tissue expression shown in brackets)

A mouse study,[13] has shown GLUT8 is up-regulated following insulin stimulation, though a more recent GLUT8 knockout mouse shows normal early embryonic development in the absence of this transporter.[14]

Links: Biochemistry - glucose transporters | GLUT1 | GLUT2 | GLUT8

Blastula Cell Communication

Two types of cell junctions have been identified located at different regions in the developing blastocyst.

Tight junctions

Located close to outer surface create a seal, isolates interior of embryo from external medium.

Gap junctions

Allow electrically coupling of the cells of epithelium surrounding the fluid-filled cavity.

Tight junction 01.jpg Gap junction 01.jpg
Adhesion EM Images: GIT epithelia EM1 | GIT epithelia EM2 | GIT epithelia EM3 | Desmosome EM
Adhesion Cartoons: Tight junction | Adherens Junction | Desmosome | Gap Junction

Blastocyst Hatching - zona pellucida lost, ZP has sperm entry site, and entire ZP broken down by uterine secretions and possibly blastula secretions.

Uterine Glands - secretions required for blastocyst motility and nutrition

Links: MBoC Figure 21-69. The blastula

Blastocyst Hatching

At about day 5 the human blastocyst "hatches" out of the protective zona pellucida. This hatching allows increased growth, access to uterine nutrient secretions and blastocyst adhesion to the uterine lining. Associated with this hatching process are a series of physical contractions.

In the blastocyst, repeated contractions occur after blastocoel formation and the frequency of contractions is greater during the hatching period than in the periods both before and after hatching.[15] Interestingly, the same researchers in this mouse study suggest that the weaker contractions (less than 20% volume reduction) seen have a role in hatching, in contrast to strong contractions (20% or more volume reduction) have the opposite effect of inhibiting hatching.

In the mouse model, the identified sites of zona pellucida shedding varied (24% mural site, 24% inner cell mass site, 17% equatorial site, and 35% other sites).[16]

Links: Blastocyst Day 5-6 Movie
<mediaplayer width='500' height='450' image="">File:Human_blastocyst_day_5-6.mp4</mediaplayer>

Human blastocyst contractions (day 5-6)[5]

Molecular Factors

  • TEA DNA- binding domain, these factors bind to the consensus TEA/ATTS cognate binding site[17]
    • TEF-3 - renamed Tead1 and Tead4
    • Tead3 - is expressed in the placental syncytiotrophoblasts
  • E-cadherin - Calcium ion-dependent cell adhesion molecule, a cell membrane adhesive protein required for morula compaction
  • epithin - A type II transmembrane serine protease, identified in mouse for compaction of the morula during preimplantation embryonic development. Expressed from 8-cell stage at blastomere contacts and co-localises in the morula with E-cadherin. PMID: 15848395
  • Na, K-adenosine triphosphatase - A sodium potassium pump that generates an osmotic gradient for fluid flow into the blastocoel
  • Zonula occludens-1 - (ZO-1) Tight junction protein involved in morula to blastocyst transformation in the mouse PMID: 18423437

Blastocyst in Other Species

Mouse Blastocyst

Early mouse development cartoon.jpg

Early mouse development model[19]

Links: Mouse Development

Bovine Blastocyst

Links: Bovine Development


  1. 1.0 1.1 Pu Zhang, Marco Zucchelli, Sara Bruce, Fredwell Hambiliki, Anneli Stavreus-Evers, Lev Levkov, Heli Skottman, Erja Kerkelä, Juha Kere, Outi Hovatta Transcriptome profiling of human pre-implantation development. PLoS ONE: 2009, 4(11);e7844 PubMed 19924284 | PMC2773928 | PLoS One
  2. 2.0 2.1
  3. Gloryn Chia Le Bin, Silvia Muñoz-Descalzo, Agata Kurowski, Harry Leitch, Xinghua Lou, William Mansfield, Charles Etienne-Dumeau, Nils Grabole, Carla Mulas, Hitoshi Niwa, Anna-Katerina Hadjantonakis, Jennifer Nichols Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst. Development: 2014, 141(5);1001-10 PubMed 24504341 | Development
  4. 5.0 5.1 5.2 Connie C Wong, Kevin E Loewke, Nancy L Bossert, Barry Behr, Christopher J De Jonge, Thomas M Baer, Renee A Reijo Pera Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat. Biotechnol.: 2010, 28(10);1115-21 PubMed 20890283 | Nat Biotechnol.
  5. Jason C Parks, Blair R McCallie, Ann M Janesch, William B Schoolcraft, Mandy G Katz-Jaffe Blastocyst gene expression correlates with implantation potential. Fertil. Steril.: 2011, 95(4);1367-72 PubMed 20864103
  6. Yojiro Yamanaka, Fredrik Lanner, Janet Rossant FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst. Development: 2010, 137(5);715-24 PubMed 20147376
  7. C Y Fong, A Bongso Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture. Hum. Reprod.: 1999, 14(3);774-81 PubMed 10221713
  8. Noriko Tanaka, Takumi Takeuchi, Queenie V Neri, Eric Scott Sills, Gianpiero D Palermo Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model. J Transl Med: 2006, 4;20 PubMed 16681851 | PMC1479373 | J Transl Med.
  9. 10.0 10.1 Christine E Bell, Michele D Calder, Andrew J Watson Genomic RNA profiling and the programme controlling preimplantation mammalian development. Mol. Hum. Reprod.: 2008, 14(12);691-701 PubMed 19043080 | Mol Hum Reprod.
  10. Noriyuki Nishioka, Shinji Yamamoto, Hiroshi Kiyonari, Hiroko Sato, Atsushi Sawada, Mitsunori Ota, Kazuki Nakao, Hiroshi Sasaki Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos. Mech. Dev.: 2007, 125(3-4);270-83 PubMed 18083014
  11. Gerald M Kidder, Andrew J Watson Roles of Na,K-ATPase in early development and trophectoderm differentiation. Semin. Nephrol.: 2005, 25(5);352-5 PubMed 16139691
  12. 13.0 13.1 M O Carayannopoulos, M M Chi, Y Cui, J M Pingsterhaus, R A McKnight, M Mueckler, S U Devaskar, K H Moley GLUT8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst. Proc. Natl. Acad. Sci. U.S.A.: 2000, 97(13);7313-8 PubMed 10860996 | PMC16542 | Proc Natl Acad Sci U S A.
  13. Mathieu Membrez, Edith Hummler, Friedrich Beermann, Jacques-Antoine Haefliger, Rebecca Savioz, Thierry Pedrazzini, Bernard Thorens GLUT8 is dispensable for embryonic development but influences hippocampal neurogenesis and heart function. Mol. Cell. Biol.: 2006, 26(11);4268-76 PubMed 16705176
  14. Sueo Niimura Time-lapse videomicrographic analyses of contractions in mouse blastocysts. J. Reprod. Dev.: 2003, 49(6);413-23 PubMed 14967891
  15. K Yamazaki, Y Kato Sites of zona pellucida shedding by mouse embryo other than muran trophectoderm. J. Exp. Zool.: 1989, 249(3);347-9 PubMed 2708952
  16. P Jacquemin, J J Hwang, J A Martial, P Dollé, I Davidson A novel family of developmentally regulated mammalian transcription factors containing the TEA/ATTS DNA binding domain. J. Biol. Chem.: 1996, 271(36);21775-85 PubMed 8702974
  17. 18.0 18.1 18.2 18.3 18.4 Maria Keramari, Janet Razavi, Karen A Ingman, Christoph Patsch, Frank Edenhofer, Christopher M Ward, Susan J Kimber Sox2 is essential for formation of trophectoderm in the preimplantation embryo. PLoS ONE: 2010, 5(11);e13952 PubMed 21103067 | PMC2980489 | PLoS One.
  18. Pawel Krupinski, Vijay Chickarmane, Carsten Peterson Simulating the mammalian blastocyst--molecular and mechanical interactions pattern the embryo. PLoS Comput. Biol.: 2011, 7(5);e1001128 PubMed 21573197 | PMC3088645 | PLoS Comput Biol.


Theodore P Rasmussen, Gareth N Corry Epigenetic pre-patterning and dynamics during initial stages of mammalian preimplantation development. J. Cell. Physiol.: 2010, 225(2);333-6 PubMed 20607796

Katie Cockburn, Janet Rossant Making the blastocyst: lessons from the mouse. J. Clin. Invest.: 2010, 120(4);995-1003 PubMed 20364097

Janet Rossant Stem cells and lineage development in the mammalian blastocyst. Reprod. Fertil. Dev.: 2007, 19(1);111-8 PubMed 17389140


Joana Santos, C Filipe Pereira, Aida Di-Gregorio, Thomas Spruce, Olivia Alder, Tristan Rodriguez, Véronique Azuara, Matthias Merkenschlager, Amanda G Fisher Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo. Epigenetics Chromatin: 2010, 3;1 PubMed 20157423

Bette J Dzamba, Karoly R Jakab, Mungo Marsden, Martin A Schwartz, Douglas W DeSimone Cadherin adhesion, tissue tension, and noncanonical Wnt signaling regulate fibronectin matrix organization. Dev. Cell: 2009, 16(3);421-32 PubMed 19289087

Bhavwanti Sheth, Rachael L Nowak, Rebecca Anderson, Wing Yee Kwong, Thomas Papenbrock, Tom P Fleming Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation. Exp. Cell Res.: 2008, 314(18);3356-68 PubMed 18817772

Noriyuki Nishioka, Shinji Yamamoto, Hiroshi Kiyonari, Hiroko Sato, Atsushi Sawada, Mitsunori Ota, Kazuki Nakao, Hiroshi Sasaki Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos. Mech. Dev.: 2007, 125(3-4);270-83 PubMed 18083014

Yojiro Yamanaka, Amy Ralston, Robert O Stephenson, Janet Rossant Cell and molecular regulation of the mouse blastocyst. Dev. Dyn.: 2006, 235(9);2301-14 PubMed 16773657

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