Talk:Blastocyst Development

Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage

Nat Biotechnol. 2010 Oct 3. [Epub ahead of print]

Wong CC, Loewke KE, Bossert NL, Behr B, De Jonge CJ, Baer TM, Pera RA.

[1] Institute for Stem Cell Biology and Regenerative Medicine, School of Medicine, Stanford University, Stanford, California, USA. [2] Department of Obstetrics and Gynecology, School of Medicine, Stanford University, Stanford, California, USA. [3] These authors contributed equally to this work. Abstract 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). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.

PMID: 20890283

(CCC permission for reuse 7oct2010)

Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst

Mesnard D, Constam DB. J Cell Biol. 2010 Oct 4;191(1):129-39. Epub 2010 Sep 27.

Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β-related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface-targeted fluorescent biosensor (cell surface-linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

PMID: 20876279

http://www.ncbi.nlm.nih.gov/pubmed/20876279

New technique to quantify the lipid composition of lipid droplets in porcine oocytes and pre-implantation embryos using Nile Red fluorescent probe

Theriogenology. 2010 Sep 10. [Epub ahead of print]

Romek M, Gajda B, Krzysztofowicz E, Kepczynski M, Smorag Z.

Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, R. Ingardena 6, 30-060 Krakow, Poland. Abstract The principal objective of this study was to develop a novel method based on confocal microscopy and a solvatochromic fluorescent dye, Nile red (NR) to quantify the main types of lipids in a single mammalian oocyte and embryo. We hypothesize that NR staining followed by the decomposition of NR-spectra identifies and quantifies the triglycerides, phospholipids, and cholesterol in a single oocyte and embryo. We analyzed the lipid droplets in porcine oocytes and pre-implantation embryos up to the hatched blastocyst stage developed in vivo and in cultured blastocysts. The emission spectrum of NR-stained mixture of different lipid types is a convolution of several component spectra. The principal component analysis (PCA) and a multivariate curve resolution-alternating least squares method (MCR-ALS) allowed to decompose the emission spectrum and quantify the relative amount of each lipid type present in mixture. We reported here that the level of the triglycerides, phospholipids and cholesterol in lipid droplets significantly decreases by 17.7%, 26.4% and 23.9%, respectively, from immature to mature porcine oocytes. The content of triglycerides and phospholipids remains unchanged in droplets of embryos from the zygote up to the morula stage. Then the triglyceride level decreases in the blastocyst by 15.1% and in the hatched blastocyst by 37.3%, whereas the amount of phospholipids decreases by 10.5% and 12.5% at the blastocyst and hatched blastocyst stages, respectively. In contrast, the content of cholesterol in droplets does not change during embryo cleavage. The lipid droplets in the blastocyst produced in vivo contain lower amounts of triglycerides (by 26.1%), phospholipids (by 14.2%) and cholesterol (by 34.8%) than those in the blastocyst cultured in NCSU-23 medium. In conclusion, our new technique is suitable to quantify the content of triglycerides, phospholipids and cholesterol in individual mammalian oocytes and embryos. Our findings indicate an important role for lipids during porcine oocyte maturation and early embryonic development, and suggest an altered lipid metabolism in cultured embryos.

PMID: 20833424

Hatching and distribution of actin filaments in mouse blastocysts whose activities of protein kinase A were suppressed by H-89

J Reprod Dev. 2010 Feb;56(1):103-9. Epub 2009 Nov 2.

Suzuki R, Niimura S.

Graduate School of Science and Technology, Niigata University, Japan. Abstract The role of actin filaments and contractions in hatching was determined in mouse blastocysts whose actin filament bundling abilities had been suppressed by H-89, an inhibitor of protein kinase A. The hatching rate of blastocysts developed from morulae in a medium containing H-89 at a concentration of 4.0 microM was 17.2%, which was significantly lower than the 76.7% of the control blastocysts developed from morulae in a medium without H-89. The rates of blastocysts starting hatching and forming a slit in the zona pellucida were significantly lower in H-89-treated blastocysts (84.4 and 21.9%) than in control blastocysts (100.0 and 90.6%). The lengths of time needed for slit formation in the zona pellucida and for completion of hatching were significantly longer in the H-89-treated blastocysts (27.4 and 43.3 h) than in the control blastocysts (6.5 and 18.8 h). Over the course of 32 h after blastocoel formation, the number of strong contractions was similar in the H-89-treated and control blastocysts, but the number of weak contractions was significantly fewer in the H-89-treated blastocysts (2.41 times) than in the control blastocysts (4.19 times). Although the distribution of actin filaments was similar in the H-89-treated and control blastocysts in the pre-hatching, hatching and post-hatching periods, the rate of H-89-treated blastocysts in which most trophectoderm cells possessed the fluorescence of actin filaments (12.7%) was significantly lower than the 95.0% of the control blastocysts in the pre-hatching period. These results suggest that actin filament-mediated movements of trophectoderm cells contribute to hatching by facilitating the protrusion of trophectoderm cells from a small hole in the zona pellucida and by enlarging the protrusion. We also suggest that the low hatching ability of the treated blastocysts is related to weak contractions with a low frequency and to strong contractions requiring a longer time for re-expansion.

PMID: 19881218 http://www.ncbi.nlm.nih.gov/pubmed/19881218

Compaction

http://www.ncbi.nlm.nih.gov/pubmed/20042384

Compaction is initiated by E-cadherin mediated cell adhesion, which is regulated post-translationally via protein kinase C

Gene replacement reveals a specific role for E-cadherin in the formation of a functional trophectoderm. Kan NG, Stemmler MP, Junghans D, Kanzler B, de Vries WN, Dominis M, Kemler R. Development. 2007 Jan;134(1):31-41. Epub 2006 Nov 30. PMID: 17138661

"During mammalian embryogenesis the trophectoderm represents the first epithelial structure formed. The cell adhesion molecule E-cadherin is ultimately necessary for the transition from compacted morula to the formation of the blastocyst to ensure correct establishment of adhesion junctions in the trophectoderm. Here, we analyzed to what extent E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, we introduced N-cadherin cDNA into the E-cadherin genomic locus. We show that the expression of N-cadherin driven from the E-cadherin locus reflects the expression pattern of endogenous E-cadherin. Heterozygous mice co-expressing E- and N-cadherin are vital and show normal embryonic development. Interestingly, N-cadherin homozygous mutant embryos phenocopy E-cadherin-null mutant embryos. Upon removal of the maternal E-cadherin, we demonstrate that N-cadherin is able to provide sufficient cellular adhesion to mediate morula compaction, but is insufficient for the subsequent formation of a fully polarized functional trophectoderm. When ES cells were isolated from N-cadherin homozygous mutant embryos and teratomas were produced, these ES cells differentiated into a large variety of tissue-like structures. Importantly, different epithelial-like structures expressing N-cadherin were formed, including respiratory epithelia, squamous epithelia with signs of keratinization and secretory epithelia with goblet cells. Thus, N-cadherin can maintain epithelia in differentiating ES cells, but not during the formation of the trophectoderm. Our results point to a specific and unique function for E-cadherin during mouse preimplantation development."

Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo. Dard N, Louvet-Vallée S, Santa-Maria A, Maro B. Dev Biol. 2004 Jul 1;271(1):87-97. PMID: 15196952

Expression of epithin in mouse preimplantation development: its functional role in compaction. Khang I, Sonn S, Park JH, Rhee K, Park D, Kim K. Dev Biol. 2005 May 1;281(1):134-44. PMID: 15848395

A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Riethmacher D, Brinkmann V, Birchmeier C. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):855-9. PMID: 7846066

Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse. Wang H, Ding T, Brown N, Yamamoto Y, Prince LS, Reese J, Paria BC. Dev Biol. 2008 Jun 1;318(1):112-25. Epub 2008 Mar 20. PMID: 18423437

Time-lapse videomicrographic analyses of contractions in mouse blastocysts

J Reprod Dev. 2003 Dec;49(6):413-23.

Niimura S.

Faculty of Agriculture, Niigata University, Japan. niimura@agr.niigata-u.ac.jp Abstract Contraction has been observed in cultured blastocysts of many mammals, but little is known about the features of the contraction and its physiological role in blastocysts. The author analyzed contractions of a large number of cultured mouse blastocysts by time-lapse videomicrography. The results revealed that blastocysts repeated contractions of different degrees during the expanded stage from 10 h after blastocoel formation, and that the number of contractions was greater during the hatching period than in the periods pre- and post-hatching. The results also showed that the time needed for both contraction and re-expansion to the size before contraction tended to lengthen in blastocysts severely contracted. It was inferred that contractions of blastocysts occur physiologically in relation to myosin light chain kinase, but not due to an increase in permeability between trophectoderm cells in association with their division, or the influence of culture. Furthermore, it was inferred that re-expansion of contracted blastocysts occurs due to active transport and accumulation of Na(+) from the trophectoderm cells into blastocoelic fluid as a result of the action of Na(+)/K(+)-ATPase activated in the membrane of trophectoderm cells. Our results suggested that contractions are also present in blastocysts developed in vivo, and that weak contractions (less than 20% volume reduction) play an important role in hatching, whereas strong contractions (20% or more volume reduction) have the effect of inhibiting hatching. From our results on contractions of various blastocysts, it seems possible to evaluate the developmental ability of embryos, i.e. embryo quality, based on contractions of blastocysts.

PMID: 14967891 http://www.ncbi.nlm.nih.gov/pubmed/14967891

Advanced embryo development during extended in vitro culture: observations of formation and hatching patterns in non-transferred human blastocysts

Hum Fertil (Camb). 2002 Nov;5(4):215-20.

Porter RN, Tucker MJ, Graham J, Sills ES.

Division of Cell Sciences, University of Southampton, Southampton S016 7PX, UK. Abstract Human embryos not chosen for fresh transfer or cryopreservation were maintained in extended in vitro culture for up to 9 days after fertilization to observe blastocyst formation and hatching features. These non-transferred embryos were derived from 64 consecutive IVF cycles, and were not cryopreserved either because of compromised morphology or because the patients did not consent to cryopreservation for personal reasons. Embryos were cultured individually to monitor daily growth until developmental arrest, and differential blastocyst formation and hatching were analysed among groups of patients and embryos. In the population studied, hatching occurred most commonly on day 7 after fertilization (range 5-9 days). A total of 301 blastocysts was observed, of which 116 (38.5%) eventually hatched in vitro irrespective of day of formation. A trend towards earlier blastocyst formation and a greater likelihood of hatching was noted in this population. Both blastocyst formation and hatching appeared negatively correlated with increasing maternal age and higher basal serum FSH concentrations on day 3 of development, although these trends did not reach statistical significance. Comparison of intracytoplasmic sperm injection (ICSI) (n = 25) and conventional insemination (n = 39) cycles showed a similar rate of blastocyst formation in both groups (54 and 52%, respectively; P > 0.05), but hatching patterns varied significantly between these groups (4.1 versus 61.6%, respectively; P < 0.0001). The discovery of marked impairment of hatching among non-transferred ICSI embryos supports the case for reconsideration of the appropriateness of assisted blastocyst hatching in selected cases.

PMID: 12477966 http://www.ncbi.nlm.nih.gov/pubmed/12477966

Regulation of blastocyst formation

Watson AJ, Barcroft LC. Front Biosci. 2001 May 1;6:D708-30. Review. PMID: 11333210

"Preimplantation or pre-attachment development encompasses the "free"-living period of mammalian embryogenesis, which directs development of the zygote through to the blastocyst stage. Blastocyst formation is essential for implantation, establishment of pregnancy and is a principal determinant of embryo quality prior to embryo transfer. Cavitation (blastocyst formation) is driven by the expression of specific sets of gene products that direct the acquisition of cell polarity within the trophectoderm, which is both the first epithelium of development and the outer cell layer encircling the inner cell mass of the blastocyst. Critical gene families controlling these events include: the E-cadherin-catenin cell adhesion family, the tight junction gene family, the Na/K-ATPase gene family and perhaps the aquaporin gene family. This review will update the roles of each of these gene families in trophectoderm differentiation and blastocyst formation. The current principal hypothesis under investigation is that blastocyst formation is mediated by a trans-trophectoderm ion gradient(s) established, in part, by Na/K-ATPase, which drives the movement of water through aquaporins (AQPs) across the epithelium into the extracellular space of the blastocyst to form the fluid-filled blastocoel. The trophectoderm tight junctional permeability seal regulates the leakage of blastocoel fluid, and also assists in the maintenance of a polarized Na/K-ATPase distribution to the basolateral plasma membrane domain of the mural trophectoderm. The cell-to-cell adhesion provided by the E-cadherin-catenin gene families is required for the establishment of the tight junction seal and the maintenance of the polarized Na/K-ATPase distribution. Blastocyst formation is therefore directly linked with trophectoderm cell differentiation, which arises through fundamental cell biological processes that are associated with the establishment of cell polarity."

http://www.ncbi.nlm.nih.gov/pubmed/1335276