Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation.
Abstract: Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.
Publication Date: 2003-08-06 PubMed ID: 12904313DOI: 10.1095/biolreprod.103.018515Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research explores the impact of in vitro production on the development and capsule formation of horse embryos. It shows that embryos produced in this way have a lower blastocyst formation rate and fail to form a vital glycoprotein capsule, an essential part of early embryo development.
Study Approach
- The researchers developed horse embryos through in vitro production (IVP), a laboratory procedure where an egg is fertilized by sperm outside the body.
- They compared embryos that had undergone IVP using two distinct methods – one where the embryos were cultured in synthetic oviduct fluid (SOF), and another where they were temporarily transferred to the oviduct of a sheep.
- As a control, they also studied embryos that had been naturally developed in the uterus of a mare.
Assessing Embryo Development
- Researchers assessed morphology, or shape and structure, of these embryos using light microscopy.
- Using multiphoton scanning confocal microscopy, they evaluated the distribution of microfilaments–structural elements within the cell–and measured apoptosis, or cell death.
Observations and Findings
- The results showed a notably higher blastocyst rate (16%) in embryos transferred to a sheep’s oviduct compared to those cultured in synthetic oviduct fluid (6.3%).
- Embryos produced by IVP were noticeably smaller and compact, displayed fewer cells, and had a poorly defined inner cell mass. These findings linked IVP with suboptimal embryonic development.
- The researchers found that IVP embryos had higher incidents of cell death and irregularly distributed microfilaments, further indicating compromised development.
Capsule Formation
- The embryos were stained with a specific antibody to examine the formation of the glycoprotein capsule that plays a crucial role in early embryo development.
- The researchers observed that despite IVP embryos producing the necessary glycoprotein for capsule formation, instead of creating a normal capsule, the glycoprotein failed to aggregate properly, thereby causing a malformed capsule.
Conclusion
- This study provides significant evidence showing that the in vitro production techniques used in this study had a negative impact on horse embryo development and capsule formation.
- The researchers concluded that more efficient ways of in vitro production of horse embryos need to be developed to better mimic natural conditions and yield a higher success rate.
Cite This Article
APA
Tremoleda JL, Stout TA, Lagutina I, Lazzari G, Bevers MM, Colenbrander B, Galli C.
(2003).
Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation.
Biol Reprod, 69(6), 1895-1906.
https://doi.org/10.1095/biolreprod.103.018515 Publication
Researcher Affiliations
- Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands. J.Tremoleda@vet.uu.nl
MeSH Terms
- Actin Cytoskeleton / ultrastructure
- Animals
- Apoptosis
- Blastocyst / cytology
- Blastocyst / physiology
- Blastocyst / ultrastructure
- Cell Culture Techniques / methods
- Cell Nucleus / physiology
- Cell Nucleus / ultrastructure
- Cells, Cultured
- Cytoskeleton / ultrastructure
- Egg Proteins / analysis
- Egg Proteins / metabolism
- Embryonic and Fetal Development
- Fallopian Tubes / transplantation
- Female
- Follicular Fluid
- Horses / embryology
- Male
- Membrane Glycoproteins / analysis
- Membrane Glycoproteins / metabolism
- Oocytes / transplantation
- Receptors, Cell Surface / analysis
- Receptors, Cell Surface / metabolism
- Sheep
- Zona Pellucida Glycoproteins
Citations
This article has been cited 15 times.- Rudolf Vegas A, Hamdi M, Podico G, Bollwein H, Fröhlich T, Canisso IF, Bauersachs S, Almiñana C. Uterine extracellular vesicles as multi-signal messengers during maternal recognition of pregnancy in the mare. Sci Rep 2022 Sep 16;12(1):15616.
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- Smits K, Goossens K, Van Soom A, Govaere J, Hoogewijs M, Vanhaesebrouck E, Galli C, Colleoni S, Vandesompele J, Peelman L. Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes 2009 Dec 11;2:246.
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