Title: ULTRASTRUCTURE OF THE EMBRYONIC STEM CELLS OF THE 8-DAY PIG BLASTOCYST BEFORE AND AFTER IN VITRO MANIPULATION: DEVELOPMENT OF JUNCTIONAL APPARATUS AND THE LETHAL EFFECTS OF PBS MEDIATED CELL-TO-CELL DISSOCIATION.
Submitted to: The Anatomical Record
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 7, 1999
Publication Date: N/A
Interpretive Summary: It has not been possible to grow epiblast cells of the pig in vitro so as to yield pig embryonic stem cell cultures that can be used for the genetic engineering as has been done in the mouse. One of the major barriers to establishing these cultures may reside in the way the pig epiblast cells are joined to each other. The objective of the study was to investigate the structure of the cells of the pig blastocyst (8-day post-fertilization embryo) with emphasis on the cell-to-cell adhesion structures of the epiblast cells. This was compared to the cellular structures found in the epiblast cells of the mouse blastocyst. Also, the consequences of calcium withdrawl from the culture environment was investigated since mouse epiblast cells apparently tolerate calcium withdrawl but pig epiblast cells may not. The major finding of the study was that mature adhesive structures are not evident between the epiblast cells of the early intact hatched pig blastocyst. Within a day or less of hatching, however, tight junctional complexes and desmosomes develop between the epiblast cells of the pig blastocyst and persist throughout the isolation and culture of the epiblast cells. In contrast, mouse epiblast cells had desmosomes and gap junctions but no tight junctional complexes. Withdrawal of calcium from the cultured pig epiblast cells for 5-10 minutes caused failure of the desmosomal connections and catastrophic loss of cytoplasm. The findings indicate that pig epiblast cells are adhered to each other in a different way than mouse epiblast cells and that a calcium withdrawal is quickly lethal to cultured pig epiblast cells.
Ultrastructural analysis of 8-day hatched pig blastocysts, inner cell mass (ICM), cultured ICM, and cultured epiblast tissue was undertaken to investigate epiblast cell-to-cell adhesion structures and their associated intracellular cytoskeletal elements. A comparison was made between early (small) and late (large spherical) 8-day blastocysts. Comparison was also made between cultured the pig epiblast tissue and cultured 129/Sv mouse epiblast tissue, the source of most mouse embryonic stem cell lines. Pig blastocyst trophectoderm and endoderm cells were connected by well developed desmosomes and tight junction complexes (TJ) with many associated microfilaments. Large numbers of phagocytosed sperm heads and tails were observed in the trophectoderm. The epiblast cells of small blastocysts had large lipid (yolk) vacuoles, and no desmosomes or TJ connections and no well organized microfilaments bands. The epiblast cells of large blastocysts were devoid of gross lipid vacuoles and were connected by desmosomes and TJ structures with some associated microfilaments. Epiblast cells in the cultured ICMs were similar in character to those in the intact large blastocyst. Isolated pig epiblasts cultured for 36 h formed a monolayer whose cells were connected by desmosomes, adherens junctions and TJ, and continuous microfilaments ran between adherens junctions parallel to cytoplasmic membranes. Mouse epiblast cells were connected by desmosomes and gap junctions but not TJ, and they had relatively scant microfilament associations. Withdrawal of Ca++ from the cultured pig epiblast cells caused extensive rupturing of the cells within minutes. The results may indicate a primary reason why pig epiblast cells have not been established as embryonic stem cells in culture.