The fine structure of human embryonic stem (ES) cell colonies was analysed by transmission electron microscopy (TEM) after 35 passages of in-vitro culture. Most cells formed compact, saucer-shaped colonies with epithelioid cells on the periphery and polygonal cells within the colony. Three morphological types of cells were identified based on their fine structure: undifferentiated cells resembling inner cell mass (ICM) cells of blastocysts; protein-synthesizing cells at the onset of cellular differentiation; and compact masses of secretory cells resembling unicellular goblet cells of the intestine. The predominant cell type was the undifferentiated ES cells resembling ICM cells of blastocysts. These cells had large nuclei containing reticulated nucleoli, well-developed rough endoplasmic reticulum (RER), Golgi complexes, elongated tubular mitochondria, lysosomes and typical centrosomes with centrioles associated with microtubules and microfilaments, organizing the cytoskeleton. Some ES cells have very large nuclei and scanty cytoplasms with fewer organelles. The isolated or attached protein-synthesizing cells at the onset of differentiation had extensive RER and large Golgi complexes. The morphologically differentiated cells formed compact colonies and resembled goblet-like cells in microstructure. They had RER and large Golgi complexes associated with secretory vesicles. The epithelioid cells at the periphery were columnar and largely polarized by centrosomes associated with Golgi complexes. Epithelioid cells in all three categories had specialized cell junctions (desmosomes), anchored by tonofilaments, and surface blebs. Isolated cells were seen on the surface, towards the centre of the colony, and their free surfaces had microvilli and larger blebs. Approximately 3-5% of all cells were mitotic, with typical bipolar spindles organized by centrosomes, pivotally located at the poles, and appeared to resemble typical somatic cells.
Cultured human, ampullary, epithelial cells obtained from fertile women undergoing hysterectomy were evaluated for the support of human embryonic cleavage and growth in vitro. Twelve patients provided 23 embryos for co-culture with subcultured ampullary cells grown in T6 + 15% patient's serum and 18 embryos for growth in T6 + 15% patient's serum alone (controls). Of embryos co-cultured with ampullary cells, 78% cleaved to the compacted embryo stage and 69% cavitated as compared with 50 and 33% respectively for controls (P less than 0.01). Only 30% of co-cultured embryos reached the expanded blastocyst and 26% underwent hatching as compared with 28% for both stages in controls. At the 2 - 4- and 6 - 8-cell stages, 91 and 87% of co-cultured embryos showed an absence or slight fragmentation as compared with 72 and 61% respectively for embryos grown in medium alone (P less than 0.01). None of the co-cultured embryos showed unequal-sized blastomeres while 22% of controls showed unequal cleavage. Embryos grown with ampullary cells cleaved slightly faster than controls. Scanning electron micrographs showed that ampullary cells collected from co-cultures were all of the secretory type with several microvilli and apical protrusions. It is clear that subcultured human ampullary cells support human embryonic cleavage and yield a reasonable number of good quality embryos up to the cavitation stage. Development past the expanded blastocyst and hatching stages seems to involve another critical phase with its own specific requirements.
Critical examination of 30 blastocysts by transmission electron microscopy at various stages of blastulation and hatching, has revealed the presence of specialized, plump, trophoblastic cells at the points of hatching, which seem to aid in initial breaking of the zona pellucida (ZP) and then widen its opening to permit the progressive emergence of the embryo in amoeboid fashion, when it acquires a characteristic dumb-bell shape. These cells are named 'zona-breaker' cells and their characteristics are described. Normally, trophoblast cells in expanding blastocysts are flattened (squamous), forming a continuous robust epithelium with specialized cell junctions. Bundles of tonofilaments anchor onto desmosomes, forming a terminal web. Proper expansion of blastocysts by intake of fluid into the blastocoele causes an increase in internal hydrostatic pressure that stretches the trophoblast epithelium leading to an enlargement of its volume two- to three-fold, consequently thinning the zona prior to hatching. This is an important prerequisite to normal hatching. The blastocysts usually hatch out at the pole opposite the inner cell mass (ICM), though a few hatch out at the embryonal pole or elsewhere. In all cases zona-breakers seem to play a vital role in the hatching process.
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