"Human metaphase II (MII) oocytes were cryopreserved by slow freezing through a two-step propanediol (PrOH) dehydration protocol (0.75-1.5 mol\/l PrOH, 0.2 mol\/l sucrose). At thawing, oocytes were sequentially transferred in three different thawing solutions (A: 1.0 mol\/l PrOH+0.3 mol\/l sucrose; B: 0.5 mol\/l PrOH+0.3 mol\/l sucrose; C: 0.3 mol\/l sucrose) and randomly fixed after thawing only in sol. A (group A), A+B (group B), A+B+C (group C). . Fresh MII oocytes were used as controls (C). Samples were then prepared for light and transmission electron (LM and TEM) microscopy, to determine the stage of freeze-thawing where may occur: a) vacuolization; b) a premature cortical granule (CG) exocytosis; c) the formation of organelle-specific associations, as mitochondria-SER (M-SER) aggregation or mitochondria-vesicle (M-V) complexes. By LM, both fresh and cryopreserved oocytes were rounded, about 100 µm in diameter, provided with a homogenous ooplasm and surrounded by a continuous zona pellucida. Vacuoles (V) were only occasionally detected in the ooplasm of C. On the contrary, a slight to moderate vacuolization was found in slow-frozen oocytes. Morphometric analysis revealed that the mean number±standard deviation (SD) of V larger than 0.5 µm in diameter\/100 µm2 was 1.05±0.01 (C), 7.2±0.01 (group A), 17.043±0.1 (group B), 9.5±0.01 (group C). By TEM, V appeared electron-negative and enclosed by a membrane often interrupted. CGs were scarce in groups A-C, respect to those found in C, and their number decreased as thawing progressed. In detail, the mean number±SD of CGs in 10 µm was 9.50±0.44 (C), 6.38±1.15 (group A), 3.17±0.29 (group B), 2.33±0.58 (group C). M-SER aggregates appeared large and with a spherical shape. They significantly decreased in number following slow-freezing. In fact, the number of M-SER in 100 μm2 was 0.87±0.05 in C, 0.203±0.031 in group A, 0.045±0.037 in group B and 41.33 ± 12.86 in group C. Differently to what observed for M-SER aggregates, the number of M-V complexes significantly increased in the ooplasm of oocyte from groups A-C. Specifically, the number of M-V was 0.009±0.007 in C; 3.063±1.244 in group A; 4.44±0.937 in group B and 2.067±0.950 in group C.. This study demontrated that: 1) vacuolization may be a recurrent form of cellular damage during slow-freezing. V start to form during freezing to increase at thawing, particularly during the PrOH step-wise dilution; 2) a premature CG exocytosis progressively occurs during the whole freeze\/thawing procedure; 3) significant variations, of opposite trend, in the number of M-SER aggregates and M-V complexes occurred during the freeze\/thawing thus suggesting a dynamic process of transition between these two forms of organelle-aggregations.. "

Patterns of vacuolization in human metaphase II oocytes after cryopreservation: Ultrastructural aspects

PALMERINI, MARIA GRAZIA;MACCHIARELLI, GUIDO;
2012-01-01

Abstract

"Human metaphase II (MII) oocytes were cryopreserved by slow freezing through a two-step propanediol (PrOH) dehydration protocol (0.75-1.5 mol\/l PrOH, 0.2 mol\/l sucrose). At thawing, oocytes were sequentially transferred in three different thawing solutions (A: 1.0 mol\/l PrOH+0.3 mol\/l sucrose; B: 0.5 mol\/l PrOH+0.3 mol\/l sucrose; C: 0.3 mol\/l sucrose) and randomly fixed after thawing only in sol. A (group A), A+B (group B), A+B+C (group C). . Fresh MII oocytes were used as controls (C). Samples were then prepared for light and transmission electron (LM and TEM) microscopy, to determine the stage of freeze-thawing where may occur: a) vacuolization; b) a premature cortical granule (CG) exocytosis; c) the formation of organelle-specific associations, as mitochondria-SER (M-SER) aggregation or mitochondria-vesicle (M-V) complexes. By LM, both fresh and cryopreserved oocytes were rounded, about 100 µm in diameter, provided with a homogenous ooplasm and surrounded by a continuous zona pellucida. Vacuoles (V) were only occasionally detected in the ooplasm of C. On the contrary, a slight to moderate vacuolization was found in slow-frozen oocytes. Morphometric analysis revealed that the mean number±standard deviation (SD) of V larger than 0.5 µm in diameter\/100 µm2 was 1.05±0.01 (C), 7.2±0.01 (group A), 17.043±0.1 (group B), 9.5±0.01 (group C). By TEM, V appeared electron-negative and enclosed by a membrane often interrupted. CGs were scarce in groups A-C, respect to those found in C, and their number decreased as thawing progressed. In detail, the mean number±SD of CGs in 10 µm was 9.50±0.44 (C), 6.38±1.15 (group A), 3.17±0.29 (group B), 2.33±0.58 (group C). M-SER aggregates appeared large and with a spherical shape. They significantly decreased in number following slow-freezing. In fact, the number of M-SER in 100 μm2 was 0.87±0.05 in C, 0.203±0.031 in group A, 0.045±0.037 in group B and 41.33 ± 12.86 in group C. Differently to what observed for M-SER aggregates, the number of M-V complexes significantly increased in the ooplasm of oocyte from groups A-C. Specifically, the number of M-V was 0.009±0.007 in C; 3.063±1.244 in group A; 4.44±0.937 in group B and 2.067±0.950 in group C.. This study demontrated that: 1) vacuolization may be a recurrent form of cellular damage during slow-freezing. V start to form during freezing to increase at thawing, particularly during the PrOH step-wise dilution; 2) a premature CG exocytosis progressively occurs during the whole freeze\/thawing procedure; 3) significant variations, of opposite trend, in the number of M-SER aggregates and M-V complexes occurred during the freeze\/thawing thus suggesting a dynamic process of transition between these two forms of organelle-aggregations.. "
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11697/89065
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