Factors affecting the survival,fertilization, and embryonic development of mouse oocytes after vitrification using glass capillaries |
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Authors: | Xiuwen Tan Enliang Song Xiaomu Liu Wei You Fachun Wan |
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Institution: | (1) Shandong Key Lab of Animal Disease Control and Breeding, Institute of Animal Husbandry and Veterinary Medicine, Shandong Academy of Agriculture Sciences, Jinan, Shandong, 250100, China |
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Abstract: | Cryopreservation of mammalian oocytes is an important way to provide a steady source of materials for research and practice
of parthenogenetic activation, in vitro fertilization, and nuclear transfer. However, oocytes cryopreservation has not been
common used, as there still are some problems waiting to be solved on the repeatability, safety, and validity. Then, it is
necessary to investigate the damage occurred from vitrification and find a way to avoid or repair it. In this study, mouse
mature oocytes were firstly pretreated in different equilibrium media, such as 5% ethylene glycol (EG) + 5% dimethyl sulfoxide
(DMSO), 10% EG + 10% DMSO, and 15% EG + 15% DMSO in TCM199 supplemented with 20% fetal calf serum (FCS), for 1, 3, and 5 min,
respectively, and then oocytes were transferred into vitrification solution (20% EG, 20% DMSO, 0.3 M sucrose, and 20% FCS
in TCM199, M2, Dulbecco’s phosphate buffered saline, and 0.9% saline medium, respectively) and immediately loaded into glass
capillaries to be plunged into liquid nitrogen. After storage from 1 h to 1 wk, they were diluted in stepwise sucrose solutions.
The surviving oocytes were stained for cortical granule, meiotic spindles, and chromosomes. Oocytes without treatments were
used as controls. The results showed that oocytes pretreated in 5% EG +5% DMSO group for 3–5 min or in 10% EG + 10% DMSO group
for 1–3 min were better than other treatments. Oocytes vitrified in TCM199 as basic medium showed higher survival and better
subsequent embryonic development than other groups. When the concentration of FCS in vitrification solution reduced below
15%, the rates of survival, fertilization, and developing to blastocyst declined dramatically. The inner diameter (0.6 mm)
of glass capillaries and amount of vitrification solution (1–3 μl) achieved more rapid cooling and warming and so reduce the
injury to oocytes. Cropreservation led to the exocytosis of cortical granule of oocytes (about 10%) and serious disturbance
of microtubules and chromosomes. With 2 h incubation, the microtubules could repolymerize and the rate of fertilization in
vitro was much higher than those of 1 and 3 h incubation groups. In conclusion, the protection of basic medium and FCS to
oocytes during cryopreservation and sufficient cooling and warming rates using glass capillaries have profound effects on
oocytes survival and subsequent embryonic development competence. The appropriate time for fertilization in vitro may be related
to the recovery of spindles after incubation and avoiding ageing in the whole process. |
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Keywords: | |
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