Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes
Introduction
Vitrification was introduced in the mid 80's as an alternative cryopreservation method to the traditional slow-freezing technology [35], [46], [47]. Since then, vitrification of mammalian embryos and oocytes has been the subject of many investigations [11], [20], [27], [41]. However, a limited progress has been made despite an extensive research over the past 20 years as the ability of cryopreserved oocytes to complete embryo development is still very low in many animal species. This might be due to a higher content of fosfolipids in the oocytes of animals than of humans [16], [37], [50]. The percentage of bovine blastocyst developing from in vitro matured (IVM) and vitrified oocytes ranges from 2.4 to 20% and is significantly lower than in the case of non-vitrified and IVM oocytes (22.4–35.6%) [11], [30], [41], [52].
It has also been reported that freezing induces hardening of the zona pellucida of the oocyte. Larman et al. [21] showed that it took six times longer to dissolve zona pellucida by chymotrypsin in mouse oocytes vitrified in the presence of calcium than in non-vitrified control oocytes. However, removing extracellular calcium from the vitrification medium, significantly reduced (by 40%) zona hardening. It has been suggested that during cryopreservation, oocytes might be artificially activated, as the cortical granules are released into cytoplasm [34] and their content fusses into zona pellucida layers prior to actual fertilization inducing zona hardening. Normally, such events as an increase in intracellular calcium and cortical granules membrane fusion are triggered by sperm-egg fusion [18].
On the other hand, there is a limited data on how the vitrification/warming process affects the functioning of genes which are related to oocyte quality and embryo development. It has been shown that follistatin, might be a marker of oocyte quality [38] as the reduced transcript abundance for follistatin in bovine oocytes was associated with a poor developmental competence of the embryos [33]. Applying immunostaining techniques, Balboula et al. [4] has demonstrated an increase in CTSB activity in poor quality IVM oocytes. CTSB influences bovine oocyte developmental competence by regulating apoptosis [3], [4], [5] through indirect activation of caspases via mitochondrial membrane degradation, leading to translocation of apoptosis-initiating components from mitochondria to cytoplasm [7]. Additionally, CTSB is involved in the turnover of proteins and plays various roles in maintaining a normal metabolism of the cells [29].
Therefore, the aim of our study was to determine the effect of vitrification procedure and the presence or absence of calcium in vitrification solution on: 1) the size of the oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) the transcripts level for FST and CTSB in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes.
Section snippets
Oocyte recovery and in vitro maturation (IVM)
Bovine ovaries were collected at a local slaughterhouse and transported to the laboratory in a phosphate buffered saline (PBS) within 1 h at 38 °C. The ovaries were washed several times in PBS supplemented with 50 IU/mL penicillin G (Sigma-Aldrich, St. Louis, USA). The oocytes were aspirated from follicles (2–6 mm in diameter) using 18 G needle attached to a 10 mL syringe. Only cumulus-oocyte complexes (COCs) surrounded by compact multiple layers of cumulus cells were selected for IVM. Groups
Statistical analysis
The results were analyzed using the Statistica 10.0 software (Statsoft, Tulsa, OK, USA and IBM SPS Statistics 21.0, Predictive Solutions). Distribution of data was verified with the Shapiro-Wilk test and the Kolmogorov-Smirnov test with the Lilliefors correction. In view of non-Gaussian distribution of data, the non-parametric Mann-Whitney U test (a two group comparison) and the Kruskal-Wallis test (a three groups comparison) were employed. The Pearson chi-square test was used to determine
Effect of vitrification on the morphology of bovine oocytes
The vitrification/warming process did not change neither the external and internal diameter of oocytes nor the thickness of zona pellucida of bovine immature and mature oocytes (Table 1). Similarly, the presence of calcium in the vitrification solutions did not have any effect on the diameter and thickness of the zona pellucida of survived oocytes compared to non-vitrified oocytes or oocytes vitrified without calcium (Table 1). The overall survival rate of bovine oocytes vitrified without
Discussion
In our study, survival rate of the vitrified oocytes is comparable to results reported earlier for the bovine oocytes regardless of the type of the cryoprotectant used [10], [23], [47], [52]. It was previously demonstrated by Vajta et al. [46] that a small final volume of vitrification solution and direct contact with the liquid nitrogen resulted in a lower percentage of damaged zonas and lysed plasma membranes in bovine oocytes. Similarly, Endoh et al. [12] showed that most of the vitrified
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgments
The study was supported by the statutory fund of the Institute of Animal Reproduction and Food Research of the Polish Academy of Science in Olsztyn. The authors wish to thank to Animal Insemination Center in Bydgoszcz for donating frozen bovine semen, Mr. M. Blitek for his assistance in collecting and transporting bovine ovaries from the slaughterhouse, Mrs. M. Karasim for the technical assistance and Mrs. I. Kieda for the English language assistance.
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2021, CryobiologyCitation Excerpt :When zygotes were vitrified in cryotop and cryolock, the rates of survival, two-cell and blastocyst development were the highest. Although both MII oocytes and zygotes have low surface to volume ratios, the difference in their responses to vitrification in different devices could be due to variations in chilling sensitivity, membrane permeability, cytoplasmic lipid content and cellular structure [1,27]. Another difference is that the DNA of an MII oocyte is suspended in the cytoplasm on the meiotic spindle while that of a zygote is held within the nuclear membrane [27].
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