Elsevier

Cryobiology

Volume 76, June 2017, Pages 18-23
Cryobiology

Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes

https://doi.org/10.1016/j.cryobiol.2017.05.001Get rights and content

Abstract

The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes.

The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P < 0.04, P < 0.001, respectively) and provoked increased mRNA FST expression in oocytes matured in vitro compared to immature oocytes (P < 0.01) and those vitrified without calcium (P < 0.004). CTSB mRNA expression was increased in immature oocytes and oocytes vitrified with calcium compare to mature oocytes (P < 0.02). The developmental potential of vitrified oocytes was impaired compared to non-vitrified oocytes, being more evident in oocytes vitrified with calcium.

In summary, vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these 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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