Relationship between bovine oocytes developmental competence and mRNA expression of apoptotic and mitochondrial genes following the change of vitrification temperatures and cryoprotectant concentrations
Introduction
Oocyte cryopreservation has important scientific significance and wide prospects in application. It can provide sufficient experimental materials for in vitro fertilization, transgenosis, and other biotechnological research. In vitrification, the smaller the volume of the cryopreservation carrier, the faster the cooling rate, whereas the higher the cryoprotective agent concentrations (CPAs), the higher the oocyte vitrification success rate [3,10]. When the volume of the cryopreservation carrier is kept constant, the oocyte vitrification success rate can be improved by increasing the cooling rate and CPAs to some extent. The basic principle of vitrification is to prevent the formation of ice crystals by increasing cooling rate or using high CPAs [42]. However, although high CPAs can reduce the formation of ice crystals in freezing, reduce the damage of ice crystals to cells, at the same time, it will also cause some damage to cells due to the osmotic pressure [39] and toxicity of cryoprotectants [13]. Vitrification has such an important characteristic, increasing the cooling rate can reduce the CPAs, thus reducing the damage caused by high CPAs [50]. Therefore, we expect to improve the oocyte vitrification success rate by increasing the cooling rate and appropriately reducing the CPAs. At present, the widely used cryogen of oocyte cryopreservation is liquid nitrogen (LN; −196 °C). The temperature difference between cryogen and vitrified sample determines the cooling rates, the larger the temperature difference, the higher the cooling rate. To our knowledge, liquid helium (LHe) is the lowest vitrification temperature (−269 °C) that can be used as cryogen. In our previous study, LHe instead of LN was used as a cryogen for bovine GV-stage oocyte vitrification. The optimum CPAs could be decreased from 6.6 to 5.6 M. After LN and LHe vitrification, the developmental capacity of oocytes and the mRNA expression levels of several maternal genes were significantly different [51,54]. We hypothesized that these differences were caused by different vitrification temperatures (VTs) and CPAs. Therefore, it is of great significance to study the effects of VT and CPAs on the mRNA expression in bovine mature oocytes after vitrification at an immature stage.
Apoptosis is the gradual regulation of the death of a type of cell through a series of cascade signals, and it plays an important role in regulating growth and development, as well as the immune response; eliminating excess or abnormal cells; and maintaining a constant number of cells and to normally survive [20]. Mitochondria, as a very important place to generate energy for eukaryotic cells, is a place for cellular respiration. The mitochondrial respiratory chain is located on the mitochondrial inner membrane, consisting of complexes I, II, III, and IV, as well as the electron transfer vector and cytochrome C. The function of the mitochondrial respiratory chain is to complete biological oxidation and oxidative phosphorylation and to produce ATP, which is a very important part of oocyte development [5,8]. In our previous experiment, we found the expression level of the apoptotic gene, P53, was higher (P < 0.05) in LN vitrification than in fresh oocyte and LHe vitrification groups, and mitochondrial swelling was observed in LN vitrification [51]. It is necessary to further study the effects of VTs and CPAs on the apoptosis- and mitochondria-related genes. In the previous study, five mitochondria-related differentially expressed genes (DEGs) were screened through RNA-seq, but apoptosis-related DEGs have not been found according to the edgeR program (P ≤ 0.05; fold change ≥ 2) [55]. The DEGs of the four vitrification groups were significantly enriched in oxidative phosphorylation and parkinson's disease pathway based on the previous RNA-seq result analysis. In order to ensure the accuracy of the experiment and fully understand the effects of VTs and CPAs on the mRNA expression of key genes in apoptosis- and mitochondria-related pathways. The 8 apoptotic genes (BAD, BAX, BCL2A1, BID, BTK, CASP3, TP53, and TP53I3), and 12 mitochondrial genes (ATP5A1, COX6B1, DERA, FIS1, ND4, NDUFA1, NDUFA4, POLG2, PRDX2, SLC25A5, TFB1M, and UQCRB) were selected for further study based on the RNA-seq results.
The present study aimed to evaluate the developmental ability of bovine oocytes under the different VTs (−269 °C and −196 °C) and different CPAs (5.6 M and 6.6 M), investigate the mRNA expression levels of 8 apoptotic genes and 12 mitochondrial genes in oocytes under the different VTs and different CPAs, confirm the DEGs related to VTs and CPAs, and preliminarily analyze the metabolic pathways that VTs and CPAs may affect in terms of oocyte development through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these DEGs. Finally, analyze the relationship between oocyte developmental ability and mRNA expression of 8 apoptosis genes and 12 mitochondrial genes following the change of vitrification temperatures and cryoprotectant concentrations.
Section snippets
The processing of oocyte LHe vitrification and LN vitrification, warming, in vitro maturation, in vitro fertilization and in vitro culture of embryo
All the processing were same as Chen [15].
Quantitative real-time reverse transcription PCR
The total cellular RNA was extracted from 20 oocytes in each group (n = 3) using TRIzol reagent (Invitrogen, USA), according to the manufacturer's instructions, and then handled in chloroform, isopropyl alcohol, and 75% alcohol successively. In the final step, it was dissolved in 3.5 μL RNase free water. The cDNA template was amplified using the PrimeScript RT reagent Kit (Takara, Dalian, China) in a 10 μL system: 2 μL of 5 × RT Reaction Buffer, 0.5 μL
Experiment 1: effect of VTs and CPAs on the developmental competence of bovine mature oocytes after vitrification at an immature stage
As shown in Table 2, the blastocyst rate in LHe 5.6 M was the highest among the four vitrified groups (13.7% vs. 9.4%, 1.3%, and 8.4%; P < 0.05). Normal morphology, maturation, cleavage and blastocyst rates in LHe 5.6 M were higher than those in LN 5.6 M (91.6% vs. 53.8%, 56.3% vs. 21.1%, 45.1% vs. 10.9%, 13.7% vs. 1.3%, respectively; P < 0.05). The blastocyst rate in LHe 5.6 M was higher than that in LHe 6.6 M (13.7% vs. 9.4%; P < 0.05). The blastocyst rate in LN 6.6 M were higher than that in
Discussion
The real-time expression of genes during oocyte maturation is very important for late embryonic development. Oocyte freezing is bound to affect its gene expressions and subsequent development ability. Therefore, detecting the changes of key gene mRNA expression levels has become an effective method for cryopreservation damage appraisal [6].
Declaration of competing interest
There are no any commercial or associative interest that represent a conflict of interest in connection with the work submitted.
Acknowledgments
This project was supported by the National Natural Science Foundation of China (Grant Nos. 31872354). Xue Li Yu conceived and designed the experiments. Zhi Yang Zhang and Meng Dan Cai performed the experiments. Yi Heng Liu, Jia Qi Liu, Shi Yu Zhao, Xiao Xia Li and Ying Hua Li contributed reagents, materials, and analytical tools. Zhi Yang Zhang analyzed the data. Zhi Yang Zhang and Xue Li Yu wrote the manuscript.
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