Abstract
[Ca2+]i is essential for mammalian oocyte maturation and early embryonic development, as those processes are Ca2+ dependent. In the present study, we investigated the effect of [Ca2+]i on in vitro maturation and reprogramming of oocytes in a lower calcium model of oocyte at metaphase II (MII) stage, which was established by adding cell-permeant Ca2+ chelator BAPTA-AM to the maturation medium. Results showed that the extrusion of the first polar body (PB1) was delayed, and oocyte cytoplasmic maturation, including mitochondrial and endoplasmic reticulum distribution, was impaired in lower calcium model. The low-calcium-model oocytes presented a poor developmental phenotype of somatic cell nuclear transfer (SCNT) embryos at the beginning of activation of zygotic genome. At the same time, oxidative stress and apoptosis were observed in the low-calcium-model oocytes; subsequently, an RNA-seq analysis of the lower-calcium-model oocytes screened 24 genes responsible for the poor oocyte reprogramming, and six genes (ID1, SOX2, DPPA3, ASF1A, MSL3, and KDM6B) were identified by quantitative PCR. Analyzing the expression of these genes is helpful to elucidate the mechanisms of [Ca2+]i regulating oocyte reprogramming. The most significant difference gene in this enriched item was ID1. Our results showed that the low calcium might give rise to oxidative stress and apoptosis, resulting in impaired maturation of bovine oocytes and possibly affecting subsequent reprogramming ability through the reduction of ID1.
Highlights
Somatic cell nuclear transfer (SCNT) is of much current interest with potential applications in the protection of endangered species, agriculture, and regenerative medicine (Wu and Hochedlinger, 2011; Tachibana et al, 2013; Iqbal et al, 2021; Son et al, 2021)
Results showed that the [Ca2+]i level was significantly decreased at 0, 8, 12, and 24 h after BAPTA-AM treatment when compared with the control group (0 h: control group 2,075.27 ± 46.09, oocyte number n = 70 vs. treatment group 1,836.79 ± 27.78, n = 80, P < 0.001; 8 h: control group 2,804.34 ± 69.74, n = 60 vs. treatment group 1,760.33 ± 72.56, n = 60, P < 0.001; 12 h: control group 4,243.22 ± 96.26, n = 80 vs. treatment group 2,685.96 ± 97.75, n = 80, P < 0.001; 24 h: control group 2,200.49 ± 52.63, n = 70 vs. treatment group 1,608.42 ± 41.56, n = 80, P < 0.001; Figure 1B)
The intracellular [Ca2+]i level of 16.5 h was significantly lower in the BAPTA-AM treatment group when compared with the control group. These results indicated that intracellular calcium ion chelating agent BAPTA-AM can successfully establish an metaphase II (MII) low-calcium model of bovine oocytes
Summary
Somatic cell nuclear transfer (SCNT) is of much current interest with potential applications in the protection of endangered species, agriculture, and regenerative medicine (Wu and Hochedlinger, 2011; Tachibana et al, 2013; Iqbal et al, 2021; Son et al, 2021). Studies have shown that BAPTA-AM can regulate the fertilization and developmental ability of vitrified bovine oocytes and improve the survival rate and in vitro maturation (IVM) rate of porcine vitrified GV oocytes, so it can be used to explore the effects of Ca2+ on bovine oocyte maturation and SCNT embryo development
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