Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (12): 2183-2192.doi: 10.3864/j.issn.0578-1752.2019.12.015

• FOOD SCIENCE AND ENGINEERING • Previous Articles    

Expression of p66Shc and Its Relationship with Cytoplasmic Redox Homeostasis in Sheep Oocytes

ZHANG Tong1,2,LI RuiLan1,2,FAN XiaoMei1,3,LIU ChunJie1,HAI RiHan1,HUO Min1,ZHANG JiaXin1()   

  1. 1 Inner Mongolia Autonomous Region Key Laboratory of Animal Genetics, Breeding and Reproduction/College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018
    2 College of Medicine, Shanxi Datong University, Datong, 037009 Shanxi
    3 Basic Medical College, Inner Mongolia Medical University, Hohhot 010110
  • Accepted:2019-04-17 Online:2019-06-16 Published:2019-06-22
  • Contact: JiaXin ZHANG E-mail:zjxcau@163.com

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Abstract:

【Objective】The aim of this study was to investigate the expression characteristics of p66Shc and its relationship with cytoplasm redox homeostasis in different quality sheep oocytes before and after maturation, which could provide a theoretical basis for revealing the molecular mechanism of p66Shc involved in the regulation of cytoplasmic redox in oocytes.【Method】The sheep cumulus-oocyte complexes (COCs) of ovaries used in this experiment were derived from the slaughterhouse. High quality and poor quality immature oocytes, conventional 24 h matured oocytes and aged oocytes (matured 30 h) were collected, respectively. The expression of p66Shc in different quality sheep oocytes before and after maturation was analyzed by quantitative real-time PCR. The p66Shc protein and mitochondria were co-located by using cellular immunofluorescence combined with MitoTracker Red probe. Simultaneously, the ROS level and the redox homeostasis of different quality oocytes were detected by fluorescence probe DCFH-DA and oocyte spontaneous fluorescence, respectively. In addition, exogenous H2O2-induced oxidative stress was used to treat high quality immature oocytes, and the expression and localization of p66Shc protein were detected and analyzed.【Result】The real-time fluorescent quantitative results showed that the expression of p66Shc mRNA in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes (P<0.05). However, there was no significant difference in the expression of p66Shc mRNA in high-quality oocytes before and after maturation (P>0.05). The co-localization results showed that the regional distribution of p66 protein was consistent with active mitochondrial distribution. The cellular immunofluorescence results showed that the expression of p66Shc protein in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes (P<0.05). Compared with high quality immature oocytes and conventional 24 h matured oocytes, poor quality immature oocytes and aged oocytes showed disordered mitochondrial distribution, decreased activity, increased ROS levels, and imbalance redox homeostasis. In addition, compared with the control group without added exogenous H2O2, exogenous H2O2-induced oxidative stress by treating high-quality immature oocytes significantly (P<0.05) upregulated the expression of p66Shc protein and induced p66Shc from the cytoplasm to the nucleus. 【Conclusion】 The results indicated that the p66Shc gene exhibited high levels of expression in poor quality immature oocytes and aged oocyte, while H2O2-induced oxidative stress significantly up-regulated the expression of p66Shc protein and affected its subcellular localization. In conclusion, the elevated expression of p66Shc perturbed cytoplasmic redox homeostasis in sheep oocytes.

Key words: sheep oocytes, p66Shc, mitochondria, ROS, redox homeostasis

Fig. 1

Different quality sheep oocytes before and after maturationa: high quality immature oocytes; b: poor quality immature oocytes; c: conventional 24 h matured oocytes; d: matured 30 h (aged oocytes)"

Table 1

The oligonucleotide primer for p66Shc and β-actin"

基因
Gene		 基因ID
Gene ID		 退火温度
Annealing temperature (℃)		 引物序列（5'-3'）
The sequence of primer		 产物长度
Product length (bp)
p66Shc 101113548 60 F: CGGGGTTTCCTACTTGGTT
R: ACGGCTACAGGGCTTTCTC		 169
β-actin 443052 60 F: GTCATCACCATCGGCAATGA
R: CGTGAATGCCGCAGGATT		 88

Fig. 2

The relative expression of p66Shc in different quality oocytes before and after maturationThe different superscripts mean significant difference (P<0.05), and the same superscripts mean no significant difference (P>0.05)"

Fig. 3

Co-localization of p66Shc protein and mitochondria in different quality oocytes before and after maturationA: High quality immature oocytes; B: Poor quality immature oocytes; C: Negative control (immature oocytes); D: Conventional 24 h matured oocytes; E: Aged oocytes; F: p66Shc protein fluorescence intensity. Green labeled p66Shc, red labeled mitochondrial, blue labeled nuclear, the scale bar represents 30 μm. The different superscripts mean significant difference (P<0.05), and the same superscripts mean no significant difference (P>0.05)"

Fig. 4

ROS levels of different quality oocytes before and after maturationThe different superscripts mean significant difference (P<0.05), and the same superscripts mean no significant difference(P>0.05)"

Fig. 5

Redox homeostasis of different quality oocytes before and after maturationFAD ++ / NAD (P) H fluorescence ratio. The different superscripts mean significant difference (P<0.05), and the same superscripts mean no significant difference (P>0.05)"

Fig. 6

Hydrogen peroxide induced oxidative stress upregulated the expression of p66ShcA: ROS level; B: FAD ++ / NAD (P) H fluorescence ratio; C1、C2: Control group (30 min or 1 h); C3、C4: treatment group (100 μM H2O2 treatment 30 min or 1 h, respectively); Green labeled p66Shc, blue labeled nuclear, white arrows point to the nucleus, The scale bar represents 30 μm. The different superscripts mean significant difference (P<0.05), and the same superscripts mean no significant difference (P>0.05)"

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