Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (11): 2285-2296.doi: 10.3864/j.issn.0578-1752.2020.11.014

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

RNA-Binding Motif Protein 3(RBM3) Expression is Regulated by Insulin-Like Growth Factor (IGF-1) for Protecting Yak (Bos grunniens) Cumulus Cells from Apoptosis During Hypothermia Stress

PAN YangYang1,WANG Meng1,RUI Xian2,WANG LiBin1,HE HongHong1,WANG JingLei1,MA Rui1,XU GengQuan1,CUI Yan1,FAN JiangFeng1,YU SiJiu1()   

  1. 1College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070
    2Lanzhou Biological Pharmaceutical Factory, China Animal Husbandry Industry Co., LTD, Lanzhou 730046
  • Received:2019-05-05 Accepted:2019-10-28 Online:2020-06-01 Published:2020-06-09
  • Contact: SiJiu YU E-mail:sjyu@163.com

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Abstract:

【Objective】 The aim of this study was to verify the relationship between insulin-like growth factor 1 (insulin-like growth factor, IGF-1) and RNA binding motif protein 3 (RBM3) during hypothermia stress and cell freezing in animals, and to investigate the mechanism of IGF-1 involved in inhibiting hypothermia injury of mammalian cells. 【Method】 Yak (Bos grunniens) cumulus cells were cultured in vitro, the effects of different concentrations (0, 50, 100 and 200 ng·mL -1) IGF-1 and hypothermia stress (25℃ and 30℃) on RBM3 expression in yak cumulus cells were detected by the methods of real-time fluorescence quantitative PCR (qRT-PCR), Western blot (WB) and immunofluorescence. The expression of RBM3 was compared when the cumulus cells treated with 100 ng·mL -1 and 0 IGF-1 for 30 h and then stressed at low temperature (25℃ and 30℃) for 8 h. The difference of apoptosis level of cumulus cells was evaluated from the groups that treated with 0 ng·mL -1 IGF-1, 100 ng·mL -1IGF-1, 100 ng·mL -1 IGF-1+RBM3 inhibitor for 30 h and then stressed at 25℃ for 8 h. The expression of apoptosis-related genes (Bax and Bcl-2) in cumulus cells from three groups was also detected at mRNA and protein levels. 【Result】 (1) The expression level of RBM3 in cumulus cells treated with IGF-1 was significantly higher than that in control group (0 ng·mL -1), which was the highest in cumulus cells from 100 ng·mL -1 IGF-1 group. The RBM3 protein could be detected in nucleus and cytoplasm of cumulus cells, which only expressed in cytoplasm under 0 and 50 ng·mL -1 treatment groups. (2) The levels of RBM3 could be increased by hypothermia stress, and which also could be enhanced when 100 ng·mL -1 IGF-1 was added before cold stress. The RBM3 protein could be detected in nucleus and cytoplasm of cumulus cells after hypothermia stress and treated with IGF-1. (3) After stress at 25℃ for 8 h, the apoptosis rate of cumulus cells treated with 100 ng·mL -1 IGF-1 was (15.94 ±2.03)%, which was significantly lower than those of cumulus cells without IGF-1 treatment (25.86 ±1.09)% or with 100 ng·mL -1 IGF-1+RBM3 inhibitor (20.14±2.65)%. The expression level of Bcl-2 was significantly higher than those in the control and IGF-1+RBM3 inhibitor groups (P<0.05), while the expression level of Bax was significantly lower than control and IGF-1+RBM3 inhibitor groups (P<0.05). 【Conclusion】RBM3 was involved in the regulation of hypothermia stress in yak cumulus cells, and IGF-1 could regulate its expression level in hypothermia stress, which helped to reducing the apoptosis of cumulus cells induced by low temperature. The results in this study provided the key information for revealing the molecular mechanism of IGF-1 and RBM3 involved in the protection of animal bodies or cells from hypothermia damage, and provided theoretical basis for the improvement of somatic and germ cell freezing techniques.

Key words: yak, RNA-binding motif protein 3(RBM3), insulin-like growth factor (IGF-1), hypothermia stress, apoptosis

Table 1

The information of primers used in Real-time PCR"

基因
Gene		 引物序列
Primer sequence		 温度
Tm (℃)		 产物大小
Product size (bp)		 GenBank 登录号
GenBank accession No.
RBM3 F:GCTTCATCACCTTCACCAATC
R:GGTATGACAATCGACCTGGAGC		 60 220 MF142258.1
Bax F:TTTGCTTCAGGGTTTCATC
R:CAGCTGCGATCATCCTCT		 59 174 NM173894.1
Bcl-2 F:CTGCACCTGACGCCCTTCAC
R:GCGTCCCAGCCTCCGTTGT		 62 236 NM001166486.1
β-actin F:CTTCAACACCCCTGCCAT
R:CTCGGCTGTGGTGGTGAAG		 60 238 JF830811

Fig. 1

Effect of different concentration of IGF-1 and CAPE on the levels of RBM3 in yak cumulus A: Effect of different concentration of IGF-1 and CAPE on mRNA levels of RBM3 in yak cumulus; B: The detection of RBM3 protein in yak cumulus cells in different groups; C: Effect of different concentration of IGF-1 and CAPE on protein levels of RBM3 in yak cumulus; Different superscripts mean significantly different (P<0.05). The same below"

Fig. 2

Effect of different of temperature on the levels of RBM3 in yak cumulus A: Effect of different of temperature on mRNA levels of RBM3 in yak cumulus; B: The detection of RBM3 protein in yak cumulus cells in different groups; C: Effect of different of temperature on protein levels of RBM3 in yak cumulus"

Fig. 3

Effect of different of temperature on the levels of RBM3 in yak cumulus treated with 100 ng·mL-1 IGF-1 A: Effect of different of temperature on mRNA levels of RBM3 in yak cumulus treated with 100 ng·mL-1 IGF-1; B: The detection of RBM3 protein in yak cumulus cells in different groups; C: Effect of different of temperature on protein levels of RBM3 in yak cumulus treated with 100 ng·mL-1 IGF-1"

Fig. 4

Compared RBM3 levels in yak cumulus stressed at different of temperature and treated with different concentration IGF-1 A: Compared RBM3 mRNA levels in yak cumulus stressed at different of temperature and treated with different concentration IGF-1; B: Detection of RBM3 protein in yak cumulus stressed at different of temperature and treated with different concentration IGF-1; C: Compared RBM3 protein levels in yak cumulus stressed at different of temperature and treated with different concentration IGF-1"

Fig. 5

Immunofluorescence detection of RBM3 protein in yak cumulus treated with different concentration IGF-1 The HSP70 protein was stained with green fluorescence; Blue fluorescence is the DAPI for nucleus labeling"

Fig. 6

Immunofluorescence detection of RBM3 protein in yak cumulus treated with different concentration IGF-1 and stressed at 25℃ for 8 h The RBM3 protein was stained with green fluorescence; Blue fluorescence is the DAPI for nucleus labeling"

Fig. 7

Detection the apoptosis ratio of yak cumulus cells in different groups A: Yak cumulus at 37℃; B: Yak cumulus stressed at 30℃ for 8 h; C: Yak cumulus stressed at 25℃ for 8 h; D: Yak cumulus treated with 100 ng·mL-1 IGF-1 and stressed at 25℃ for 8 h; E: Yak cumulus treated with 100 ng·mL-1 IGF-1+CAPE and stressed at 25℃ for 8 h"

Table 2

Apoptosis rates of yak cumulus cells in different groups"

组别 Groups 细胞凋亡率 Rate of apoptosis (%)
37℃ 7.36±0.65d
30℃ 16.23±2.81c
25℃ 25.86±1.09a
100 ng·mL-1 IGF-1 + 25℃ 15.94±2.03c
100 ng·mL-1 IGF-1+CAPE+ 25℃ 20.14±2.65b

Fig. 8

Bcl-2 and Bax mRNA levels in yak cumulus from different groups and stressed at 25℃ for 8 h A: Bcl-2 mRNA levels in yak cumulus; B: Bax mRNA levels; C: Ratio of Bcl-2 to Bax mRNA"

Fig. 9

Bcl-2 and Bax protein levels in yak cumulus from different groups and stressed at 25℃ for 8 h A: Detection of Bcl-2 and Bax protein levels in yak cumulus treated with different concentration IGF-1, CAPE and stressed at 25℃ for 8 h; B: Bcl-2 protein levels; C: Bax protein levels; D: Ratio of Bcl-2 to Bax"

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