Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (8): 1667-1675.doi: 10.3864/j.issn.0578-1752.2022.08.016

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

Effects of Soy Isoflavones on the Proliferation and Apoptosis of Yak Ovarian Granulosa Cells

WANG JiaMin1,3,SHI JiaChen1,2,MA FangFang1,2,CAI Yong4,QIAO ZiLin1,3()   

  1. 1Gansu Tech Innovation Center of Animal Cell, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030
    2College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030
    3Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030
    4Department of Experiment & Teaching, Northwest Minzu University, Lanzhou 730030
  • Received:2021-02-19 Accepted:2021-07-27 Online:2022-04-16 Published:2022-05-11
  • Contact: ZiLin QIAO E-mail:87032164@qq.com;670267497@qq.com

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Abstract:

【Background】Soybean isoflavones, mainly including genistein and daidzein, could exert estrogen-like effects, scavenge free radicals and promote cell proliferation. Granulosa cells are an important cell population in follicles and its physiological state is directly related to ovarian function, and the apoptosis of granulosa cells causes oocyte atresia. Yak (Bos grunniens) is one of the endemic species in Qinghai-Tibet plateau, but its reproductive rate is so low, however, the mechanism is still unclear. Ovary granulosa cell is an ideal model to study the regulating mechanism of female animal reproduction. 【Objective】The aim this study was to investigate the effects of soybean isoflavones on the proliferation and apoptosis of granulosa cells of yak ovary, and to provide evidence for the mechanism of soybean isoflavones.【Method】The yak ovarian granulosa cells were isolated and cultured. Immunohistochemistry staining was used to check FSHR for ovarian granulosa cell authenticating. MTT assay was used to detect cell proliferation, then the growth curve was drawn. Ovarian granulosa cells treated with different concentration of genistein and daidzein (0, 1 000, 2 000, 3 000, 4 000 and 5 000 pg·mL-1), the optimal concentration of genistein or daidzein for was selected to treat the second-generation granulosa cells for 48 h, then, the cell culture medium was collected and used to detect the concentration of estradiol and progesterone secreted by granulosa cells by ELISA. At the same time, the cells were collected to extract total RNA and to research the expression of proliferation-related gene AKT1 and apoptosis-related genes Bcl-2, Bax, Bad, Fas, FasL, Caspase-3 and p53 by qRT-PCR.【Result】The yak ovarian granulosa cells were typical epithelial-like cells. After inoculated for 2 h, the granulosa cells began to adhere to the wall. After 12 h, the cells appeared aggregation and growth. After 24 h, the cells were larger and showed long fusiform, star-shaped or polygonal. After cultured for 48 h, the cells looked like paving appearance. Immunohistochemistry staining showed FSHR positive indicated that the cultured cells were ovarian granulosa cells. MTT assay showed that the growth curve of the yak ovarian granulosa cells was S-shape: the growth was slow in 24 h and the cells were in the latent growth stage, and then, which was rapidly proliferated at 24-48 h and entered the exponential growth phase. The granulosa cells, in the plateau phase, steadily proliferated during 48-120 h. After 120 h, the density of living cells began to decline, and the cells entered the phase of degeneration. The second-generation of granulosa cells grew faster and the exponential proliferation phase at 24 h, so the second-generation of granulosa cells was selected for this experiment. MTT assay showed that treated with 3 000 pg·mL-1 genistein or daidzein for 48h, the cell viability were significantly promoted (P<0.01), the secretion of estradiol were induced treated with daidzein, but the progesterone secretion were markedly inhibited treated with genistein. The results of qRT-PCR showed that 3 000 pg·mL-1 genistein or daidzein significantly up-regulated the expression of AKT1, Bcl-2, Bad, p53, Fas and Bcl-2/Bax (P<0.01), down-regulated the expression of Bad and FasL. In addition, genistein significantly down-regulated the expression of Caspase-3 and daidzein significantly up-regulated it (P<0.01). 【Conclusion】The yak ovarian granulosa cells were isolated and cultured to provide an effective cell model for further study on the yak female reproduction regulating mechanism. The results indicated that genistein and daidzein inhibited the progesterone secretion of yak ovarian granulosa cells, promoted cell proliferation, and protected cells from apoptosis by up-regulating Bcl-2, p53, Fas and Bcl-2/Bax and down-regulating Bad and FasL.

Key words: genistein, daidzein, yak, ovarian granulosa cells, proliferation, apoptosis

Table 1

The information of primers used in Real-time PCR"

基因 Gene GenBank登录号 GenBank accession No. 引物序列 Primer sequence 产物长度 Product size (bp)
β-actin NM_173979.3 F：AGGCTGTGCTGTCCCTGTATG
R：GCTCGGCTGTGGTGGTAAA		 207
AKT1 XM_024981593.1 F：GCGTGACCATGAATGAGTTT
R：AACGGGTGCCGGGAGTTCTG		 196
Bcl-2 NM_001166486.1 F：CTGCACCTGACGCCCTTCAC
R：GCGTCCCAGCCTCCGTTGT		 236
Bax XM_015458140.2 F：TTTGCTTCAGGGTTTCATC
R：CAGCTGCGATCATCCTCT		 173
Bad XM_024987322.1 F：GGAGTCGTCACAGCTCCTACC
R：GTGAAACTCGTCGCTCATCCT		 161
p53 XM_010815982.3 F：CCTCCCAGAAGACCTACCCT
R：CTCCGTCATGTGCTCCAACT		 221
Fas NM_174662.2 F：GGGATCTGGGTTCACTTGTC
R：ACTGGTGCTCACGATACAGG		 157
FasL NM_001098859.2 F：GCATACAGCATCATCTTTGGAG
R：AGGAACTGTAGACCTGCTGGAC		 642
Caspase-3 NM_004346.4 F：TTCTTGTACGCATATTCTAC
R：TTCTATTGCTACCTTTCG		 162

Fig. 1

Yak ovarian granulosa cells cultured A: Cultured for 24 h ×40; B: Cultured for 48 h ×100; C: FSHR Immuno-histochemical SP staining ×400; D: Growth curve"

Fig. 2

Effects of genistein and daidzein on the proliferation of yak ovarian granulosa cells The different lowercase letters indicate significant differences (P<0.05);The different uppercase letters indicate extremely significant differences (P<0.01). The same as below"

Fig. 3

Effects of genistein and daidzein on the E2 and P4 secretion of the yak ovarian"

Fig. 4

Effects of genistein and daidzein on the proliferation and apoptosis related genes of the yak ovarian granulosa cells"

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