Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (23): 4940-4949.doi: 10.3864/j.issn.0578-1752.2020.23.018

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

Effect of KISS1 Interference on the Function of Porcine Granulosa Cells in Porcine Ovary

LU SiYu1(),HE YingTing1,ZHOU XiaoFeng1,XIN XiaoPing1,ZHANG AiLing2,YUAN XiaoLong1,ZHANG Zhe1,LI JiaQi1()   

  1. 1Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/National Engineering Research Center for Breeding Swine Industry/College of Animal Science, South China Agricultural University, Guangzhou 510642
    2Guangdong Development Center of Applied Ecology and Ecological Engineering in Universities/Biology and Food Engineering Institute,Guangdong University of Education, Guangzhou 510310
  • Received:2019-11-28 Accepted:2020-10-29 Online:2020-12-01 Published:2020-12-09
  • Contact: JiaQi LI E-mail:siyu_lu19@163.com;jqli@scau.edu.cn

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Abstract:

【Background】 As the basic structural and functional unit of ovary, the main functions of follicles are ovulation and hormone secretion. Granulosa cells proliferation can promote the development of follicles, while the excessive apoptosis of granulosa cells will inhibit the development of follicles, induce follicular atresia, reduce the estrus frequency and affect the reproductive capacity of female animals. Recent studies have found that the KISS1 plays an important role in ovary. 【Objective】 This study was aimed to explore the effects of KISS1 on apoptosis, cell cycle and estrogen secretion of porcine granulosa cells, through interfering the expression of KISS1, so as to provide some useful information in molecular regulation mechanism of KISS1 on porcine granulosa cells. 【Method】 To investigate the effect of knockdown KISS1 on the transcription of key genes on phosphatidylinositol 3-kinase (PI3K) signaling pathway in porcine granulosa cells, KISS1-siRNA were designed and transfected to porcine granulosa cells in vitro, and then quantitative real time PCR (qRT PCR) was applied to detect the transcription of KISS1. Then, flow cytometry, Annexin V-FITC, and ELISA were utilized to explore the effects of KISS1-siRNA on granulosa cell cycle, apoptosis, and secretion of E2, respectively. Finally, qRT-PCR was applied to explore the effects of KISS1-siRNA on estrogen receptor and estrogen signaling pathway. 【Result】 In porcine granulosa cells, interfering KISS1 gene could significantly decrease the transcription of AKT1(P<0.05), while PIK3CG, PI3CI and PDK1 decreased with no significant difference (P>0.05). The mRNA levels of the genes, involving in activation of PI3K signaling pathway like FOXO3, TSC2, and BAD, were also decreased with no significant difference (P>0.05). Transfecting KISS1-siRNA to porcine granulosa cells could significantly promote cell apoptosis (P<0.01), arrest cell cycle at G0/G1 phase (P<0.01), and decrease the E2 secretion (P<0.01). After knockdown KISS1, the transcription of estrogen receptor ESR1 and ESR2, as well as Star, CYP17, 3B-HSD, 17B-HSD and CYP19A of estrogen pathway were also decreased significantly (P<0.05). 【Conclusion】 This study confirmed that KISS1 gene could participate in PI3K and estrogen signaling pathways. Interfering KISS1 could promote porcine granulosa cells apoptosis, arrest cell cycle at G0/G1 phase (P<0.01), and decrease the E2 secretion. It suggested that KISS1 played an important role in the division, growth and estrogen secretion of porcine granulosa cells.

Key words: sows, granulosa cells, KISS1, PI3K, E2

Table 1

Quantitative primer"

引物名称 Primer name Gene ID 引物序列 Primer sequence 产物大小 Size (bp)
qRT-KISS1 100145896 F: AACCAGCATCTTCTCACCAGG 192
R: CTTTCTCTCCGCACAACGC
qRT-PIK3CG 396979 F: AACGGGCTTTGAGATAGTGAA 184
R: AAGTTGCTTGGTTGGTGGATA
qRT-PIK3C1 5290 F: CAAGTGAGAATGGTCCGAATG 152
R: GTGGAAGAGTTTGCCTGTTTT
qRT-PDK1 100286871 F: AAATCACCAGGACAGCCAATA 190
R: CTTCTCGGTCACTCATCTTCAC
qRT-AKT1 100126861 F: CTGCACAAACGAGGCGAGT 89
R: CGCTCCTTGTAGCCGATGAA
qRT-FOXO3 733621 F: ACAAACGGCTCACTCTGTCCCA 85
R: GAACTGTTGCTGTCGCCCTTATC
qRT-TSC2 100505408 F: CGAGGTGGTGTCCTACGAGAT 115
R: GAGCAGGCGTTCAATGATGTT
qRT-BAD 100521065 F: AGTCGCCACTGCTCTTACCC 172
R: TCTTGAAGGAACCCTGGAAATC
qRT-ESR1 10013276 F: GATGCCTTGGTCTGGGTGAT 124
R: AGTGTTCCGTGCCCTTGTTA
qRT-ESR2 396697 F: AAGGGAAAAGGAGGATGGGACA 202
R: CAGATAGGGACTGCGTGGAGGT
qRT-Star 396597 F: GGAAAAGACACAGTCATCACCCAT 121
R: CAGCAAGCACACACACGGAAC
qRT-CYP17 100157047 F: AAGCCAAGACGAACGCAGAAAG 228
R: TAGATGGGGCACGATTGAAACC
qRT-3BHSD 445539 F: GGGGCTTCTGTCTTGATTCCA 284
R: GGTTTTCAGTGCTTCCTTGTGC
qRT-17BHSD 397574 F: CCCAACGCAGGAGACTCAAAAT 149
R: CCAGAGCCCATAACGAAGACAGA
qRT-CYP19A 403332 F: GCTGGACACCTCTAACAACCTCTT 91
R: TTGCCATTCATCAAAATAACCCT

Fig. 1

Interference effect of porcine KISS1 in granulosa cells * P<0.05,** P<0.01。下同 The same as below"

Fig. 2

Interfering KISS1 inhibits PI3K signaling pathway A. PI3K signaling pathway activates expression of related genes. B. PI3K signaling pathway inhibits the expression of related genes"

Fig. 3

Interfering KISS1 promotes granulosa cell apoptosis A. Flow cytometry of KISS1-siRNA transfection. B. Flow cytometry of Scrambled-siRNA transfection. C. Interfering KISS1 promotes granulosa cell apoptosis"

Fig. 4

Interfering KISS1 arrests granulosa cells in interphase"

Fig. 5

Interfering KISS1 inhibits granulosa cell secretion of E2"

Fig. 6

Gene transcription level of estrogen secretion related pathway A. Interfering KISS1 inhibits ESR1 and ESR2 expression; B. Interfering KISS1 inhibits the expression of estrogen pathway genes"

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