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. 2022 May 12:10:873095.
doi: 10.3389/fcell.2022.873095. eCollection 2022.

A Novel SNP in the Promoter Region of IGF1 Associated With Yunshang Black Goat Kidding Number via Promoting Transcription Activity by SP1

Kunyu Li  1   2 , Yufang Liu  1   2 , Xiaoyun He  1 , Lin Tao  1 , Yanting Jiang  3 , Rong Lan  3 , Qionghua Hong  3 , Mingxing Chu  1
Affiliations

A Novel SNP in the Promoter Region of IGF1 Associated With Yunshang Black Goat Kidding Number via Promoting Transcription Activity by SP1

Kunyu Li et al. Front Cell Dev Biol. .

Abstract

IGF1, a member of the insulin-like growth factor (IGF) superfamily, is also known as the growth-promoting factor (somatomedin C). IGF1 is involved in vertebrate growth and development, immunity, cell metabolism, reproduction, and breeding. However, there are relatively few studies on the relationship between IGF1 and goat reproduction. In this study, a new transcription factor SP1 bound to the IGF1 g. 64943050T>C promoted granulosa cell (GC) proliferation. A mutation g.64943050T>C located in the promoter region of IGF1 was identified. Association analysis revealed that the kidding number in the first and second litters and the average number of first three litters of the CC genotype (2.206 ± 0.044, 2.254 ± 0.056, and 2.251 ± 0.031) were significantly higher than those in the TC genotype (1.832 ± 0.049, 1.982 ± 0.06, and 1.921 ± 0.034) and TT genotype (1.860 ± 0.090, 1.968 ± 0.117, and 1.924 ± 0.062) (p < 0.05). The kidding number in the third litter of the CC genotype (2.355 ± 0.057) was significantly higher than that in the TT genotype (2.000 ± 0.107) (p < 0.05). Then, the function of this mutation was validated by the dual-luciferase reporter assay and EMSA. The results showed that the luciferase activity of IGF1-mutant-C was significantly higher than that of IGF1-Wild-T (p < 0.05). The EMSA also showed that the binding ability of IGF1-mutant-C was higher than that of IGF1-Wild-T (p < 0.05). Subsequently, the transcription factor SP1 was predicted to bind to the mutation of IGF1 (g.64943050T>C). Overexpression of SP1 promotes the expression of IGF1 in the primary granulosa cells (GCs). The results of the CCK-8 assay and the expression of GC proliferation factors (CDK4, cyclin D1, and cyclin D2) demonstrated that SP1 promoted GC proliferation by regulating IGF1 expression. Our results suggested that the IGF1 g.64943050T>C was significantly associated with the kidding number of Yunshang black goats, and SP1 as a transcription factor of IGF1 binding to the mutation T>C regulated the expression of IGF1. Furthermore, SP1 promoted goat GC proliferation by regulating the expression of IGF1, which provides a new insight for the goat fertility trait.

Keywords: Goat; IGF1; Sp1; granulosa cell proliferation; kidding number; single-nucleotide polymorphism (SNP).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Detection of the expression of different genotypes of IGF1 in goat ovaries. Bars represent the mean ± SEM of at least three repeats. *p < 0.05; **p < 0.01.
FIGURE 2
FIGURE 2
Effect of mutations on the promoter activity of IGF1. IGF1-Mutant-C represents the mutant type, IGF1-Wild-T represents the wild type, and PGL3-basic represents control. They were transfected into 293T cells. The results are expressed as the mean ± SEM (n = 3) in arbitrary units based on firefly luciferase activity normalized against Renilla luciferase activity. A t-test was conducted using SPSS 25.0 to detect the differences. Bars represent the mean ± SEM of at least three repeats. *p < 0.05; **p < 0.01.
FIGURE 3
FIGURE 3
Binding of different biological probes to ovarian nucleoprotein. (A) Binding of the probe to the nucleoprotein. These different probes were incubated with ovary nuclear extracts, and DNA–protein complexes were visualized by autoradiography. DNA–protein complex: DNA and transcription factor SP1 binding complex. (B) Sample gray value.
FIGURE 4
FIGURE 4
Transcription factor SP1 affects the expression of IGF1. (A) Transfection efficiency of the vector in granular cells. (B) Three SP1 interference efficiency graphs were represented using RT-qPCR to detect the expression level of SP1. (C,D) SP1 overexpression and interference vectors were transfected into goat granulosa cells, and the expression levels of SP1 and IGF1 were detected by RT-qPCR. Bars represent the mean ± SEM of at least three repeats. *p < 0.05, **p < 0.01.
FIGURE 5
FIGURE 5
Transcription factor SP1 regulates the proliferation of granulosa cells. (A) After SP1 was overexpressed, the cell counting kit-8 (CCK-8) was used to determine the proliferation curve of granulosa cells. (B) After interfering with SP1, Cell Counting Kit-8 (CCK-8) was used to determine the proliferation curve of granular cells. (C) Expression abundance of cell proliferation–related genes (CDK4, cyclinD1, and cyclinD2) was detected after overexpression of SP1 by RT-qPCR. (D) Expression abundance of cell proliferation–related genes (CDK4, cyclinD1, and cyclinD2) was detected after interference with SP1 by RT-qPCR. (E) Granulosa cells stained positive for 5-ethynyl-2-deoxyuridine (EdU) were detected with an EdU kit after SP1 overexpression and inhibition, EdU (red), Hoechst (blue). (F,G) Fold change of granulosa cell proliferation rate after SP1 overexpression and inhibition. Bars represent the mean ± SEM of at least three repeats. *p < 0.05; **p < 0.01.
FIGURE 6
FIGURE 6
Flow chart of the IGF1 g.64943050T>C mutation regulating goat reproduction through the transcription factor SP1.
See this image and copyright information in PMC

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