Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (18): 3998-4007.doi: 10.3864/j.issn.0578-1752.2021.18.017

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

Identification of circINHBB During Follicular Atresia and Its Effect on Granulosa Cell Apoptosis

MA MengNan(),WANG HuiMing,WANG MiaoMiao,YAO Wang,ZHANG JinBi,PAN ZengXiang()   

  1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095
  • Received:2020-06-29 Accepted:2021-05-13 Online:2021-09-16 Published:2021-09-26
  • Contact: ZengXiang PAN E-mail:18795975502@163.com;owwa@njau.edu.cn

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Abstract:

【Background】The follicular development is related to the reproductive capacity and production performance of mammals. Follicular development includes the growth and development of oocytes, the initiation and growth of the primordial follicles, and the development of primary follicles. In addition, these biological processes are closely related to the proliferation and apoptosis processes of follicle granulosa cells. The main inducement of follicular atresia is the apoptosis of granulosa cells, which is extremely complex and regulated by various cytokines. Previous studies have shown that non-coding RNAs (ncRNAs) are involved in various biological processes as regulators, including cell proliferation and apoptosis. Circular RNA (circRNA) is a new type of ncRNAs, widely existing in organisms. The circRNA is involved in the regulation of various physiological processes, but there are few studies in the field of livestock reproduction, especially, its expression, distribution and biological function in the pig ovary and follicle are rarely studied. Inhibin (INH) is a gonadal glycoprotein hormone, which is mainly produced by granulosa cells of ovarian follicles in female animals. It is an important factor to control ovulation in mammals. Previous studies have shown that the coding gene of the precursor of INH βsubunit in porcine follicles may form a circular RNA, i.e. circINHBB. 【Objective】In this study, the sequence structure and cell distribution of circINHBB in porcine follicular tissue were verified, and its expression difference in healthy and atresia follicles analyzed to explore the effect of circINHBB on cell apoptosis in porcine granulosa cells cultured in vitro, so as to broaden the research ideas of circRNAs in the field of livestock breeding, and provide reference for improving the reproductive capacity of livestock. 【Method】 Firstly, the porcine follicles were collected, amplified by PCR and sequenced by Sanger sequencing to verify the sequence structure of circINHBB. Secondly, the follicles were divided into two groups, including healthy and atretic, and the expression difference of circINHBB was detected by qRT PCR in each group. Then, the FISH experiment was used to verify the distribution of circINHBB in the granulosa cells. Finally, the porcine ovarian granulosa cells were cultured in vitro, circINHBB was knocked down by siRNA, and the effect of circINHBB on granulosa cell apoptosis was detected by flow cytometry. 【Result】Sanger sequencing confirmed the existence of circINHBB in porcine follicles, and it was a circular RNA formed by reverse splicing of the INHBB mRNA 5’UTR. qRT PCR results confirmed that the expression level of circINHBB was higher in healthy follicles, but significantly decreased in atresia follicles. FISH further verified the distribution of circINHBB in the cytoplasm of porcine granulosa cells. After siRNA knockdown, the expression of circINHBB was significantly decreased, and the apoptosis rate of granulosa cells increased significantly. 【Conclusion】The circular structure of circINHBB was verified in the porcine ovary, and its distribution in cytoplasm suggested that circINHBB might be involved in post-transcriptional regulation. The fact that the expression of circINHBB was lower in atresia follicles, and knockdown of circINHBB could significantly increase the level of apoptosis, indicating that circINHBB had a significant inhibitory effect on the apoptosis of porcine granulosa cells. Thus, it might be an active regulatory factor in the growth and development of follicles. This study explored the role of circRNA in follicular atresia and granulosa cell apoptosis, which was an important supplement to the regulatory mechanism of follicular atresia.

Key words: circRNA, circINHBB, follicular atresia, porcine granulosa cell, apoptosis

Table 1

Judgment criteria for porcine healthy and early atresia follicles"

卵泡类型
Follicle type		 孕酮/雌二醇
P4/E2		 颗粒细胞密度
GC density（cells/µL）		 大小
Size（mm）		 颜色
Colour		 通透度
Clarity		 血管数量
Number of blood vessels
健康卵泡 HF ≤1.3 ≤25000 4-5 红 Red 透亮Transparent 多 Many
早期闭锁卵泡 EA ≥1.8 ≥50000 4-5 黄/白Yellow/White 浑浊 Turbid 少 Less

Table 2

Primer and PCR reaction conditions"

基因 Gene 登录号 Acc.No. 引物(5'- 3') Primer (5'- 3') 产物长度 Product length（bp）
circINHBB AY116585.1 F: AGGGCGAGACGCTCAGGT 223
R: GCACGGGTCGGTTCAGAAG
GAPDH AF017079 F: GGACTCATGACCACGGTCCAT 220
R: TCAGATCCACAACCGACACGT

Fig. 1

Appearance of porcine healthy (H) and early atresia (EA) follicles"

Table 3

Sample information of ovary"

样品编号
Sample No.		 孕酮/雌二醇
P4/E2		 颗粒细胞密度
GC density (cells/μL)		 颜色
Colour		 通透度
Clarity		 血管数量
Number of blood vessels
H1 0.30 22000 红 Red 透亮 Transparent 多 Many
H2 0.66 20250 红 Red 透亮 Transparent 多 Many
H3 0.95 19500 红 Red 透亮 Transparent 多 Many
H4 1.09 19750 红 Red 透亮 Transparent 多 Many
H5 1.22 14000 红 Red 透亮 Transparent 多 Many
H6 1.29 16500 红 Red 透亮 Transparent 多 Many
EA1 1.96 50000 黄 Yellow 浑浊 Turbid 少 Less
EA2 2.13 50000 白 White 浑浊 Turbid 少 Less
EA3 3.72 62500 黄 Yellow 浑浊 Turbid 少 Less
EA4 5.39 62500 黄 Yellow 浑浊 Turbid 少 Less
EA5 8.66 100000 白 White 浑浊 Turbid 少 Less
EA6 16.20 75000 白 White 浑浊 Turbid 少 Less

Fig. 2

Identification of circINHBB (A) Schematic diagram of circRNA primer design; (B) Electrophoresis analysis of circINHBB PCR product; (C) Electropherogram of circINHBB Sanger sequencing and splicing site of circINHBB"

Fig. 3

The localization of circINHA detected by FISH CircINHA was labelled by red fluorescence, Nuclei were stained by DAPI. Scale bar, 50 μm"

Fig. 4

circINHBB expression in healthy and early atrestic follicles ”*” means P<0.05"

Fig. 5

Knockdown of circINHBB promotes apoptosis of porcine granulosa cells A: circINHBB was knockdown by si-circINHBB effectively; b: si-circINHBB had no effect on INHBB mRNA expression; c: si-circINHBB promoted the apoptosis of porcine granulosa cells"

Table 4

circINHBB interacting miRNA"

结合的miRNA
Bound miRNA		 结合类型
Combination type		 自由能
Free Energy		 分数
Fraction
ssc-miR-10391 7mer-m8 -20.4 36
ssc-miR-342 7mer-m8 -16.9 32
ssc-miR-432-5p 7mer-m8 -18.8 38
ssc-miR-744 7mer-m8 -28.0 39
ssc-miR-9810-3p 7mer-m8 -12.4 24
ssc-miR-9824-5p 7mer-m8 -20.8 33
ssc-miR-122-5p 7mer-A1 -14.8 33
ssc-miR-1249 7mer-A1 -16.7 26
ssc-miR-181d-3p 7mer-A1 -13.7 18
ssc-miR-2411 7mer-A1 -12.9 33

Fig. 6

The regulation mechanism that circINHBB may be involved in circINHBB may participate in gene expression regulation by ① competitive splicing with linear mRNA or ② specific binding to miRNAs"

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