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On-line Access: 2021-11-16

Received: 2021-04-18

Revision Accepted: 2021-05-27

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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Zhinan ZHOU

https://orcid.org/0000-0001-6078-938X

Xiang CHEN

https://orcid.org/0000-0001-6495-6851

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Journal of Zhejiang University SCIENCE B 2021 Vol.22 No.11 P.893-905

http://doi.org/10.1631/jzus.B2100366


Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats


Author(s):  Zhinan ZHOU, Xiang CHEN, Min ZHU, Weiwei WANG, Zheng AO, Jiafu ZHAO, Wen TANG, Lei HONG

Affiliation(s):  Key Laboratory of Plateau Mountain Animal Genetics, Breeding and Reproduction of Ministry of Education, Key Laboratory of Animal Genetics, Breeding and Reproduction of Guizhou Province, College of Animal Science, Guizhou University, Guiyang 550025, China

Corresponding email(s):   XChen2@gzu.edu.cn

Key Words:  Cathepsin D (CTSD), Litter size trait, Granulosa cells, Cell apoptosis, Cell cycle, Cell proliferation


Zhinan ZHOU, Xiang CHEN, Min ZHU, Weiwei WANG, Zheng AO, Jiafu ZHAO, Wen TANG, Lei HONG. Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats[J]. Journal of Zhejiang University Science B, 2021, 22(11): 893-905.

@article{title="Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats",
author="Zhinan ZHOU, Xiang CHEN, Min ZHU, Weiwei WANG, Zheng AO, Jiafu ZHAO, Wen TANG, Lei HONG",
journal="Journal of Zhejiang University Science B",
volume="22",
number="11",
pages="893-905",
year="2021",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B2100366"
}

%0 Journal Article
%T Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats
%A Zhinan ZHOU
%A Xiang CHEN
%A Min ZHU
%A Weiwei WANG
%A Zheng AO
%A Jiafu ZHAO
%A Wen TANG
%A Lei HONG
%J Journal of Zhejiang University SCIENCE B
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%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2100366

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T1 - Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats
A1 - Zhinan ZHOU
A1 - Xiang CHEN
A1 - Min ZHU
A1 - Weiwei WANG
A1 - Zheng AO
A1 - Jiafu ZHAO
A1 - Wen TANG
A1 - Lei HONG
J0 - Journal of Zhejiang University Science B
VL - 22
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SP - 893
EP - 905
%@ 1673-1581
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PB - Zhejiang University Press & Springer
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DOI - 10.1631/jzus.B2100366


Abstract: 
cathepsin D (CTSD), the major lysosomal aspartic protease that is widely expressed in different tissues, potentially regulates the biological behaviors of various cells. Follicular granulosa cells are responsive to the increase of ovulation number, hence indirectly influencing litter size. However, the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated. This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues. Moreover, western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats. Subsequently, the effects of CTSD knockdown on cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific traits, including bone morphogenetic protein receptor IB (BMPR-IB), follicle-stimulating hormone (FSHR), and inhibin α (INHA), were determined in granulosa cells. Results showed that CTSD was expressed in corpus luteum, follicle, and granulosa cells. Notably, CTSD expression in the monotocous group was significantly higher than that in the polytocous group. In addition, CTSD knockdown could improve granulosa cell proliferation, inhibit cell apoptosis, and significantly elevate the expression of proliferating cell nuclear antigen (PCNA) and B cell lymphoma 2 (Bcl-2), but it lowered the expression of Bcl-2-associated X (Bax) and caspase-3. Furthermore, CTSD knockdown significantly reduced the ratios of cells in the G0/G1 and G2/M phases but substantially increased the ratio of cells in the S phase. The expression levels of cyclin D2 and cyclin E were elevated followed by the obvious decline of cyclin A1 expression. However, the expression levels of BMPR-IB, FSHR, and INHA clearly increased as a result of CTSD knockdown. Hence, our findings demonstrate that CTSD is an important factor affecting the litter size trait in goats by regulating the granulosa cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific trait.

组织蛋白酶D沉默调节颗粒细胞的生物学行为并影响山羊的产仔性状

目的:探究组织蛋白酶D(Cathepsin D,CTSD)对颗粒细胞生物学行为及山羊产羔性状的调控机制。
创新点:以中国贵州省优良地方品种黔北麻羊为对象,以CTSD为候选基因,以卵泡颗粒细胞为模型,首次探明了CTSD沉默对山羊颗粒细胞生物学行为的影响及其对山羊产羔性状的调控机理,对进一步挖掘、创新和利用中国山羊品种资源等方面具有重要意义。
方法:使用免疫组化法对CTSD在卵巢中进行定位分析;应用蛋白质印迹法(western blotting)技术探究CTSD在单、多羔山羊卵巢中的表达差异;使用细胞计数试剂(cell counting kit-8,CCK-8)技术检测CTSD沉默对细胞增殖的影响;使用流氏细胞仪检测CTSD沉默对颗粒细胞凋亡及周期的影响;使用定量逆转录聚合酶链式反应(qRT-PCR)技术探究CTSD沉默对细胞增殖因子(增殖细胞核抗原(proliferating cell nuclear antigen,PCNA))、细胞凋亡标志基因(B细胞白血病/淋巴瘤2(B cell leukemia/lymphoma 2,Bcl-2)、Bcl-2相关X(Bcl-2-associated X,Bax)、半胱天冬酶3(caspase-3))、细胞周期蛋白(细胞周期蛋白A1(cyclin A1)、细胞周期蛋白D2(cyclin D2)、细胞周期蛋白E(cyclin E))及多胎性状候选基因(骨形态发生蛋白受体IB(bone morphogenetic protein receptor, type 1B,BMPR-IB)、促卵泡素受体(follicle stimulating hormone receptor,FSHR)、抑制素α(inhibin subunitα,INHA))表达的影响;使用western blotting检测CTSD敲低对多胎性状候选基因BMPR-IBFSHRINHA在翻译水平表达的影响。
结论:CTSD沉默可能通过一系列因素的表达水平(包括PCNABcl-2Baxcaspase-3cyclin A1cyclin D2cyclin E)来调控颗粒细胞的增殖、凋亡和细胞周期进展,进而影响颗粒细胞的生物学功能。此外,CTSD可能通过调控多产性状的候选基因,包括BMPR-IBFSHRINHA来影响卵泡发育和排卵,从而间接影响山羊的产仔数。

关键词:组织蛋白酶D(CTSD);产羔性状;颗粒细胞;细胞凋亡;细胞周期;细胞增殖

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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