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.
摘要
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 subunit α, 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-IB、FSHR、INHA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PCNA、Bcl-2、Bax、caspase-3、cyclin A1、cyclin D2和cyclin 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-IB、FSHR和INHA来影响卵泡发育和排卵, 从而间接影响山羊的产仔数.
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Acknowledgments
This study was supported by the National Key R&D Program of China (No. 2017YFD0501904), the National Natural Science Foundation of China (No. 31760652), and the Major Projects of Science and Technology in Guizhou Province (QK-major projects 2016-3002), China.
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Zhinan ZHOU and Xiang CHEN conceived and designed the experimental program. Zhinan ZHOU, Min ZHU, Weiwei WANG, and Zheng AO wrote and edited the manuscript. Zhinan ZHOU, Wen TANG, and Lei HONG performed the experiments. Zhinan ZHOU and Jiafu ZHAO revised the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all data in this study and take responsibility for the integrity and security of the data.
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Zhinan ZHOU, Xiang CHEN, Min ZHU, Weiwei WANG, Zheng AO, Jiafu ZHAO, Wen TANG, and Lei HONG declare that they have no conflict of interest.
The animal handling procedures in this study adhered to the Animal Welfare Guidelines of the Animal Protection and Use Committee of Guizhou University, Guiyang, China (approval number: EGZU-2017T010). All animal handling procedures were performed to ensure minimal suffering.
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Zhou, Z., Chen, X., Zhu, M. et al. Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats. J. Zhejiang Univ. Sci. B 22, 893–905 (2021). https://doi.org/10.1631/jzus.B2100366
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DOI: https://doi.org/10.1631/jzus.B2100366