Full Text:   <1854>

Summary:  <867>

CLC number: 

On-line Access: 2021-11-16

Received: 2021-04-18

Revision Accepted: 2021-05-27

Crosschecked: 0000-00-00

Cited: 0

Clicked: 2978

Citations:  Bibtex RefMan EndNote GB/T7714


Zhinan ZHOU


Xiang CHEN


-   Go to

Article info.
Open peer comments

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


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",
publisher="Zhejiang University Press & Springer",

%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
%J Journal of Zhejiang University SCIENCE B
%V 22
%N 11
%P 893-905
%@ 1673-1581
%D 2021
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2100366

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
IS - 11
SP - 893
EP - 905
%@ 1673-1581
Y1 - 2021
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B2100366

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.


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。
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。


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


[1]AoZ, WuX, ZhouJ, et al., 2019. Cloned pig fetuses exhibit fatty acid deficiency from impaired placental transport. Mol Reprod Dev, 86(11):1569-1581.

[2]BootsCE, JungheimES, 2015. Inflammation and human ovarian follicular dynamics. Semin Reprod Med, 33(4):270-275.

[3]BowmanLL, KondratevaES, TimofeyevMA, et al., 2018. Temperature gradient affects differentiation of gene expression and SNP allele frequencies in the dominant Lake Baikal zooplankton species. Mol Ecol, 27(11):2544-2559.

[4]BradfordGE, 1972. Genetic control of litter size in sheep. J Reprod Fertil Suppl, 15:23-41.

[5]BrooksS, TylerCR, CarnevaliO, et al., 1997. Molecular characterisation of ovarian cathepsin D in the rainbow trout, Oncorhynchus mykiss. Gene, 201(1-2):45-54.

[6]CaiHF, ChenZ, LuoWX, 2014. Associations between polymorphisms of the GFI1B gene and growth traits of indigenous Chinese goats. Genet Mol Res, 13(1):872-880.

[7]ChenFL, WangN, YangDQ, et al., 2016. Herp depletion arrests the S phase of the cell cycle and increases estradiol synthesis in mouse granulosa cells. J Reprod Dev, 62(2):159-166.

[8]ChouCH, ChenMJ, 2018. The effect of steroid hormones on ovarian follicle development. Vitam Horm, 107:155-175.

[9]CuiZF, LiuLB, ZhaoXL, et al., 2019. Analysis of expression and single nucleotide polymorphisms of INHA gene associated with reproductive traits in chickens. BioMed Res Int, 2019:8572837.

[10]de StasioR, BorrelliL, KilleP, et al., 1999. Isolation, characterization and molecular cloning of cathepsin D from lizard ovary: changes in enzyme activity and mRNA expression throughout ovarian cycle. Mol Reprod Dev, 52(2):126-134.https://doi.‍org/10.1002/(SICI)1098-2795(199902)52:‍2<126::AID-MRD2>3.0.CO;2-O

[11]DubeyV, LuqmanS, 2017. Cathepsin D as a promising target for the discovery of novel anticancer agents. Curr Cancer Drug Targets, 17(5):404-422.

[12]FengX, LiFZ, WangF, et al., 2018. Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep. Biosci Rep, 38(2):BSR20171350.

[13]FranciscoCC, LuisCLJ, MarinaEBJ, et al., 2019. Effect of temperature and pH on the secondary structure and denaturation process of jumbo squid hepatopancreas cathepsin D. Protein Pept Lett, 26(7):532-541.

[14]FuchimotoDI, MizukoshiA, SchultzRM, et al., 2001. Posttranscriptional regulation of cyclin A1 and cyclin A2 during mouse oocyte meiotic maturation and preimplantation development. Biol Reprod, 65(4):986-993.

[15]GeLH, XuYS, XiaWS, et al., 2018. Synergistic action of cathepsin B, L, D and calpain in disassembly and degradation of myofibrillar protein of grass carp. Food Res Int, 109:481-488.

[16]GougeonA, 1996. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev, 17(2):121-155.

[17]GreenfeldCR, BabusJK, FurthPA, et al., 2007. BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries. Reproduction, 133(1):107-116.

[18]GwonSH, KimHK, BaekHJ, et al., 2017. Cathepsin B & D and the survival of early embryos in red spotted grouper, Ephinephelus akaara. Dev Reprod, 21(4):457-466.

[19]HanP, XinHY, PengJY, et al., 2017. Identification and expression of X-linked inhibitor of apoptosis protein during follicular development in goat ovary. Theriogenology, 98:30-35.

[20]HsuehAJW, KawamuraK, ChengY, et al., 2015. Intraovarian control of early folliculogenesis. Endocr Rev, 36(1):1-24.

[21]HuHY, JiaQ, ZhouB, et al., 2020. Comparative analysis of the ovarian transcriptome reveals novel insights into fertility differences in Large White sows. Genes Genomics, 42(7):715-725.

[22]KrancW, BudnaJ, KahanR, et al., 2017. Molecular basis of growth, proliferation, and differentiation of mammalian follicular granulosa cells. J Biol Regul Homeost Agents, 31(1):1-8.

[23]LeyriaJ, FrutteroLL, Ligabue-BraunR, et al., 2018. DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae): purification, bioinformatic analyses and the significance of its interaction with lipophorin in the internalization by developing oocytes. J Insect Physiol, 105:28-39.

[24]LingYH, XiangH, LiYS, et al., 2014. Exploring differentially expressed genes in the ovaries of uniparous and multiparous goats using the RNA-Seq (Quantification) method. Gene, 550(1):148-153.

[25]LiuJH, YangM, JingL, et al., 2018. Silica nanoparticle exposure inducing granulosa cell apoptosis and follicular atresia in female Balb/c mice. Environ Sci Pollut Res, 25(4):3423-3434.

[26]LiuJL, YangL, TianHY, et al., 2016. Cathepsin D is involved in the oxygen and glucose deprivation/reperfusion-induced apoptosis of astrocytes. Int J Mol Med, 38(4):1257-1263.

[27]LvFH, AghaS, KantanenJ, et al., 2014. Adaptations to climate-mediated selective pressures in sheep. Mol Biol Evol, 31(12):3324-3343.

[28]MahdaviM, NanekaraniS, HosseiniSD, 2014. Mutation in BMPR-IB gene is associated with litter size in Iranian Kalehkoohi sheep. Anim Reprod Sci, 147(3-4):93-98.

[29]MartinsFS, SaraivaMVA, Magalhães-PadilhaDM, et al., 2014. Presence of growth hormone receptor (GH-R) mRNA and protein in goat ovarian follicles and improvement of in vitro preantral follicle survival and development with GH. Theriogenology, 82(1):27-35.

[30]McGeeEA, HsuehAJW, 2000. Initial and cyclic recruitment of ovarian follicles. Endocr Rev, 21(2):200-214.

[31]MinarowskaA, MinarowskiL, KarwowskaA, et al., 2007. Regulatory role of cathepsin D in apoptosis. Folia Histochem Cytobiol, 45(3):159-163.

[32]RDVSMorais, ThoméRG, LemosFS, et al., 2012. Autophagy and apoptosis interplay during follicular atresia in fish ovary: a morphological and immunocytochemical study. Cell Tissue Res, 347(2):467-478.

[33]PanZX, ZhangJB, LinF, et al., 2012. Expression profiles of key candidate genes involved in steroidogenesis during follicular atresia in the pig ovary. Mol Biol Rep, 39(12):10823-10832.

[34]PengJY, GaoKX, XinHY, et al., 2016. Molecular cloning, expression analysis, and function of decorin in goat ovarian granulosa cells. Domest Anim Endocrinol, 57:108-116.

[35]PradeepPK, LiXL, PeegelH, et al., 2002. Dihydrotestosterone inhibits granulosa cell proliferation by decreasing the cyclin D2 mRNA expression and cell cycle arrest at G1 phase. Endocrinology, 143(8):2930-2935.

[36]SalesCF, MeloRMC, PinheiroAPB, et al., 2019. Autophagy and Cathepsin D mediated apoptosis contributing to ovarian follicular atresia in the Nile tilapia. Mol Reprod Dev, 86(11):1592-1602.

[37]SoedeNM, LangendijkP, KempB, 2011. Reproductive cycles in pigs. Anim Reprod Sci, 124(3-4):251-258.

[38]StrzalkaW, ZiemienowiczA, 2011. Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation. Ann Bot, 107(7):1127-1140.

[39]SuiMX, WangHH, WangZW, 2015. Molecular cloning, polymorphisms, and expression analysis of the RERG gene in indigenous Chinese goats. Genet Mol Res, 14(4):14936-14946.

[40]TangHL, HuangXJ, WangJ, et al., 2019. circKIF4A acts as a prognostic factor and mediator to regulate the progression of triple-negative breast cancer. Mol Cancer, 18:23.

[41]TuloneC, UchiyamaY, NovelliM, et al., 2007. Haematopoietic development and immunological function in the absence of cathepsin D. BMC Immunol, 8:22.

[42]XiaY, WangQ, HeXD, et al., 2020. Cloning and expression analysis of the follicle-stimulating hormone receptor (FSHR) gene in the reproductive axis of female yaks (Bos grunniens). Domest Anim Endocrinol, 70:106383.

[43]YangZW, YangXY, LiuGB, et al., 2020. Polymorphisms in BMPR-IB gene and their association with litter size trait in Chinese Hu sheep. Anim Biotechnol, ahead-of-print.

[44]ZhangH, WuZM, YangYP, et al., 2019. Catalpol ameliorates LPS-induced endometritis by inhibiting inflammation and TLR4/NF-κB signaling. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 20(10):816-827.

[45]ZhangP, WangJ, LangH, et al., 2018. Knockdown of CREB1 promotes apoptosis and decreases estradiol synthesis in mouse granulosa cells. Biomed Pharmacother, 105:1141-1146.

[46]ZhangT, RawsonDM, TostiL, et al., 2008. Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 °C using controlled slow cooling. Cryobiology, 56(2):138-143.

[47]ZhangXD, HuangL, WuT, et al., 2015. Transcriptomic analysis of ovaries from pigs with high and low litter size. PLoS ONE, 10(10):e0139514.

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2022 Journal of Zhejiang University-SCIENCE