CTSD对黔北麻羊卵泡颗粒细胞的调控机制及功能分析

doi:10.11843/j.issn.0366-6964.2021.05.014

Abstract

Abstract: The aim of this study was to analyze the regulatory mechanism of cathepsin D (CTSD) on the follicular granulosa cells of Qianbei Ma goat and to explore its effect mechanism on litter size traits.In this study, 36-week-old, healthy, polytocous female Qianbei Ma goat (n=5) were used as the research object, follicular granulosa cells from ovarian tissues collected after slaughter were isolated and cultured. The eukaryotic expression vector pEGFP-N3-CTSD was constructed, and the eukaryotic expression efficiency at the transcription and translation levels after introducing it into cells was examined; CCK-8 assay was used to detect the effect of recombinant plasmid on the proliferation of granulosa cells in different time periods. The effects of recombinant plasmid on the apoptosis and cycle of granulosa cells were detected by flow cytometry; Subsequently, RT-qPCR was used to detect the effect of recombinant plasmids on the expression levels of apoptosis-related genes Bcl-2, Bax, Caspase-3, cell cycle-related factors Cyclin A1, Cyclin D2, and Cyclin E mRNA at the cellular level. Finally, the prolificacy traits candidate genes BMPR-IB, FSHR, INHA were used as functional genes to verify the effect of recombinant plasmids on their mRNA and protein expression at the transcription and translation levels. The results of double enzyme digestion and sequencing confirmed that the eukaryotic expression vector pEGFP-N3-CTSD with CTSD gene of Qianbei Ma goat was successfully constructed, and the expression of CTSD in granulosa cells could be extremely significantly increased at the transcription and translation levels (P<0.01); The cell proliferation test results showed that the up-regulation of CTSD in granulosa cells could inhibit cell proliferation, and the inhibition efficiency on cell proliferation was extremely significant at 12, 24, 48 and 72 h (P<0.01); The results of apoptosis detection showed that the overexpression of CTSD could significantly promote the apoptosis of granulosa cells(P<0.01) and significantly down-regulate the expression of anti-apoptosis gene Bcl-2 (P<0.05), significantly up-regulate the expression of apoptosis-related genes Bax and Caspase-3 (P<0.01). In addition, the results of cell cycle detection showed that the recombinant plasmid pEGFP-N3-CTSD could significantly up-regulate the number of cells at G0/G1 and G2/M phases (P<0.01), and significantly down-regulate the number of cells at S phase(P<0.01), significantly increase the expression of cell cycle related factors Cyclin A1(P<0.01), and significantly decrease the expression of Cyclin D2 (P<0.01). The results of RT-qPCR and Western blot detection showed that the up-regulation of CTSD in granulosa cells could significantly down-regulate the expression of prolificacy trait candidate genes and proteins BMPR-IB, FSHR and INHA at transcriptional and translational levels (P<0.01).In this study, it was found that the high expression of CTSD could inhibit cell proliferation, promote cell apoptosis, change the cycle process of granulosa cells, change the expression of apoptosis-related factors Bcl-2, Bax, Caspase-3 and cell cycle-related factors Cyclin A1, Cyclin D2, Cyclin E, and significantly reduce the expression of prolificacy trait candidate genes and proteins BMPR-IB, FSHR and INHA in granulosa cells(P<0.01). It is suggested that CTSD may regulate the biological behavior of granulosa cells and affect the expression of candidate genes of prolific traits in goats by changing the expression of related factors at the cell level, and then indirectly become an important factor affecting litter size traits in goats. This study laid a foundation for further exploring the regulation mechanism of CTSD on litter size traits and the mechanism of its effect on granulosa cells in goats.

Key words: CTSD, Qianbei Ma goat, granulosa cell, regulation mechanism, litter size traits

CLC Number:

S827.3

ZHOU Zhinan, CHEN Xiang, ZHANG Yan, YANG Peifang, HUI Maomao, TANG Wen, HONG Lei. Regulatory Mechanism and Functional Analysis of CTSD on Follicular Granulosa Cells of Qianbei Ma Goat[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1278-1292.

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Regulatory Mechanism and Functional Analysis of CTSD on Follicular Granulosa Cells of Qianbei Ma Goat

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