可变剪接体WNT4-β对山羊卵泡颗粒细胞增殖和激素分泌的影响

doi:10.11843/j.issn.0366-6964.2022.10.020

Abstract

Abstract: The aim of this study was to investigate the effect of an alternative splicing (WNT4-β) of WNT4 on the follicular granulosa cells proliferation of goat. In this study, 20 healthy ewes from 4 to 6 months of age were selected, goat ovarian tissues were collected, follicular granulosa cells were isolated in vitro and cultured. The expression location of WNT4-β was determined by immunofluorescence staining. Overexpression or interference of WNT4-β in goat granulosa cells, RT-qPCR and Western blot were used to detect the expression of key marker factors (ROA1 and RHOA) in the WNT signaling pathway, and granulosa cell proliferation marker genes cyclin-D2 and CDK4. CCK-8 was used to detect the granulosa cells proliferation. ELISA was used to analyze the changes of reproductive hormone levels in granulosa cells. Immunofluorescence staining result showed that WNT4 was expressed only in follicular granulosa cells of goat, but not in oocyte cells. WNT4-β and granulosa cell proliferation factors cyclin-D2 and CDK4 mRNA significantly increased (P<0.01) after overexpression of WNT4-β, and significant increase in protein expression levels (P<0.05). The expression levels of WNT signaling pathway marker factors ROA1 and RHOA mRNA significantly increased (P<0.05), and β-catenin protein levels significantly increased (P<0.05). The mRNA expression of WNT4-β, cyclin-D2, CDK4, ROA1 and RHOA significantly reduced after interfering with WNT4-β (P<0.05). WNT4-β, cyclin-D2, CDK4 and β-catenin proteins expression significantly reduced (P<0.05). The CCK-8 result showed that overexpression of WNT4-β promoted proliferation of granulosa cells (P<0.05). ELISA results showed that estradiol (E₂) levels significantly increased (P<0.05) and progesterone (P₄) levels increased but not significantly (P>0.05) in granulosa cells after overexpression of WNT4-β. The opposite result was got after interfering with WNT4-β, the granulosa cell proliferation was inhibited (P<0.05) and the levels of E₂ and P₄ significantly reduced (P<0.05). In conclusion, the WNT4 alternative splicing WNT4-β promotes follicular granulosa cell proliferation and ovulation-related steroid hormone secretion in goats by regulating the WNT signaling pathway, and this study provides a theoretical basis for revealing the molecular function of WNT4 in reproductive traits in goats.

Key words: goat, follicular development, granulosa cell proliferation, alternative splicing, WNT4-β

CLC Number:

S827.3

WANG Peng, HAN Haiyin, LI Wentao, LIU Ziyi, CHU Mingxing, LIU Yufang. Effect of Alternative Splicing WNT4-β on Follicular Granulosa Cell Proliferation and Hormone Secretion in Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3480-3489.

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Effect of Alternative Splicing WNT4-β on Follicular Granulosa Cell Proliferation and Hormone Secretion in Goats

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