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This study aimed to investigate the mechanism by which knockdown of androgen receptor （AR） in sheep affects the development of ovarian granulosa cells （GC）. The healthy， adult sheep was selected as experimental subjects， employing surgical procedures to obtain ovarian tissue， from which granulosa cells were subsequently isolated and characterized.After silencing AR expression in granulosa cells （GCs）， cell proliferation and estradiol （E2） production were assessed in sheep ovarian GCs through CCK-8 and ELISA techniques.Furthermore， after AR knockdown， key differentially expressed genes （DEGs） affecting ovarian granulosa cell development were identified through transcriptome sequencing （RNA-seq）. These genes were subjected to functional enrichment analysis， and signaling pathways involved in granulosa cell development， along with their associated DEGs， were validated using RT-qPCR and Western blot assays.The results demonstrated that the study successfully isolated and identified granulosa cells， and that knockdown of AR significantly reduces the proliferation activity of these cells.More importantly， after AR was knocked down， 1 187 differentially expressed genes was identified through transcriptome sequencing， with 465 genes upregulated and 722 genes downregulated.Based on these differentially expressed genes， key signaling pathways involved in granulosa cell development were identified， including the PI3K-Akt pathway and the estrogen signaling pathway. The key genes PI3K， AKT， p-AKT， ESR2， CYP19A1， BAX， BAX/BCL2， and Caspase3 were further validated.Following AR silencing， the expression levels of critical genes within the PI3K-Akt signaling cascade—specifically PI3K， AKT， and p-AKT were significantly reduced （P<0.05）. Similarly， key genes associated with estrogen biosynthesis， ESR2 and CYP19A1， exhibited significant downregulation （P<0.05）. Conversely， genes linked to cellular proliferation， including BAX， the BAX/BCL2 ratio， and Caspase3， showed a significant increase in expression （P<0.05）. In summary， these findings suggest that knocking down AR in sheep ovarian granulosa cells may influence cell proliferation and hormone synthesis through the PI3K-Akt signaling pathway and the estrogen signaling pathway， thereby participating in the process of cellular development.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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