SummaryThis study aimed to: (i) characterize cultured granulosa cells (GCs) from different follicle sizes morphologically and molecularly; and (ii) select a suitable model according to follicular size that maintained GC function during culture. Buffalo ovaries were collected from a slaughterhouse and follicles were classified morphologically into: first group ≤ 4 mm, second group 5–8 mm, third group 9–15 mm and fourth group 16–20 mm diameter. GC pellets were divided into two portions. The first portion served as the control fresh pellet, and the secondwas used for 1 week for GC culture. Total RNA was isolated, and qRT-PCR was performed to test for follicle-stimulating hormone receptor (FSHR), cytochrome P450 19 (CYP19), luteinizing hormone/choriogonadotropin receptor (LHCGR), proliferating cell nuclear antigen (PCNA), apoptosis-related cysteine peptidase (CASP3), anti-Müllerian hormone (AMH), and phospholipase A2 group III (PLA2G3) mRNAs. Estradiol (E2) and progesterone (P4) levels in the culture supernatant and in follicular fluids were measured using enzyme-linked immunosorbent assay (ELISA). Basic DMEM-F12 medium maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was 0.001 ≤ P ≤ 0.01 and decreased at the end of culture compared with the fresh pellet. There was a fine balance between expression patterns of the proliferation marker gene (PCNA) and the proapoptotic marker gene (CASP3). AMH mRNA was significantly increased (P < 0.001) in cultured GCs from small follicles, while cultured GCs from other three categories (5–8 mm, 9–15 mm and 16–20 mm) showed a clear reduction (P < 0.001). Interestingly, the relative abundance of PLA2G3 mRNA was significantly (P < 0.001) increased in all cultured GCs. E2 and P4 concentrations were significantly (P < 0.001) decreased in all cultured groups. Primary cultured GCs from small follicles could be a good model for better understanding follicular development in Egyptian buffaloes.

中文翻译：

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不同滤泡大小的原代培养水牛颗粒细胞与其体内对应物的表达谱比较

摘要本研究旨在： (i) 从形态和分子上表征来自不同卵泡大小的培养颗粒细胞 (GC) (ii) 根据卵泡大小选择合适的模型，在培养过程中保持 GC 功能。从屠宰场收集水牛卵巢，并在形态上将卵泡分类为：第一组≤4 mm，第二组 5-8 mm，第三组 9-15 mm，第四组直径 16-20 mm。GC颗粒被分成两部分。第一部分作为对照新鲜沉淀，第二部分用于GC培养1周。分离总 RNA，并进行 qRT-PCR 以检测促卵泡激素受体（FSHR), 细胞色素 P450 19 (CYP19), 促黄体激素/绒毛膜促性腺激素受体 (长对撞机), 增殖细胞核抗原 (PCNA)、凋亡相关的半胱氨酸肽酶 (CASP3), 抗苗勒管激素 (AMH) 和磷脂酶 A2 组 III (PLA2G3) mRNA。使用酶联免疫吸附试验 (ELISA) 测量培养上清液和卵泡液中的雌二醇 (E2) 和孕酮 (P4) 水平。基本 DMEM-F12 培养基保持培养的 GC 的形态外观。相对丰度FSHR,CYP19， 和长对撞机mRNA 为 0.001 ≤磷≤ 0.01 并且与新鲜颗粒相比在培养结束时下降。增殖标记基因的表达模式之间存在良好的平衡（PCNA) 和促凋亡标记基因 (CASP3)。AMHmRNA显着增加（磷< 0.001) 在小卵泡培养的 GCs 中，而其他三类（5-8 mm、9-15 mm 和 16-20 mm）培养的 GCs 明显减少（磷< 0.001)。有趣的是，相对丰富的PLA2G3mRNA 显着 (磷< 0.001) 在所有培养的 GC 中增加。E2 和 P4 浓度显着 (磷< 0.001) 在所有培养组中下降。来自小卵泡的原代培养的 GC 可能是更好地了解埃及水牛卵泡发育的良好模型。