| 摘要: |
| 【目的】研究猪有腔卵泡发育和闭锁中颗粒细胞凋亡与卵泡液类固醇激素的变化,为了解猪有腔卵泡发育与闭锁的调控机理提供参考。【方法】从猪卵巢分离完整有腔卵泡，按质量分为健康卵泡、早期闭锁卵泡和晚期闭锁卵泡3类；再按卵泡直径大小分为大卵泡组（＞5 mm）、中卵泡组（3～5 mm）和小卵泡组（＜3 mm）3组，制作完整有腔卵泡的石蜡切片，HE染色后观察其形态学变化。应用Annexin Ⅴ-FITC/PI双染流式细胞仪检测壁层颗粒细胞凋亡情况，放射免疫测定法（RIA）分析卵泡液中雌二醇（E2）和孕酮（P4）浓度的变化。【结果】不同大小健康卵泡内均存在少量的凋亡颗粒细胞，卵泡闭锁时颗粒细胞凋亡率显著增加，小卵泡表现尤为明显；晚期闭锁小卵泡中约有60%的颗粒细胞发生凋亡。健康大卵泡组E2浓度显著高于中、小卵泡组，P4浓度与中卵泡组无差异，但显著低于小卵泡组。卵泡闭锁时不同大小卵泡均表现出E2浓度显著下降和P4浓度显著增加。中、小健康卵泡的卵泡液中P4/E2＜5，而健康大卵泡中P4/E2＜1；早期闭锁大卵泡卵泡液中P4/E2＞1，而早期闭锁中、小卵泡中P4/E2在5～15；当P4/E2＞15时，卵泡则处于晚期闭锁状态。【结论】颗粒细胞凋亡是卵泡闭锁的主要诱因，但卵泡闭锁程度因卵泡大小而异，小卵泡似乎更易发生闭锁；卵泡液中P4/E2可作为判定有腔卵泡质量的一个标准。 |
| 关键词: 猪 有腔卵泡 颗粒细胞凋亡 孕酮 雌二醇 |
| DOI： |
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| 基金项目:国家高技术研究与发展计划项目（2008AA101003）；国家基础研究发展规划（973）项目（2007CB947403）；教育部博士点基金项目(200807120022) |
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| Analysis of the granulosa cells apoptosis and the changes of hormone during porcine follicular development and atresia |
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LIN Pengfei1,2, JU Shiqiang1, LUO Biping,et al1
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1.College of Veterinary Medicine ,Nanjing Agricultural University;2.College of Veterinary Medicine ,Northwest A&F University
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| Abstract: |
| 【Objective】 This study was to analyse the relationship between the apoptotic granulosa cells and the levels of steroids of porcine follicle during development and atresia,so as to supply experimental data for further revealing of the regulatory mechanism of follicular development and atresia.【Method】 The whole antral follicles were isolated from porcine ovaries and classified as follows:healthy follicles,early atretic follicles and progressed atretic follicles,then divided into 3 groups according to the follicular diameter:large follicles (＞5 mm),medium follicles (3-5 mm) and small follicles (＜3 mm).The paraffin section was prepared for porcine intact antral follicle,and the morphological characteristic of porcine antral follicles was observed using HE staining.The percentage of apoptotic granulosa cells was examined using Annexin Ⅴ-FITC/PI double-labeling,so was the concentrations of estradiol (E2) and progesterone (P4) in follicular fluid of cultured above different-sized follicles using radioimmunoassay.【Result】 There were limited numbers of apoptotic granulosa cells in the healthy follicles of different sizes,and the percentage of the apoptotic granulosa cells was significantly increased during follicular atresia,especially in small follicles.About 60% of granulosa cells became apoptosis in progress atretic small follicles.In healthy large follicles,the concentrations of E2 of follicular fluid was significantly higher than that in the medium and small follicles,the concentration of P4 showed no difference with the medium follicles but was significantly lower than that in the small follicles.The concentrations of estradiol (E2) significantly declined and progesterone (P4) concentrations significantly increased between healthy and early atretic follicles regardless of follicle size.P4/E2 was less than 5 in follicular fluid of healthy medium and small follicles,but the P4/E2 was less than 1 in follicular fluid of healthy large follicles.P4/E2 was more than 1 in follicular fluid of early atretic large follicles,however,P4/E2 was between 5 to 15 in early atretic medium and small follicles,when the ratio was over 15,then the follicles became the progress atretic state.【Conclusion】 Granulosa cells apoptosis is the main cause of follicular atresia,the degree of which varies with the size of follicles,and small follicles seem to be easier atretic than medium and large follicles.The concentrations P4/E2 of follicular fluid can be determined as a standard of the quality of antral follicles. |
| Key words: swine antral follicles granulosa cells apoptosis estradiol progesterone |
