Gene expression profiling of differentially expressed genes in granulosa cells of bovine dominant follicles using suppression subtractive hybridization
Gene expression profiling of differentially expressed genes in granulosa cells of bovine dominant follicles using suppression subtractive hybridization
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DOI：
10.1095/biolreprod.103.021709
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发表时间：
2004-02-01
影响因子：
3.6
通讯作者：
Lussier, JG
中科院分区：
文献类型：
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作者：
Fayad, T;Lévesque, V;Lussier, JG
牛中超过3至4 mm的窦卵泡的发育在发情周期中出现2至3次的波形。每一波都在3- 4-mm阶段周期性地募集多个卵泡，然后选择一个卵泡成为优势卵泡（DF）。参与卵泡优势过程的分子决定因素仍然知之甚少。本研究的目的是比较基因表达的颗粒细胞（GC）之间的生长优势卵泡从第5天的发情周期和nonselected小卵泡（小于或等于4 mm）使用抑制性消减杂交（SSH）的方法，以确定候选基因差异表达的DF的GC。从DF cDNA中减去小卵泡cDNA（DF-SF），并用于建立DF GC-扣除cDNA文库。共鉴定出42个非冗余cDNA。先前鉴定的基因，如CX43，CYP 19，INHBA和SERPINE 2的检测支持我们的实验模型的有效性和SSH作为分析方法的使用。对于选定的基因，如ApoER 2，CPD，CSPG 2，14-3-3-β-D，NR 5A 2/SF 2，RGN/SMP 30和SERPINE 2，通过虚拟北方印迹或逆转录酶-聚合酶链反应比较基因表达谱，结果证实，与小卵泡相比，优势卵泡GC中它们的mRNA增加或诱导。我们的结论是，我们已经确定了新的基因（已知的和未知的），在牛GC上调，可能会影响卵泡生长，优势，或两者兼而有之。
Development of antral follicles beyond 3 to 4 mm in cattle appears as a wave pattern that occurs two to three times during the estrous cycle. Each wave presents a cyclic recruitment of multiple follicles at the 3- to 4-mm stage, followed by the selection of a single follicle that becomes the dominant follicle (DF). The molecular determinants involved in the follicular dominance process remain poorly understood. The objective of the current study was to compare gene expression in granulosa cells (GCs) between growing dominant follicles from Day 5 of the estrous cycle and nonselected small follicles (less than or equal to 4 mm) using the suppression subtractive hybridization (SSH) approach to identify candidate genes differentially expressed in GCs of the DF. Small follicle cDNAs were subtracted from DF cDNAs (DF-SF) and used to establish a DF GC-subtracted cDNA library. A total of 42 nonredundant cDNAs were identified. Detection of previously identified genes such as CX43, CYP19, INHBA, and SERPINE2 supported the validity of our experimental model and the use of SSH as the method of analysis. For selected genes such as ApoER2, CPD, CSPG2, 14-3-3 epsilon, NR5A2/SF2, RGN/SMP30, and SERPINE2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction, and results confirmed an increase or induction of their mRNA in GCs of dominant follicles compared with that of small follicles. We conclude that we have identified novel genes (known and unknown) that are up-regulated in bovine GCs that may affect follicular growth, dominance, or both.