Ndiaye Kalidou, Fayad Tania, Silversides David W, Sirois Jean, Lussier Jacques G
Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6.
Biol Reprod. 2005 Aug;73(2):324-33. doi: 10.1095/biolreprod.104.038026. Epub 2005 Apr 13.
Molecular determinants and mechanisms involved in ovarian follicular growth, ovulation, and luteinization are not well understood. The objective of this study was to identify genes expressed in bovine granulosa cells (GC) of dominant follicles (DF) and downregulated after hCG-induced ovulation, using the suppression subtractive hybridization (SSH). GC were collected from DF at Day 5 of the estrous cycle and from ovulatory follicles (OF) obtained 23 h following injection of hCG. A subtracted cDNA library (DF-OF) was generated and screened using unsubtracted (DF, OF) and subtracted (DF-OF, OF-DF) cDNAs as complex (32)P-probes. A total of 32 nonredundant cDNAs were identified: 23 cDNAs matched with sequences of known biological function and 9 cDNAs with complete or partial sequences of undefined biological function. Detection of genes known to be downregulated during the periovulatory period in the bovine species, such as CPD, CYP11A1, CYP19A1, FSHR, LRP8/ ApoER2, and SERPINE2, validated the physiological model and analytical techniques used. For a subset of genes, such as ARFGAP3, CYP11A1, CYP19A1, FSHR, FST, GJA1, IDH3, INHBA, LHCGR, LHCGR lacking exon 10, PRC1, PRG1, RPA2, SCD, and TRIB2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction from follicles obtained at different developmental stages. Results confirmed a downregulation of the respective mRNAs in GC of OF compared with that of DF. We conclude that we have identified novel genes that are downregulated by hCG in bovine GC of DF during the periovulatory period, which may contribute to follicular growth, ovulation, and/or luteinization.
卵巢卵泡生长、排卵和黄体化过程中涉及的分子决定因素和机制尚未完全明确。本研究的目的是利用抑制性消减杂交技术（SSH），鉴定在优势卵泡（DF）的牛颗粒细胞（GC）中表达且在人绒毛膜促性腺激素（hCG）诱导排卵后下调的基因。在发情周期第5天从DF收集GC，并在注射hCG后23小时从排卵卵泡（OF）中收集GC。构建了一个消减cDNA文库（DF-OF），并使用未消减的（DF、OF）和消减的（DF-OF、OF-DF）cDNA作为复杂的（32）P探针进行筛选。共鉴定出32个非冗余cDNA：23个cDNA与已知生物学功能的序列匹配，9个cDNA具有未明确生物学功能的完整或部分序列。检测牛物种在围排卵期已知下调的基因，如CPD、CYP11A1、CYP19A1、FSHR、LRP8/ApoER2和SERPINE2，验证了所使用的生理模型和分析技术。对于一部分基因，如ARFGAP3、CYP11A1、CYP19A1、FSHR、FST、GJA1、IDH3、INHBA、LHCGR、缺少外显子10的LHCGR、PRC1、PRG1、RPA2、SCD和TRIB2，通过虚拟Northern印迹或逆转录聚合酶链反应，比较了从不同发育阶段获得的卵泡中的基因表达谱。结果证实，与DF相比，OF的GC中相应mRNA下调。我们得出结论，我们已经鉴定出在围排卵期hCG作用下，DF的牛GC中下调的新基因，这些基因可能有助于卵泡生长、排卵和/或黄体化。
