Analysis of gene expression in granulosa cells of ovine antral growing follicles using suppressive subtractive hybridization

• Published in: Animal reproduction science Vol. 115; no. 1; pp. 39 - 48
• Main Authors: Chen, A. Qin, Wang, Zheng Guang, Xu, Zi Rong, Yu, Song Dong, Yang, Zhi Gang
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2009; [Amsterdam]: Elsevier Science
• Subjects: Animals; antral follicles; cDNA libraries; diameter; DNA Primers; DNA, Complementary - genetics; ewes; Expressed Sequence Tags; Female; follicular development; Follicular Phase; Gene expression; Gene Expression Regulation; genes; Glutathione Transferase - genetics; Granulosa cells; Granulosa Cells - physiology; hybridization; in vitro studies; Inhibin-beta Subunits - genetics; Isoenzymes - genetics; Membrane Proteins - genetics; messenger RNA; Nucleic Acid Hybridization; ovarian follicles; Ovary; ovulation; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA - genetics; RNA - isolation & purification; RNA, Messenger - genetics; screening; Serpins - genetics; Sheep; SSH; suppressive subtractive hybridization; Granulosa cells; Ovary; SSH; Gene expression
• ISSN: 0378-4320; 1873-2232
• DOI: 10.1016/j.anireprosci.2008.10.022

Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5 mm) and small follicles (SF, 3–5 mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.