West-Farrell Erin R, Xu Min, Gomberg Monica A, Chow Yee Hoong, Woodruff Teresa K, Shea Lonnie D
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, USA.
Biol Reprod. 2009 Mar;80(3):432-9. doi: 10.1095/biolreprod.108.071142. Epub 2008 Nov 12.
Folliculogenesis is a coordinated process, and the genes that regulate development are difficult to investigate in vivo. In vitro culture systems permit the assessment of individual follicles during development, thereby enabling gene expression patterns to be monitored during follicle development. Mouse multilayered secondary follicles (150-180 microm in diameter) were cultured in three-dimensional matrices of varying physical properties for up to 8 days. During this period of follicle growth in vitro, antrum formation and steroid production were monitored, and mRNA was isolated. The expression levels of genes (Star, Cyp11a1, Cyp17a1, Hsd3b1, Cyp19a1, Fshr, Lhcgr, Aqp7, Aqp8, Aqp9, and Hif1a) were measured and correlated to follicle developmental status. Follicles that developed an antrum and produced appropriate levels of estrogen and progesterone had unchanging expression of Star, Aqp7, Aqp8, and Hif1a and a 34-fold increase in Cyp19a1 expression at Day 8 of culture and had elevated Lhcgr at Days 6 and 8 of culture. Follicles that were healthy but did not form an antrum or produce appropriate levels of steroids, however, demonstrated increasing levels of Star, Aqp7, Aqp8, and Hif1a and a 15-fold increase in Cyp19a1 at Day 8 of culture, and Lhcgr levels were not elevated until Day 8 of culture. To our knowledge, this study provides the first temporal analysis of gene expression using individual culture in alginate hydrogels that correlates growth and steroidogenesis during follicle development and identifies expression patterns in healthy follicles and in developmentally disadvantaged follicles.
卵泡发生是一个协调的过程，而调控其发育的基因在体内难以进行研究。体外培养系统可在卵泡发育过程中对单个卵泡进行评估，从而能够监测卵泡发育期间的基因表达模式。将直径为150 - 180微米的小鼠多层次级卵泡在具有不同物理特性的三维基质中培养长达8天。在卵泡体外生长的这段时间里，监测卵泡腔形成和类固醇生成，并分离mRNA。测定基因（Star、Cyp11a1、Cyp17a1、Hsd3b1、Cyp19a1、Fshr、Lhcgr、Aqp7、Aqp8、Aqp9和Hif1a）的表达水平，并将其与卵泡发育状态相关联。形成卵泡腔并产生适量雌激素和孕酮的卵泡，其Star、Aqp7、Aqp8和Hif1a的表达不变，在培养第8天时Cyp19a1表达增加34倍，且在培养第6天和第8天时Lhcgr升高。然而，健康但未形成卵泡腔或未产生适量类固醇的卵泡，其Star、Aqp7、Aqp8和Hif1a水平不断升高，在培养第8天时Cyp19a1增加15倍，且Lhcgr水平直到培养第8天才升高。据我们所知，本研究首次在藻酸盐水凝胶中使用个体培养对基因表达进行了时间分析，该分析将卵泡发育过程中的生长与类固醇生成相关联，并确定了健康卵泡和发育不良卵泡中的表达模式。
