Laser Capture Microdissection isolation of preovulatory granulosa cells from WT and bERKO ovaries

Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER) b is highly expressed in ovarian granulosa cells and mice lacking ERb (bERKO) are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and while informative, provides confounding results due to the heterogeneous cell types present including granulosa, theca and oocytes and exposure to in vitro conditions. Herein, we isolated preovulatory granulosa cells from WT and ERb-null mice using laser capture microdissection to examine the genomic transcriptional response downstream of PMSG (mimicking FSH) and PMSG/hCG (mimicking LH) stimulation. This allows for a direct comparison of in vivo granulosa cells at the same stage of development from both WT and ERb-null ovaries. ERb-null granulosa cells showed altered expression of genes known to be regulated by FSH (Akap12 and Runx2) as well as not previously reported (Arnt2 and Pou5f1) in WT granulosa cells. Our analysis also identified 304 genes not previously associated with ERb in granulosa cells. LH responsive genes including Abcb1b and Fam110c show reduced expression in ERb-null granulosa cells; however novel genes including Rassf2 and Megf10 were also identified as being downstream of LH signaling in granulosa cells. Collectively, our data suggests that granulosa cells from ERb-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation We used microarray to compare the gene expression profiles of wiltype (WT) and Erb-null (bERKO) preovulatory granulosa cells as they respond to either PMSG or PMSG+hCG treatments. Laser microdissection was used to collect a purified population of granulosa cells only from preovulatory follicles. We chose to compre the response to PMSG or PMSG+hCG of granulosa cells collected from either WT and bERKO preovulatory follicles. We chose to collect cells 48h after mice were treated with PMSG to compare the gene expression profile ot preovulatory granulosa cells. We also studied the response of these cells to LH (or hCG) as we collected cells 4h after mice were treated with hCG (peak of transcriptional response to hCG).明确排卵通路相关基因的时空表达模式，对于解析每种雌激素受体（estrogen receptor, ER）在调控卵泡发生与排卵过程中的功能至关重要。雌激素受体β（estrogen receptor β, ERβ）在卵巢颗粒细胞中高表达；缺失ERβ的小鼠（bERKO）因排卵效率低下而生育能力受损。既往研究多聚焦于分离的颗粒细胞或体外培养的卵泡，尽管此类研究具备一定参考价值，但因样本中包含颗粒细胞、膜细胞与卵母细胞等多种异质性细胞群，且处于体外培养环境中，往往会得到干扰性的实验结果。本研究通过激光捕获显微切割（laser capture microdissection, LCM）技术，从野生型（Wild Type, WT）与ERβ敲除小鼠体内分离排卵前颗粒细胞，以探究孕马血清促性腺激素（Pregnant Mare Serum Gonadotropin, PMSG，模拟促卵泡激素FSH）以及PMSG联合人绒毛膜促性腺激素（human Chorionic Gonadotropin, hCG，模拟促黄体生成素LH）刺激后，下游的基因组转录应答情况。该方法可实现对野生型与ERβ敲除卵巢中，同一发育阶段的体内颗粒细胞的直接比较。ERβ敲除的颗粒细胞中，已知受FSH调控的基因（Akap12与Runx2）的表达发生改变，同时还出现了野生型颗粒细胞中此前未被报道的基因（Arnt2与Pou5f1）的异常表达。本研究的分析还鉴定出304个此前未被发现与颗粒细胞中ERβ相关的基因。包括Abcb1b与Fam110c在内的LH应答基因在ERβ敲除的颗粒细胞中表达量下调；此外，本研究还鉴定出包括Rassf2与Megf10在内的新型基因，可作为颗粒细胞中LH信号通路的下游靶点。综合来看，本研究数据表明，ERβ敲除卵巢来源的颗粒细胞可能无法正常分化，且不能对促性腺激素刺激做出恰当应答。本研究采用基因芯片（microarray）技术，对比野生型与ERβ敲除排卵前颗粒细胞在分别经PMSG或PMSG+hCG处理后的基因表达谱。研究采用激光显微切割技术，仅从排卵前卵泡中分离得到纯度均一的颗粒细胞群。本研究旨在对比野生型与ERβ敲除排卵前卵泡来源的颗粒细胞对PMSG或PMSG+hCG的应答反应。我们选择在小鼠经PMSG处理48小时后收集细胞，以对比排卵前颗粒细胞的基因表达谱。同时，我们在小鼠经hCG处理4小时后收集细胞（此时为hCG转录应答的峰值时段），以研究这些细胞对LH（或hCG）的应答情况。