EGFR及其配体在控制家鸡卵泡颗粒细胞增殖、分化、凋亡过程中的作用机理研究

It is generally believed that in vertebrates, ovarian follicle development is tightly controlled by pituitary gonadotropins: follicle-stimulating hormone (FSH) and luteinizing hormone (LH). However, increasing evidences show that many autocrine/paracrine factors also play important roles in this process. Among these factors, epidermal growth factor (EGF) receptor and its ligands attract much attention. In our recent studies, Heparin-binding EGF-like growth factor (HB-EGF) was detected to be abundantly expressed in the oocytes of chicken ovary and HB-EGF treatment could significantly stimulate the proliferation of granulosa cell from chicken growing follicles and suppress kit ligand (KL) expression in cultured granulosa cells. These findings led us to hypothesize that growing oocytes may act as a signaling center by secreting EGFR ligands to control the proliferation, differentiation, and apoptosis of the surrounding granulosa cells, thus playing an active role in the regulation of ovarian follicle development in chickens. To substantiate this hypothesis, using chicken as an experimental model, present study aims to: 1) investigate the spatio-temporal expression patterns of EGFR and its multiple ligands in growing ovarian follicles; 2) reveal how EGFR and its ligands are involved in controlling the proliferation, differentiation and apoptosis of granulosa cells from developing follicles; 3) elucidate how pituitary gonadotropins, EGFR ligands, and other autocrine/paracrine factors coordinate to regulate the proliferation and differentiation of granulosa cells from ovarian follicles. Clearly, the answers to these questions will not only help us to elucidate the regulatory roles of EGFR and its ligands in ovarian follicle development of chickens, but also provide important clues to the conserved roles of EGFR ligands in the ovary of vertebrates.

脊椎动物卵泡发育受垂体促性腺激素控制。愈来愈多证据表明，卵巢产生的众多'旁分泌和自分泌'因子亦对卵泡发育起重要作用。在这些因子中，EGFR及其配体受到广泛关注。近来，我们在家鸡生长卵母细胞中检测到HB-EGF高表达，同时，体外实验证实HB-EGF可有效刺激来自家鸡各级卵泡颗粒细胞增殖，并抑制其KL基因表达。这些发现使我们推测：卵母细胞作为信号中心，可通过分泌EGFR配体来调节周围颗粒细胞增殖、分化、凋亡，进而有效控制卵泡发育。为验证该假说，本研究拟用家鸡为模型，采用原位杂交等多种研究技术，1）研究EGFR及其配体在发育卵巢中的时空表达图谱；2）揭示EGFR及其配体如何影响各级卵泡颗粒细胞增殖、分化与凋亡？3）阐释EGFR配体与垂体激素以及其它'自分泌/旁分泌'因子对颗粒细胞增殖、分化的调节。这些研究结果有望为阐明EGFR及其配体对家鸡卵泡发育的调节机制奠定基础。

脊椎动物卵泡发育受垂体促性腺激素控制。愈来愈多证据表明，卵巢产生众多‘旁分泌和自分泌’因子亦对卵泡发育起重要作用。在这些因子中，EGFR及其配体受到广泛关注。近来，我们在家鸡生长卵母细胞中检测到HB-EGF高表达，同时，体外实验证实HB-EGF可有效刺激来自家鸡各级卵泡颗粒细胞增殖。这些发现使我们推测：卵母细胞作为信号中心，可通过分泌EGFR配体来调节周围颗粒细胞增殖、分化、凋亡，进而控制卵泡发育。为验证该假说，本研究用家鸡卵巢为模型， 1）研究EGFR配体在发育卵巢中的时空表达图谱，发现EGFR及其三种配体HB-EGF, AREG，EPG在卵巢中高表达，且HB-EGF和AREG的表达具明显阶段特异性。提示：HB-EGF和AREG可作为‘旁分泌、自分泌’因子，参与家鸡卵泡发育调节； 2）发现EGFR三种配体(TGF-α, HB-EGF和EPG)均可刺激各级卵泡颗粒细胞增殖、抑制其凋亡；3）发现垂体促性腺激素(FSH和LH)对颗粒细胞EGFR配体(HB-EGF, AREG, EPG)表达的刺激效应不明显；4）鉴定到两个新型垂体激素，GRP和CART肽。其中，GRP可高效刺激颗粒细胞增殖，并诱导EGFR配体(HB-EGF)的表达。暗示GRP或与EGFR配体，协同调节卵泡颗粒细胞增殖，控制卵泡发育；5）依赖转录组及qPCR技术在各级卵泡颗粒细胞(6mm, F5, F1)鉴定到系列差异表基因，这些基因参与颗粒细胞增殖与分化调控，且其表达或受EGFR信号通路影响；6)其他 ‘旁分泌、自分泌’因子，如IGF2、 WNT4等,或协同EGFR配体，参与颗粒细胞增殖与分化的调节。这些结果为探讨EGFR配体在卵巢中的作用机理奠定基础；同时，亦为建立EGFR配体与垂体激素、卵巢 ‘旁分泌、自分泌’因子等对家鸡卵泡发育的协同调控网络奠定基础。此外，本研究也为探究EGFR配体在脊椎动物卵泡发育中的作用机制提供线索。