【作者】 葛楚天；

【导师】 张才乔； J.Petitte；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2010， 博士

【摘要】 家禽常被用作发育生物学研究的动物模型,很多研究致力在更深层次揭示家禽发育的机理,尤其是生殖系统的发育,在内源性调节的基础上试图通过外源性调控,以提高家禽的繁殖性能。本实验以艾维因鸡胚为材料,分离了胚胎期原始生殖细胞(primordial germ cell,PGC),并进行原代和传代培养,研究了表皮生长因子(EGF)、雌激素(17β-estradiol)和人参皂甙对PGC增殖的作用及其胞内信号传导机制,同时还探索了维甲酸(RA)在PGC减数分裂启动过程中的调控作用。为改善家禽繁殖性能的营养调控、转基因家禽的制备提供理论依据和实验平台。1.鸡胚PGC的分离、培养及鉴定在体视显微镜下用玻璃针分离19期3.5d鸡胚生殖嵴部位(去除头突、心脏、尿囊膜和尾部,取后肠前1/3的背外侧组织)或28期5.5d鸡胚性腺(从中肾中剥离出来),胰蛋白酶+EDTA消化分散组织,用含10%胎牛血清和10 ng/ml白血病抑制因子(LIF)的KO-DMEM培养液体外共培养PGC和体细胞的方式建立原代培养,然后传代培养于鸡胚成纤维细胞饲养层上进行进一步培养。同时我们从13-15期鸡胚血管中分离提取PGC进行原代和饲养层上传代培养。传至3-5代后,分离PGC集落,用碱性磷酸酶(AKP)、过碘酸雪夫氏(PAS)化学反应及c-kit、SSEA-1,3,4、EMA-1等免疫细胞化学鉴定PGC,用BrdU掺入法检测细胞的增殖活性。并采用RT-PCR方法检测干细胞多能性相关基因(Pou5fl, Sox2, Nanog, AP和Tert),生殖细胞特异性基因(c-kit, Vasa和Dazl)和减数分裂标记基因(Stra8和Scyp3)。上述结果表明PGC-体细胞原代共培养再传代于饲养层上培养的模型是可以用于PGC增殖活性调控的研究。2.EGF对鸡胚PGC增殖的影响及其机理的研究本实验研究了EGF对鸡胚PGC增殖的影响及其相关的信号传递途径。结果表明：经过EGF(10-100 ng/m1)处理后,PGC的克隆数目和面积呈时间和剂量依赖性显著增加。EGF激活蛋白激酶C (PKC),但是这种作用被EGF受体磷酸化抑制剂AG1478及细胞内钙离子螯合剂EGTA所阻断。此外,EGF还能促进细胞核因子NF-κB (p65)核转位及IkBα的降解,但此效应均可被AG1478, EGTA,H7(PKC抑制剂)及SN50(NF-κB特异性抑制剂)所抑制。实验还表明EGF诱导的PGC促增殖作用分别被AG1478, EGTA, H7及SN50显著抑制。EGF能促进细胞周期蛋白Cyclin Dl/周期蛋白依赖激酶CDK6, Cyclin E/CDK2及凋亡抑制因子Bcl-2的表达：同时,下调促凋亡因子Bax的表达,抑制细胞凋亡蛋白酶3/9的活性,并且这些作用都被AG1478, EGTA, H7和SN50阻断。从而说明EGFR, PKC, NF-κB信号通路介导EGF诱导的DNA合成及抗凋亡作用。以上结果表明：在体外培养条件下,EGF能过Ca2+/PKC, NF-κB信号途径促进鸡胚PGC的增殖,揭示EGF信号通路在调控鸡胚胎生殖细胞发育中起着重要的作用。3.雌激素对鸡胚PGC增殖调控机理的研究我们研究了17p-雌二醇(E2)对鸡胚PGC增殖的影响,以及GPR30介导的具体的信号转导途径。结果表明,向0日龄鸡蛋卵黄中注射E2,在5天性腺(27期)中我们发现SSEA-1阳性细胞数明显高于对照组(注射DMSO)。在体外培养模型中,我们首次发现第5天性腺PGC表达GPR30,但并不表达传统的雌激素受体ERα和ERβ。1-100 nM E2以剂量依赖和时间依赖模式显著增加PGC克隆团的数量和面积。Western blot分析显示E2能激活PGC中Akt活性,GSK3β和β-连环蛋白的磷酸化水平,这些刺激作用分别被AG1478, LY294002(P13K抑制剂),KP372-1(Akt抑制剂)和GPR30 SiRNA所阻断,但是ER抑制剂ICI182,780并没有明显的抑制作用。我们还发现E2对PGC克隆团数目和面积的正调控作用被AG1478, LY294002, KP372-1和GPR30 SiRNA抑制,但被GPR30激活剂G-1和GSK3β抑制剂BIO所提高。Real-time RT-PCR结果显示,E2显著上调PGC中细胞周期蛋白cyclinDl/E,CDK2/6和原癌基因c-fos, c-myc的mRNA丰度,但是这种上调作用被AG1478、Y294002、KP372-1和GPR30 SiRNA抑制。综上所述,E2通过GPR30, EGFR, PI3K/Akt和GSK3β/β-连环蛋白信号级联反应刺激鸡胚性腺PGC的增殖。这些发现提示,E2/GPR30信号在性腺分化前生殖细胞发育中扮演重要的调控作用。4.人参皂甙对鸡胚PGC增殖的调控及其机制的研究我们就人参皂甙(ginsenosides)对鸡胚PGC的促增殖作用及所涉及的NF-kB信号途径进行研究。从3.5-4天的鸡胚中分离PGC,在添加5%胎牛血清和10 ng/ml的LIF的DMEM培养液中进行原代培养24小时。传代培养于饲养层上的PGC,用人参皂甙单独处理,或联合PKC抑制剂H7或激活剂佛波酯(PMA)处理24h。此外,通过Western blot分析了NF-κB的核转移和IκBα的降解水平。结果表明,1-100μg/ml人参皂甙以剂量依赖方式显著增加了PGC克隆团的数量和面积,但是这种正调控作用明显被10-6M的H7抑制。同时PKC免疫组化结果显示,经过人参皂甙处理后,PGC克隆团中大部分PKC转移到细胞膜上着色,说明PKC已被激活。并且,1-10μg/ml人参皂甙刺激NF-κB (p65)从PGC胞质向细胞核转移。然而,人参皂甙诱导的NF-κB核转移和IκBα降解作用被10-6M的H7显著阻断。以上结果揭示,人参皂甙能通过激活PKC/NF-κB信号途径促进鸡胚PGC的增殖。5.维甲酸(RA)对鸡胚PGC减数分裂的影响本实验我们以在体外长期培养的血液PGC为研究对象,探索RA在调控鸡胚胎期生殖细胞减数分裂中是否起着保守作用,及其RA能否直接作用于PGC上。结果表明,在27期鸡胚性腺体外培养体系中添加RA能明显使减数分裂相关基因Stra8, Sycp3和Dmcl的mRNA表达水平上调。在血液PGC无饲养层的培养模型中,RA急剧上调雌性和雄性PGC中Stra8, Sycp3和Dmcl的mRNA表达水平。流式分析结果显示,PGC经过RA处理4天后,29.5%雄性PGC和58.7%雌性PGC处于亚G1期(1n),说明此部分细胞已经进入减数分裂期。吉姆萨染色结果表明雄性PGC和雌性PGC在经RA诱导进入减数分裂偶线期和粗线期的能力不一样,雌性PGC在RA的诱导下更易进入偶线期和粗线期。综上所述,我们首次报道了RA能直接作用于PGC上从而刺激PGC进入减数分裂期,其中雌性PGC更易进入减数分裂。以上实验结果表明：从不同时期鸡胚分离出来的PGC进行原代共培养,再传代于鸡胚成纤维细胞饲养层上所建立的传代PGC培养模型可用于PGC增殖和分化调控的研究,经AKP、PAS及c-kit和SSEA-1、3、4免疫细胞化学染色等多种方法证实了PGC的原始性。在此模型上发现内源性因子EGF和外源性植物提取物人参皂甙在PGC体外扩增中的有丝分裂原作用及其分子机理；首次报道新型雌激素受体GPR30在鸡胚性腺组织和PGC中的表达情况,及其在雌激素诱导的鸡胚PGC增殖中的介导作用；同时首次证实了RA能直接促进鸡胚胎期生殖细胞进入有丝分裂。这些发现将为提高家禽繁殖性能的营养调控、制备嵌合体和转基因家禽提供理论指导和实验平台。更多还原

【Abstract】 Poultry species has always been used as animal model for the research in developmental biology for profound elucidation of the mechanisms underlining poultry development, especially the reproductive system. The reproductive performance of poultry could be greatly improved by external manipulation based on endogenous regulating mechanisms. In this study primordial germ cells (PGCs) were isolated from Avian chicken embryo, and then subcultured on feed layer after initial primary culture with somatic cells. In addition, the effect of EGF, E2 and Ginsenosides on proliferation of cultured PGCs were evaluated, together with the underlining mechanisms. Meanwhile, we also investigate the effect of RA on the initiation of meiosis in chicken PGCs. These studies will provide theoretic guidance and experimental platform for improving poultry reproductive performance and preparation of transgenic poultry.1. Isolation, culture and identification of chicken PGCsPGCs were collected from stage 14 chicken embryonic blood, stage 19 embryonic genital ridges or stage 27 embryonic gonads with a fine glass needle under a microsurgery scope. For primary culture, cell suspension containing both PGCs and somatic cells was seeded onto gelatin-treated 35 mm culture plates at a density of 1×106/well in DMEM supplemented with 5% fetal calf serum (FCS), 10ng/ml leukemia inhibitory factor (LIF),10ng/ml human basic fibroblast growth factor (bFGF), 0.1mmol/L MEM nonessential amino acids, 0.1mmol/L 2-mercaptoethanol, 2mmol/L L-glutamine (GIn), 100U/ml penicillin and 100μg/ml streptomycin. The seeded cells were then maintained at 38.5℃in 5% CO2/95% air with 60%-70% relative humidity until the PGCs colonized as a primary culture. To trace the origin of the colonies, the primary formed colonies were picked up with a fine glass needle, dissociated with 0.25% trypsin-EDTA 5 days after plating and then subjected to RT-PCR analysis for expression of PGC-specific markers. For further subculture, colonies that were positive for PGC markers were picked up and treated with 0.25% trypsin-EDTA to achieve single cell suspension and reseeded onto 6-well dishes. After three passages, staining of periodic acid-Schiff regent (PAS), stage-specific embryonic antigens (SSEA-1, SSEA-3 and SSEA-4) immunocytochemistry, and the expression of the pluripotency-associated genes cPouV, cNanog and Sox2; germ cell-specific genes c-kit, Vasa, Dazl, Stra8 and Scyp3 analysis all confirmed the characteristics of cultured PGC. The above results indicated that the primary and subculture models of PGCs could be used for studies about regulation of PGC proliferation.2. Effects of EGF on proliferation of chicken PGCsIn the present study, the effects of EGF and the EGF-related signaling pathway on proliferation of chicken primordial germ cells (PGC) were investigated. Results showed that EGF (10-100 ng/ml) increased the number and area of PGC colonies in a time and dose-dependent manner. EGF also activated PKC, a process that was inhibited by AG1478 (an EGFR tyrosine kinase inhibitor) and EGTA (an intracellular Ca2+ chelator). In addition, the degradation of IκBαand nuclear factor NF-κB (p65) translocation were observed after EGF treatment, which were significantly blocked by pretreatment with AG1478, EGTA, H7 or SN50 (NF-κB-specific inhibitor). Furthermore, we found that EGF-induced cell proliferation was significantly attenuated by AG1478, EGTA, H7 and SN50, respectively. On the other hand, inhibition of EGFR, Ca2+/PKC or NF-kB abolished the EGF-stimulated increase in the expression of cyclins CCND1 and CCNEI, cyclin-dependent kinase 6 (CDK6), CDK2 and BCL2, and restored the EGF-induced inhibition of BAX expression and caspase3/9 activity, indicating that EGFR, PKC and NF-kB signaling cascades were involved in EGF-stimulated DNA synthesis and anti-apoptosis action. In conclusion, EGF stimulated proliferation of chicken PGCs via activation of Ca2+/PKC involving NF-κB signaling pathway. These observations suggest that EGF signaling is important in regulating germ cell proliferation in the chicken embryonic gonad.3. Involvement of GPR30 on E2-induced proliferation of chicken PGCsIn the present study, The effect of 17β-Estradiol (E2) on chicken primordial germ cells (cPGC) was evaluated and the involvement of GPR30 as well as the underlying signaling pathway were investigated. Results showed that after injection of 100μg/ml E2 into dayO eggs, the percentage of SSEA-1 positive cells in day5.0 embryonic gonad was increased. Using the cPGC culture in vitro system, we first demonstrated that cPGCs express GPR30, however, both ERa and ERβwere not detected. Treatment with (1-100 nM) E2 significantly increased the area and number of cPGC colonies in a time-and dose-dependent manner. E2 also activated Akt, a process that was inhibited by AG1478 (EGFR inhibitor), LY294002 (PI3K inhibitor) or silencing GPR30 expression. However, ER inhibitor ICI182780 had no obvious inhibitory effect on this response. In addition, the phosphorylation of GSK3P and P-catenin translocation were observed after E2 treatment, which were significantly blocked by pretreatment with AG1478, LY294002, KP372-1(Akt inhibitor) and was enhanced by GSK3 inhibitor BIO. Furthermore, we found that E2-induced cell proliferation was significantly attenuated by AG1478, LY294002, KP372-1 or silencing GPR30 and was accelerated by BIO or GPR30 agonist G-1. On the other hand, E2 increased expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2 and protooncogenes (c-fos and c-myc). Increases of these cell cycle regulators and protooncogenes were abolished by inhibition of GPR30, EGFR, PI3K/Akt, or GSK3β/β-catenin. In conclusion, E2 stimulated proliferation of chicken PGCs via GPR30, EGFR, PI3K/Akt, and GSK3β/β-catenin signaling cascades. These observations suggest that E2/GPR30 signaling might play an important role in regulating germ cell development in the chicken embryonic gonad of early stage.4. Effects of ginsenosides on PGC in vitro expansionThe effect of ginsenosides on proliferation of chicken primordial germ cells (PGCs) was evaluated and involvement of NF-κB in the signaling pathway was investigated. PGCs were isolated from the genital ridge of 3.5-4 day embryos and cultured in DMEM supplemented with 5%FCS and 10 ng/ml LIF. PGCs subcultured on chicken embryonic fibroblast feeder were challenged with ginsenosides alone or in combination with PKC inhibitor H7 or activator phorbol 12-myristate 13-acetate (PMA) for 24h. Moreover, the translocation of NF-κB and degradation level of IκBαwas investigated by Western blotting analysis. Results show that PGCs were identified by periodic acid-Schiff, alkaline phosphatase histochemistry as well as c-kit, SSEA-1 and Oct-4 immunocytochemistry. Treatment with ginsenosides at 1-100μg/ml significantly increased the number and area of PGC colonies in a dose-dependent manner. However, this proliferating effect was obviously attenuated by combined treatment of H7(10-6-10-8M). Similarly, PKC staining of PGC colonies was more intensive after ginsenosides treatment compared with the control group. In addition, treatment with ginsenosides at 1-10μg/ml stimulated the translocation of NF-κB (p65). However, the NF-κB translocation and the degradation of IκBαwere significantly blocked by combined treatment with H7 (10-6M). These results indicated that ginsenosides promote proliferation of chicken PGCs through activation of PKC-involved NF-κB signaling pathway.5. Effect of RA on meiosis initiation of chicken PGCsHere we used the successful long-term culture of chicken PGCs, to investigate if retinoid acid (RA) play a conserved role in regulating entry into meiosis, and does RA act directly on PGCs or indirectly. Results showed that, using organ culture in vitro, addition of RA in stage 27 chicken gonads of both sexes siginificantly increase the mRNA expression of premeiotic gene Stra8, as well as meiotic markers Sycp3 and Dmcl. Using purified chicken PGCs culture with or without feeder layer, RA dramatically upregulated the Stra8 in both male and female PGCs, in parallel to similar increases in expressions of Dmcl and Sycp3. Flow cytometry analysis showed that, after 4-day RA treatment,29.5% male PGCs and 58.37% female PGCs were at sub G1 phase, indicating cells had entered meiosis. Statistatical analysis of giemsa stained cells showed the difference between female and male PGCs in the capability to enter into meiosis induced by RA and reach the zygotene/pachytene stage, male PGCs appeared lower capability to progress beyond zygotene/pachytene. In conclusion, we reported here for the first time that RA could induced chicken PGCs of both sexes to enter meiosis, and female PGCs respond more intensively to RA.The above results indicated that the primary and subculture models of PGCs from chicken embryos of different stages could be used to study the effects of endogenous and exogenous factors on proliferation and differentiation of cultured PGCs. PGCs were characterized by staining of AKP, PAS, c-kit and SSEA-1,3,4 immunocytochemistry. The pluripotency of PGCs was also demonstrated. EGF and GS were found to promote PGC proliferation through PKC-NF-κB signaling pathway. We first reported the involvement of the novel estrogenic receptor,GPR30 in the E2-induced proliferation of chicken PGC. We also first demonstrated the onset of meiosis in chicken PGC induced by RA. These findings provide theoretic guidance and experimental platform for improving poultry reproductive performance and preparation of chimeras and transgenic poultry.更多还原