【作者】 张大雷；

【导师】 张才乔；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2007， 博士

【摘要】 哺乳动物的生殖细胞发育是非常复杂的过程,受到多种激素、生长因子以及外源营养物质的调控。植物性生物活性物质人参皂甙(ginsenosides, GS)和大豆黄酮(daidzein, DAI)具有多种生物学功能,如抗氧化作用、雌激素样作用以及提高繁殖性能等,因而成为当前相关研究和开发的热点之一。然而,GS和DAI对生殖细胞发育的作用尚不清楚。因此,本实验以体外培养的小鼠A型精原细胞和卵母细胞为实验模型,研究GS和DAI对A型精原细胞增殖和卵母细胞减数分裂成熟的作用并探讨其机理,为植物性生物活性物质对哺乳动物生殖细胞发育的营养调控作用提供理论指导。1.小鼠A型精原细胞增殖的激素调控出生6天的ICR小鼠的睾丸组织用胶原酶分散成单个细胞,建立精原细胞-体细胞体外无血清共培养系统。经c-kit免疫细胞化学鉴定表明所分离和培养的生殖细胞为A型精原细胞。增殖细胞核抗原免疫组织化学检测表明添加了10μg/ml胰岛素、5μg/ml转铁蛋白和3×10-8mol/L亚硒酸钠的DMEM培养液(ITS培养液)可维持A型精原细胞的存活和增殖。本实验利用所建立的培养模型研究了卵泡刺激素(follicle-stimulating hormone, FSH)、表皮生长因子(epidermal growth factor, EGF)、睾酮(testosterone, T)和雌二醇(17β-estradiol, E2)对小鼠A型精原细胞增殖的影响,从而验证该培养模型在外源因素对生殖细胞发育影响研究上的适用性。EGF及其受体(EGFR)免疫细胞化学结果表明EGF和EGFR主要表达于精原细胞。单独的FSH (1～100 ng/ml)或EGF(1～10ng/ml)显著促进精原细胞的增殖。FSH(10ng/ml)联合EGF (0.1 ng/ml)具有加性效应,但与更高剂量的EGF (1～10 ng/ml)联合处理则降低了FSH的刺激作用。此外,T (10-8～10-6mol/L)和E2(10-8～10-6mol/L)均能促进小鼠A型精原细胞体外增殖,雄激素受体阻断剂氟硝丁酰胺(flutamide, FLU)或雌激素受体阻断剂他莫昔芬(tamoxifen, TMX)可分别抑制T或E2的促增殖作用,这说明T和E2是通过体细胞的介导而发挥其促进精原细胞增殖的作用。以上结果表明生殖细胞-体细胞共培养模型中的精原细胞对促性腺激素和性激素以及生长因子具有较好的反应性,FSH和T及E2可通过共培养模型中的体细胞分泌的旁分泌因子间接作用于生殖细胞,EGF直接作用于生殖细胞,从而促进精原细胞的增殖。这说明本实验建立的培养模型是一种评价A型精原细胞体外增殖调控的合适模型,并可用于哺乳动物精原细胞发育调控机理的研究。2.人参皂甙对小鼠A型精原细胞增殖的影响利用精原细胞-体细胞共培养体系来评价GS对小鼠A型精原细胞增殖的调控作用。精原细胞培养72 h后,GS (1.0～10μg/ml)能够显著刺激A型精原细胞的增殖,精原细胞集落的数目和面积均显著高于对照组。蛋白激酶C (protein kinase C, PKC)的激活剂佛波酯(phorbol 12-myristate 13-acetate, PMA,10-8～10-7mol/L)显著促进精原细胞的增殖,这一作用可被PKC的抑制剂H7所抑制。此外,腺苷酸环化酶激活剂福司克林(forskolin, FSK,10-6～10-5 mol/L)也可刺激小鼠A型精原细胞的增殖,这一作用可被蛋白激酶A(protein kinase A, PKA)抑制剂H89所抑制。这些结果表明PKA和PKC信号转导途径的激活均能促进A型精原细胞的增殖。此外,GS和PMA具有协同作用。H7可显著抑制GS诱导的A型精原细胞的增殖。然而,H89对GS诱导的A型精原细胞增殖无显著影响,说明GS的作用并不是通过PKA信号途径介导的。以上的结果表明PKA和PKC信号通路在小鼠A型精原细胞的增殖过程中起着重要的调控作用,GS可能通过PKC介导的信号转导途径促进小鼠A型精原细胞的增殖。3.大豆黄酮对多氯联苯引起的小鼠睾丸生殖细胞损伤的缓解作用A型精原细胞在体外培养时呈集落生长,而3周龄小鼠的生殖细胞呈单个生长,更利于散在细胞形态变化的观察,因此,本实验利用体外培养的小鼠睾丸生殖细胞研究了典型内分泌干扰物多氯联苯Aroclor 1254 (A 1254)对生殖细胞损伤的机理,并探讨了DAI对A1254引起的睾丸生殖细胞氧化损伤的缓解作用及其作用机制。DAI是一类广泛分布在豆类植物中的多酚类化合物,具有抗氧化作用。多氯联苯(polychlorinated biphenyls，PCBs)是一类难分解的氯代芳烃族环境污染物,对人和动物的各种组织都有很强的毒性作用,特别易诱发发育异常和生殖系统的损伤。本实验用活性氧自由基产生系统次黄嘌呤(hypoxanthine, HX)/黄嘌呤氧化酶(xanthine oxidase, XO)系统来评价DAI的抗氧化作用。结果表明,A1254(20～30μg/ml)以及HX/XO系统(10-5 mol/L; 2.5×10-3IU/ml)产生的活性氧对睾丸生殖细胞具有严重的损伤作用,导致细胞破裂,存活的细胞数目明显减少。DAI (10μg/ml)可明显缓解A1254或HX/XO系统引起的生殖细胞的氧化损伤,使存活的细胞数目显著增加,并恢复A1254引起的超氧化物歧化酶(superoxide dismutase, SOD)活性和谷胱甘肽(glutathione,GSH)水平的下降以及丙二醛(malondialdehyde, MDA)生成的增加。结果说明DAI具有抗氧化活性,能够缓解A1254引起的生殖系统氧化损伤,因此DAI对于A1254引起的生殖毒性具有重要的缓解作用。4.人参皂甙对小鼠卵母细胞成熟的影响本实验利用小鼠卵母细胞体外培养模型研究GS对卵母细胞减数分裂成熟的影响,从而进一步评价GS对生殖健康的作用。处于生发泡(germinal vesicle, GV)期的卵丘卵母细胞复合体(cumulus oocyte complexes, COC)和去卵丘的卵母细胞(denuded oocytes, DO)取自于性未成熟的ICR小鼠。生发泡破裂(germinal vesicle breakdown, GVBD)和第一极体(first polar body, PB1)排出的情况作为判断卵母细胞减数分裂成熟的指标。HX (4mmol/L)可抑制卵母细胞的自发成熟。FSH能克服HX的抑制,显著诱导COC恢复减数分裂成熟。以上的结果说明HX维持卵母细胞减数分裂阻滞的体外培养系统是能够模拟体内环境研究外源因素对卵母细胞发育成熟影响的合适模型。本实验利用该培养模型研究了GS对卵母细胞成熟的影响,结果表明1.0～10μg/ml的GS可克服HX维持的减数分裂阻滞,促进COC恢复减数分裂成熟,GVBD发生率和PB1排出率显著提高,而GS对DO没有作用。此外,GS(1.0μg/ml)和FSH(0.1 IU/ml)具有协同作用。TMX和Nω-硝基-L-精氨酸甲酯(Nω-nitro-L-arginine methyl ester, L-NAME,一种NO合酶抑制剂)被用来研究GS诱导卵母细胞减数分裂成熟的机制。TMX对GS诱导的卵母细胞减数分裂无显著影响,说明GS并不是通过雌激素样作用介导的。L-NAME (1mmol/L)可显著抑制GS诱导的减数分裂成熟,GVBD发生率和PB1排出率显著低于GS处理组。免疫组织化学染色的结果也表明GS能增加卵丘细胞上诱导型一氧化氮合成酶(inducible nitric oxide synthase, iNOS)的表达,L-NAME则可抑制GS引起的iNOS表达的增加。结果表明GS可能通过NO/iNOS体系以旁分泌的方式促进小鼠卵母细胞的成熟。结论：本实验建立了小鼠雄性的A型精原细胞和雌性的卵母细胞体外培养模型,发现添加胰岛素、亚硒酸钠和转铁蛋白的ITS培养体系能够很好地维持精原细胞的存活。多种生殖激素(FSH、T和E2)和生长因子EGF均能够促进A型精原细胞的增殖。此外,PKA和PKC信号转导途径的激活也可促进精原细胞的增殖。GS通过PKC信号途径促进A型精原细胞的增殖。此外,植物性抗氧化剂DAI可以通过抗氧化活性缓解A1254引起的生殖细胞的氧化损伤。GS可作用于卵丘细胞,通过NO/iNOS的旁分泌体系促进卵母细胞的减数分裂成熟。这些结果将为哺乳动物生殖细胞发育的营养调控以及动物繁殖性能的提高提供理论基础。更多还原

【Abstract】 The development of mammalian germ cells requires complex regulation of multiplicate hormones, growth factors and extrinsic nutritional substances. Ginsenosides (GS) and daidzein (DAI) have multiple biological functions, including antioxidative activity, estrogenic activity and reproductive regulation. However, the actions of GS and DAI on reproduction remain unclear. Therefore, the present study adopted type A spermatogonia and oocytes of mouse to evaluate effects of GS or DAI on spermatogonial proliferation and oocyte meiotic maturation and provided evidences for nutritional regulation of vegetal biological active substances on development of germ cells.1. Regulations of hormones on mouse type A spermatogonial proliferation.The serum-free germ-somatic cell coculture model was established to evaluate effects of hormones and growth factors on type A spermatogonial proliferation. Testicular cells of 6-day-old ICR mouse were dispersed by collagenase and c-kit immunocytochemistry identified type A spermatogonial cells. Immunocytochemical staining for proliferating cell nuclear antigen (PCNA) showed that ITS medium supplemented with insulin (10μg/ml), transferring (5μg/ml) and selenite (3x10-8 mol/L) could maintain spermatogonial cell survival and proliferation. The present study evaluated the effects of follicle-stimulating hormone (FSH), epidermal growth factor (EGF), testosterone (T) and 17β-estradiol (E2) on mouse type A spermatogonial proliferation. EGF and epidermal growth factor receptor (EGFR) were mainly expressed on type A spermatogonial cells. FSH (1～100 ng/ml) or EGF (1～10 ng/ml) treatment alone significantly stimulated spermatogonial cell proliferation. Furthermore, FSH (10 ng/ml) could synergize with EGF (0.1 ng/ml) to promote cell proliferation, but higher EGF at 1～10 ng/ml inhibited FSH-induced stimulating effect. T (10-8～10-6 mol/L) and E2 (10-8～10-6 mol/L) promoted proliferation of type A spermatogonia. Flutamide (FLU, an testosterone receptor antagonist) inhibited T-stimulated spermatogonial proliferation, and tamoxifen (TMX, an estrogen receptor a antagonist) inhibited the action of E2. These results showed that the germ-somatic cell coculture model allowed evaluating regulation of gonadotropin, sex steroids and growth factors on spermatogonial proliferation. FSH, T and E2 exerted stimulating actions through a paracine pathway involving indirect effects on somatic cells and EGF promoted spermatogonial proliferation through direct effects on spermatogonia. The above results indicated that the serum-free germ-somatic cell coculture model was appropriate to evaluate effects of extrinsic factors on type A spermatogonial proliferation. Furthermore, the model would be available to study the regulating mechanism of spermatogonial development.2. Effect of ginsenosides on proliferation of type A spermatogoniaThe serum-free germ-somatic cell coculture model was used to evaluate the effect of GS on proliferation of type A spermatogonia. After 72 h culture, Ginsenosides (1.0～10μg/ml) significantly stimulated proliferation of spermatogonia assessed by increased the number and area of spermatogonial colonies. Activation of protein kinase C (PKC) by Phorbol 12-myristate 13-acetate (PMA) elicited proliferation on spermatogonia at 10’8 to 10-7mol/l and PKC inhibitor H7 inhibited this effect. Likewise, forskolin (FSK), an activator of protein kinase A (PKA) exerted proliferating action on type A spermatogonia at concentration of 10-6-10-5 mol/1 and PKA inhibitor H89 inhibited this effect, suggesting activation of PKC and PKA involved type A spermatogonial proliferation. Furthermore, GS (1.0μg/ml) could synergize with PMA to promote spermatogonial proliferation. GS-stimulated spermatogonial proliferation was suppressed by combined treatment with H7. However, H89 did not influence GS-stimulated spermatogonial proliferation, suggesting the effect was not mediated by PKA pathway. These results indicated that activation of protein kinases A and C promoted proliferation of mouse type A spermatogonia and the proliferating effect of GS might be mediated by a mechanism involving the PKC signaling transduction pathway.3. Attenuating effect of daidzein on polychlorinated biphenyls-induced toxicity in mouse testicular germ cellsType A spermatogonial cells form colonies in culture. However, germ cells of 3-week-old mouse grow dispersively in vitro, which accord facilities for observation of cell morphological change. Therefore, the present study adopted mouse testicular germ cells to evaluate the attenuating effects of DAI on A1254-induced oxidative damage in testicular germ cells, with emphasis on the protective mechanism. DAI belongs to the most common isoflavones existed in leguminous plants and possesses multiple pharmacological effects. The most important biological action of DAI contributes to the antioxidant activity. Polychlorinated Biphenyls (PCBs) are members of environmental contaminations, which belong to the halogenated hydrocarbon class and impose toxic effects on normal development and reproductive system of human and animal. PCBs were ubiquitous in the environment due to the persistent and lipophilic characters. Cells were exposed to the reactive oxygen species (ROS)-producing system hypoxanthine/xanthine oxidase (HX/XO) to determine the antioxidant action of DAI. Results showed that A1254 (20～30μg/ml) and HX/XO (10-5M; 2.5×10-3IU/ml) exerted toxic effects on germ cells, induced oxidative damage and cell death. DAI obviously attenuated the oxidative damage induced by PCB and HX/XO system, increased the cell number, and restored A1254-induced decrease in superoxide dismutase (SOD) activity and glutathione (GSH) level and A1254-induced increase in malondialdehyde (MDA) formation. These results indicated DAI exerted antioxidant activity and attenuated A1254-induced oxidative damage in mouse testicle germ cells. Therefore, DAI would play critical role in preventing PCBs-induced generative toxicity.4. Effect of ginsenosides on mouse oocyte maturationMouse oocyte culture model was established to determine the role of GS on meiotic maturation of murine oocytes and further evaluate our understanding of effects of GS on generative health. Cumulus oocyte complexes (COC) and denuded oocytes (DO) in germinal vesicle (GV) stage were isolated from pregnant mare serum gonadotropin (PMSG)-primed immature mice. Germinal vesicle breakdown (GVBD) and first polar body (PB1) extrusion were used as the mark of oocyte meiotic maturation. Oocytes in GV stage could undergo spontaneous maturation after they cultured for 24 h in 10% fetal calf serum (FCS) medium. Hypoxanthine (HX, 4 mmol/L) maintained meiotic arrest and inhibited mouse oocytes from resuming meiosis in vitro maturation. Follicle-stimulating hormone (FSH) induced mouse oocytes to overcome the HX-maintained and resume meiosis in COC. The result showed this model was appropriate to evaluate effects of extrinsic factors on oocyte development. GS (1.0-10μg/ml) stimulated meiotic maturation in COC assessed by increased GVBD and PB1 extrusion. No effect of GS was observed on DO. In addition, GS (1.0μg/ml) could synergize with FSH (0.1 IU/ml) to promote oocyte meiotic maturation in COC. TMX did not influence GS-induced meiotic maturation, suggesting the effect was not mediated by estrogen. However,1 mmol/L Nω-nitro-L-arginine methyl ester (L-NAME, a nitric oxide (NO) synthase inhibitor) reduced the GS-induced oocyte maturation in COC. Immunocytochemical analysis demonstrated that GS enhanced expression of inducible nitric oxide synthase (iNOS) in the cumulus cells. L-NAME reduced the immunocytochemically detected increase in iNOS following ginsenoside exposure. These results suggested that GS might stimulate murine oocytes meiotic maturation through a paracine pathway involving the NO/iNOS system.Conclusion:Mouse type A spermatogonial cell and oocyte culture models were established to determine the roles of vegetal biological active substances on development of germ cells. These results showed ITS medium supplemented with insulin, transferrin and selenite could maintain spermatogonial cell survival. FSH, T, E2, EGF and activation of PKA and PKC could promote type A spermatogonial proliferation. GS promoted type A spermatogonial proliferation by a mechanism involving the PKC signaling transduction pathway. In addition, DAI exerted antioxidant activity and attenuated A1254-induced oxidative damage in mouse testicle germ cells. GS might stimulate murine oocytes meiotic maturation through a paracine pathway involving the NO/iNOS system. These results will provide theoretic guidance for nutritional regulation of germ cell development and improvement of reproductive performance.更多还原