生殖基本知识

Chapter2哺乳类生殖生物学基础2.1.基本概念配子：精子sperm,卵母细胞（卵子）oocyte(egg)受精卵胚胎embryo胎儿fetusday11(Mouse)after9wk(human)生殖内分泌学生殖生理学♀♂♂性内分泌系统睾丸结构精子发生结构---运动射精性周期绝经受精排卵卵泡发生乳腺生物学泌乳---哺乳胎儿---母体妊娠子宫分娩妊娠基因表达着床胎盘性别决定卵巢生理子宫生物学性别分化♀性生殖系统结构---生理生殖激素生殖激素ReproductiveEndocrinology激素来源、靶器官和功能Hormones:Sources,TargetsandFunctionsSourceHormoneTargetPhysiologicEffect下丘脑GnRHPituitary,tissuesStimulatesFSH/LHsecretionAnteriorpituitary腺垂体Follicle-stimulatinghormone(FSH)OvaryandTestesGrowthofovarianfolliclesorseminiferoustubulesLuteinizinghormone(LH)OvaryandTestesProductionofestrogenandprogesteroneortestosteroneposteriorpituitaryOxytocin催产素UterusandMammaryStimulatescontractionandsecretionofmilkPinealgland松果腺MelatoninGonads,pigmentcellsControlbiorhythmsinfluencereproductionOvaryEstrogenestradiolGeneraluterusDevelop/maintainfemalecharacteristicsstimulategrowthofuterineliningProgestrogenUterus,breastStimulatedevelopmentofuterineliningPlacentaChorionicgonadotropinAnteriorpituitaryStimulatesreleaseofFSHandLHTestes睾丸TestosteroneGeneralandreprod-uctivestructuresDevelopsandmaintainsmalesexcharacteristicspromotesspermatogenesisRelationofthepituitaryglandtothebrainandhypothalamus♀生殖周期中激素的反馈调节♀发情周期PhysiologyofReproduction1、雄性生殖器官与精子（sperm）产生精子合成和分泌类固醇激素、神经肽、鸦片肽、蛋白水解酶等。精原干细胞、精子载体睾丸注射GENESpermandspermvector2、Femalereproductiveorgans1.输卵管oviduct；2卵巢；3子宫3、Ovary冷冻与移植Mouse:Szteinetal,2019;JacksonLab;Shawetal,2000;Manipulatingthemouseembryo,3rdedscow：Shawetal,2000;AgcaY,2000Sheep:Salleetal,20191.为什么进行卵巢冷冻和移植？①基础研究②患癌症的女性，放疗和高剂量的化疗都能导致不孕2.标准：developmenttotermrat卵巢移植Mice：Jones&Krohn;Parrott,1960;Sheep:Gosdenetal,1994免疫问题MonkeyBRENDA：Leeetal,Nature,2019,428:137-84、卵泡发育与卵子成熟mouseRelationshipbetweenoocyteandfolliclecellsTraditionalopinion:1.在卵子发生过程中，卵泡细胞养育卵母细胞2.卵泡细胞通过排卵发生将卵母细胞输送到输卵管中RecentStudies：1.卵泡发生过程中，卵母细胞具有主动的关键作用2.卵母细胞与卵泡细胞间建立的双向通信，是卵子的获得受精力，以及早期胚胎发育是必需的Ovulation：卵母细胞旁分泌的GDF9、BMP-15和卵巢外LH主导Oocyte-granulosacellcommunicationisessentialfornormalgrowthanddevelopmentofboththeoocyteandthefollicle成年动物原始卵泡再生---Rewritetextbook！femalemammalsproducenoneweggsafterbirthmouseovariesharborapreviouslyundiscoveredtypeofstemcellthatcanformneweggsthroughadulthood.newJohnsonetal,2019Germ-cellproliferationinjuvenileandyoungadultovariesa示卵巢表面上皮可能的GermlineStemCells(arrowheads)，这些细胞(b)表达生殖细胞特有的MVH蛋白(mousevasahomologue,免疫偶联黄色产物，b)。这些细胞保持着正常的有丝分裂生长过程（c）.采用BrdU(red)和MVH(green)双染色方法，证明这些生长的生殖干细胞存在于幼年（d）和青年母鼠（e）的卵巢上。F图是用苏木精和伊红染色的d图卵巢组织。分裂前期(g)和中期(h)的阳性干细胞（绿色）Meiosisofoocyte小鼠成熟Oocyte的参数卵母细胞中mRNA的储存Oocytegrowth(40m85m)andaccumulationofoocyteRNAaccumulationofoocytet-PAmRNAduringoocytegrowth(40oocytes/lane)OocytegrowthandaccumulationofoocyteRNA(~300-foldincrease,12m85m)卵母细胞的IVM(invitromaturation)•Media•Hormone•GrowthFactor•aa卵母细胞的冷冻与解冻vitrificationThawingFreezing卵子形态结构DictyatestageovulationM-IIstageZonaZona1.小鼠ZP1，ZP2，ZP3分别为200，120和83KD的糖蛋白。2.由生长的卵母细胞连续合成和分泌生成Zona。糖基化在高尔基体中完成。小鼠透明带1.ZP1：维持透明带的立体结构。2.ZP2:精子第二受体。3.ZP3：精子第一受体、诱导精子的顶体反应。a.主肽链402aa,44KD,b.3-4条N-oligos(稳定蛋白质结构，防止内源性酶的降解)，c.6条丝氨酸/苏氨酸（Ser/Thr）连接的O-oligos.d.编码基因：5号染色体，单拷贝，8个exon，exon7高度变异，由其编码的25个氨基酸残基及其糖基化位点决定了受体功能和透明带反应，保证了受精过程的种间特异性。ZP2ZP2ZP2ZP2ZP2ZP2ZP2ZP2ZP2ZP2ZP2ZP3ZP3ZP3ZP3ZP3ZP3ZP3ZP3ZP3ZP1ZP3ZP3ZP3ZP2ZP1－6.2m－ZP3的生物学功能之一1.主肽链诱导精子发生顶体反应2.O-oligos末端的非还原性单糖（可能为-半乳糖或N-乙酰葡糖胺），参与识别精子表面的透明带结合蛋白（ZBP，zonabindingprotein），介导透明带与精子的第一次结合，行使精子受体的功能。ZP3的生物学功能之二Zonareactionandblockspermpenetrationtechniqueofretroviralvector5、FertilizationOutlineofmammalianfertilizationgametesarriveatfertilizedregionStep1spermpenetratesthecumuluscellslayerPH-20glycoproteinofspermholdingtheactivityofhyaluronidaseStep2spermrecognizes,binds,andpenetratesthroughthezonaofoocyte①识别、结合②AcrosomereactionofspermAcrosomereaction,penetrationStep2spermrecognizes,binds,andpenetratesthroughthezonaofoocyteStep2spermrecognizes,binds,andpenetratesthroughthezonaofoocyteZonareactionStep3.识别和融合Step4Corticalreactionandpolyspermyblock融合时的变化：阻止多精子入卵融合同时，质膜电位快速去极化，胞内游离〔Ca2+〕瞬时升高，pH值升高，皮质颗粒的释放卵母细胞的激活变化融合后的变化：“唤醒”卵母细胞，完成周期–形态：PB2的排出、精子核膜崩解、精子染色体去浓缩、雌雄原核形成、双原核向卵中央迁移、原核核膜崩解，进入第一次有丝分裂。–生理：〔Ca2+〕升高和/或Ca2+振荡、受精膜电位的形成、pH值升高–生化：母源mRNA的翻译、功能蛋白的修饰、细胞周期因子的活性变化、DNA合成的启动。卵母细胞激活的关键•胞内游离〔Ca2+〕升高是关键信号–钙池中Ca2+动员的模式Ca2+振荡–胞外Ca2+的流入是维持Ca2+振荡必需的–Ca2+振荡时，Ca2+泵动员1个Ca2+将耗掉1～2ATP，生物体采用这样一个效率低下的信号机制，意义何在？精子激活卵母细胞的初始信号•受体假说•激活因子假说PIP2.磷脂酰肌醇酯；PLC.磷脂酶C；IP3.三磷酸肌醇；DAG.甘油二酯G?PTKCa2+IP3DAGPKC???CG释放、PB2排出、双原核形成………PIP2.磷脂酰肌醇酯；PLC.磷脂酶C；IP3.三磷酸肌醇；DAG.甘油二酯截短c-kit?寡聚oscillin?Ca2+IP3DAGPKC??CG释放、PB2排出、双原核形成………?NO?Furtherchange:pronuclearformationandPB2extrusionMicroinjectionMicewithoutafather•Inmammals,genomesfrombothparentsaregenerallyneededtomakeviableoffspring.Butchangingtheexpressionof‘imprinted’genescanrenderthefather’scontributiondispensable.Loebel&Tam,2019MicewithoutafathernormalgeneexpressionofH19/Igf2Loebel&Tam,2019TheH19andIgf2genesarefoundonthesamemousechromosomeandareoppositely‘imprinted’:innormalembryos,H19isexpressedonlyfromthematernalchromosomeandIgf2onlyfromthepaternalchromosome.Onthematernalchromosome,theproteinCTCFbindstothedifferentiallymethylateddomain(DMD),blockingtheaccessofenhancerstoIgf2.Th