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1、Chapter 2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 fetusday 11(Mouse)after 9 wk(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,精子,结构-运动,射精,性周期 绝经,受精,卵子,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,Reproductive Endocrinology,激素来源、靶器官和功能,
2、Relation of the pituitary gland to the brain and hypothalamus,生殖周期中激素的反馈调节,发情周期,Physiology of Reproduction,1、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GENE,Sperm and sperm vector,2、Female reproductive organs,输卵管oviduct；2 卵巢；3 子宫,3、Ovary 冷冻与移植,Mouse:Sztein et al,1998;Jackson Lab;Sh
3、aw et al,2000;Manipulating the mouse embryo,3rd edscow：Shaw et al,2000;Agca Y,2000Sheep:Salle et al,2003,为什么进行卵巢冷冻和移植？基础研究患癌症的女性，放疗和高剂量的化疗都能导致不孕标准：development to term,rat,卵巢移植,Mice：Jones Sheep:Gosden et al,1994,免疫问题,Monkey BRENDA：Lee et al,Nature,2004,428:137-8,4、卵泡发育与卵子成熟,mouse,Relationship between
4、 oocyte and follicle cells,Traditional opinion:在卵子发生过程中，卵泡细胞养育卵母细胞卵泡细胞通过排卵发生将卵母细胞输送到输卵管中,Recent Studies：卵泡发生过程中，卵母细胞具有主动的关键作用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-granulosa cell communication is essential for normal growth and development of both the o
5、ocyte and the follicle,成年动物原始卵泡再生-Rewrite textbook！,female mammals produce no new eggs after birth,mouse ovaries harbor a previously undiscovered type of stem cell that can form new eggs through adulthood.,new,Johnson et al,2004,Germ-cell proliferation in juvenile and young adult ovaries,a示卵巢表面上皮可能的
6、Germline Stem Cells(arrowheads)，这些细胞(b)表达生殖细胞特有的MVH蛋白(mouse vasa homologue,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）,Meiosis of oocyte,小鼠成熟Oocyte的参数,卵母细胞中mRNA的储存,Oocyte growth(40m 85m)and accumulation o
7、f oocyte RNA,accumulation of oocyte t-PA mRNA during oocyte growth(40 oocytes/lane),Oocyte growth and accumulation of oocyte RNA(300-fold increase,12 m 85 m),卵母细胞的IVM(in vitro maturation),MediaHormoneGrowth Factoraa,卵母细胞的冷冻与解冻 vitrification,Thawing,Freezing,卵子形态结构,Dictyate stage,ovulation,M-II stage
8、,Zona,Zona,小鼠ZP1，ZP2，ZP3分别为200，120和83KD的糖蛋白。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,ZP1：维持透明带的立体结构。ZP2:精子第二受体。ZP3：精子第一受体、诱导精子的顶体反应。主肽链402aa,44KD,3-4条N-oligos(稳定蛋白质结构，防止内源性酶的降解)，6条丝氨酸/苏氨酸（Ser/Thr）连接的O-oligos.编码基因：5号染色体，单拷贝，8个exon，exon 7 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。,ZP2,ZP2,Z
9、P2,ZP2,ZP2,ZP2,ZP2,ZP2,ZP2,ZP2,ZP2,ZP3,ZP3,ZP3,ZP3,ZP3,ZP3,ZP3,ZP3,ZP3,ZP1,ZP3,ZP3,ZP3,ZP2,ZP1,6.2 m,ZP3的生物学功能之一,主肽链诱导精子发生顶体反应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，zona binding protein），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,Zona reaction and block sperm penetration,technique of ret
10、roviral vector,5、Fertilization,Outline of mammalian fertilization,gametes arrive at fertilized region,Step 1 sperm penetrates the cumulus cells layer,PH-20 glycoprotein of sperm holding the activity of hyaluronidase,Step 2 sperm recognizes,binds,and penetrates through the zona of oocyte,识别、结合Acrosom
11、e reaction of sperm,Acrosome reaction,penetration,Step 2 sperm recognizes,binds,and penetrates through the zona of oocyte,Step 2 sperm recognizes,binds,and penetrates through the zona of oocyte,Zona reaction,Step 3.识别和融合,Step 4 Cortical reaction and polyspermy block,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
12、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12 ATP，生物体采用这样一个效率低下的信号
13、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,PIP2,PLC-beta,PLC-gamma,G,?,PTK,Ca2+,IP3,DAG,PKC,?,?,?,CG释放、PB2排出、双原核形成,受体学说,PIP2.磷脂酰肌醇酯；PLC.磷脂酶C；IP3.三磷酸肌醇；DAG.甘油二酯,PIP2,PLC？,截短 c-kit?寡聚oscillin?,Ca2+,IP3,DAG,PKC,?,?,CG释放、PB2排出、双原核形成,激活因子学说,?,NO,?,Further change:pronuclear
14、formation and PB2 extrusion,Microinjection,Mice without a father,In mammals,genomes from both parents are generally needed to make viable offspring.But changing the expression of imprinted genes can render the fathers contribution dispensable.,Loebel&Tam,2004,Mice without a fathernormal gene express
15、ion of H19/Igf2,Loebel&Tam,2004,The H19 and Igf2 genes are found on the same mouse chromosome andare oppositely imprinted:in normal embryos,H19 is expressed only from the maternal chromosome and Igf2 only from the paternal chromosome.On the maternal chromosome,the protein CTCF binds to the different
16、ially methylated domain(DMD),blocking the access of enhancers to Igf2.Thus,H19 is expressed instead.On the paternal chromosome,the DMD is methylated(represented by CH3),and so CTCF cannot bind;the enhancers therefore have access to Igf2,which is expressed,whereas H19 is silenced.,Mice without a fath
17、er,Loebel&Tam,2004,Kono et al.1 combined the chromosomes from a fully grown egg(which has all maternal imprints)with the chromosomes from a non-growing egg from which the DMD and H19 were deleted(H1913).These deletions mimicked the absence of paternal H19 activity and enabled Igf2 expression,leading
18、 to viable adults.,Normal,Kono et al,Parthenogenetic mice developed to adulthood,Kono et al,2004,Normal reproduction,6、Embryogenesis,Morphological aspects of preimplantation embryos,SEM Morphology of embryos,Development of human preimplantation embryo,Inner cell mass and trophoblast cell layer,Molec
19、ular events during embryogenesis,Gametes nucleus decondensation基因组DNA的启动时相Genomic imprinting功能基因的转录XistTertOct-4（stem cells，略）,:Completion of meiosis,Pronuclei development,1).gametes nucleus decondensation,:Decondensation of the sperm nucleus in ooplasm,1).gametes nucleus decondensation,DNA-Histone
20、DNA-Protamine,Sperm nucleus Y,:Decondensation of the sperm nucleus in ooplasm,1).gametes nucleus decondensation,Sperm nucleus decondensation is independent of egg activation,whereas the transformation of a decondensed sperm nucleus to a pronucleus is dependent on egg activationSperm nucleus deconden
21、sation is earlier than that of oocyte nucleus,concept,2)Maternal to zygotic transition,MZT:maternal to zygotic transition ZGA:zygotic gene activation or,embryonic genome activation.由母体遗传的蛋白质和转录本直接发动的发育程序，被新的发育程序代替，新功能基因开始表达。是胚胎继续发育必需的过程，高等动物比低等动物早,Biological functions of the maternal to zygotic tran
22、sition,2)Maternal to zygotic transition,To destroy oocyte-specific transcripts,such as that for the RNA binding protein MSY2(占卵子总蛋白的2%，维持卵子中 mRNA的稳定)to replace maternal transcripts that are common to the oocyte and early embryo,e.g.actin,with zygotic transcriptsto promote the dramatic reprogramming
23、in the pattern of gene expression that is coupled with the generation of novel transcripts that are not expressed in the oocyte,maternal mRNAs prior to genome activation,2).Maternal to zygotic transition,By the mid murine two-cell stage,many embryonic genes are switched on.Coincidentally,much of the
24、 maternally inherited mRNA appears to be degraded rapidly,but maternally coded proteins can persist beyond this time.,2-细胞期的小鼠胚胎中，90%的maternal mRNAs are degraded,Time of genome activation,2).Maternal to zygotic transition,mouse：2-cell stage(Schultz,1993)bovine,ovine and human：4-to 8-cell stages(Telf
25、ord et al.,1990).,1-cell murine embryo is transcriptionally active and that RNA polymerase I,II and III are functional.,Methylation reprogramming in preimplantation embryos,3).Genomic imprinting,黑：甲基化imprinted genes红：母源的去甲基化的非印迹基因组兰：父源的去甲基化的非印迹基因组,受精后，父源基因组(兰)的去甲基化机制马上激活。对于母源基因组（红）来说，去甲基化机制是被动的，依赖于D
26、NA复制。囊胚分化出胚胎细胞系（EM）和胚胎外细胞系（EX）时，父源和母源基因组同时开始恢复甲基化过程。早期发育过程中，甲基化的印迹基因和一些重复序列（上虚线表示）不会去甲基化；同样，非甲基化的印迹基因（下虚线表示）也不会甲基化。,Concept and biological function,3).X-chromosome inactivation,Leon hypothesis:雌性的一条X染色体随机失活由X染色体上的X-inactivation centre(Xic)控制着X失活的开始、程度、以及哪一条X染色体的失活Xic同时产生Xist transcript,a remarkable
27、RNA覆盖在X染色体的cis部位，发动沉默Xist RNA coating过程使X染色体发生一系列的染色质变化，包括 Polycomb group proteins的复原过程X失活初期脆弱，需要随后的DNA methylation等epigenetic marks进一步来稳定 近期研究发现，小鼠的X失活不仅仅是在附植前阶段完成分裂期的胚胎中，所有细胞中的父源 X最初被灭活，随后在不同的细胞亚类中被选择性的激活，来组织胚胎，最后才发生随机性的失活,X inactivation during pre-implantation mouse development,3).X-chromosome in
28、activation,Xp:paternal genomeXm:maternal genomeHeard,2004,7、implantation and placentation,胎生是哺乳动物在自然选择中出现的有效繁殖模式。哺乳动物子宫是该模式的承担者,是精子获能、胚泡着床、胚胎发育和分娩的场所，因而它是一个多功能的器官。,精子获能,胚泡,着床,胚胎,分娩,发育,胚胎植入“窗口”：a)胚胎滋养层具备可侵入能力，同时，b)子宫内膜具备可接受性的这一特定而短暂的时期,“窗口”期：人 D5-7(D19-21)小 鼠 D4.5 Panda 不定，延缓型,Molecular signaling dur
29、ing implantation,非接受期子宫对囊胚反应迟钝。通过调控子宫内的多种因子，E2 and P4可以将子宫的状态由非接受期转变至接受期。反过来，子宫方面的catecholestrogen也可以将胚泡激活至有植入能力的状态。粘附过程中，子宫内的一系列信号过程 引起胚泡的植入，随后间质细胞开始蜕膜化。胚泡粘附过程中，还存在配体-受体、粘附分子(integrins and other cell-surface molecules)的作用.,Apposition Adhesion,Norwitz E.,et al.,N Engl J Med,345：14001408,胞饮突pinopodes
30、,STB微绒毛,Norwitz E.,et al.,N Engl J Med,345:14001408,CTB,STB,愈合中的子宫上皮,子宫内膜毛细血管,子宫内膜腺体,Norwitz E.,et al.,N Engl J Med,345:14001408,母体血窦sinusoid,CTB,STB,滋养层腔(lacuna),人胎盘内血液循环示意图,血窦,脐静脉,脐动脉,绒毛,子宫螺旋动脉的末端开口于绒毛间形成血窦，胎盘绒毛浸润在血窦内；而胚胎的脐动脉和静脉分支进入每一根绒毛内，胎儿与母体之间进行开放式的血液循环。,人足月胎盘示意图,8、Sexual determination&differen
31、tiation,PGC细胞起源PGC细胞迁移Sry基因的表达及雄性发育方向的确立Genome reprogramming生殖管道的发育雌性发育,图解：（左图）6.0 dpc阶段，源于胚胎外部外胚层（浅蓝色部分）趋化细胞（predisposes cells）的信号，作用于胚盘（棕色）上层细胞epiblast的PGCc前体，发动生殖细胞系的命运；（右图）接着，所有的PGCs前体向原条(Primitive steak)迁移，边迁移边增值，到达胚胎外的区域。7.0 dpc阶段，新生成的胚胎外中胚层（金黄色）形变，构成胚外体腔（exocoelomic cavity，白色），一些PGCs前体停止迁移，成簇
32、排列在生殖细胞系诞生的部位。,PGC细胞的起源,PGC migration,7.25 dpc,PGCs细胞在后羊膜褶中胚层中(posterior amniotic fold)确立,成簇排列；7.0-8.5 dpc,PGCs细胞位于原条(primitive streak)后部的下层,并且混入尿囊(allantois)基层,成簇排列,约有75个细胞左右,此时胚胎处于前体节阶段(presomite stage).8.5 dpc前数小时,表达组织非特异的碱性磷酸酶活性(tissue-nonspecific Alkaline phosphatase,TNAP),高度转录Oct3/4(又称POU5f1)m
33、RNA.PGCs细胞继续向生殖嵴迁移的过程中,碱性磷酸酶的活性仍然维持.8.0 dpc,小群体PGCs出现,总数约200左右,位于后肠(hindgut)上皮中.随后,PGCs细胞沿肠管内胚层(gut entoderm)以阿米巴方式迁移,进入肠系膜(mesentery)中胚层,最后定居于生殖嵴腔上皮.10.5-11.5 dpc期间,可以在生殖嵴上皮检测到PGCs细胞.PGCs迁移至尿囊后,细胞周期为16小时,13 dpc时,数量达到25000左右.,小鼠XX和XY生殖腺双向分化通路的分子基础,10.5-11.5 dpc时,数种因子(Sf1,Wt1,Lhx9,M33,Emx2,Igf1r/Ir/I
34、rr)参与生殖腺双潜能细胞的增殖和防止细胞凋亡的生化过程;10.5-12.5 dpc期间,GATA4/FOG2 and WT1+KTS因子与XY生殖腺中Sry基因的激活有关,Sry蛋白通过调控Sox9,Fgf9和Dax1因子,实现生殖腺向雄性方向发育,诱导体细胞向Sertoli细胞定向分化,随后在Pdgfra,Dhh,成,唯一知道的与12.5 dpc早期卵巢发育有关的两个因子是Wnt4和Fst.,左：Y特异基因，右：X 同源基因.黄色为广泛表达基因；蓝色为testis-specific genes；tooth-bud-specific genes in green,and brain-spec
35、ific genes in red.,Genetic map of the non-recombining region of the human Y chromosome,Location of SRY on Y chromosome,Sry gene,原始生殖腺向睾丸方向分化的时间明显早于向卵巢分化的时间,由此预测,雄性发育方向的启动是一个主动性的过程,在开始卵巢发育之前,雄性发育信号主导着双重潜能的生殖腺细胞向睾丸方向分化.雌性小鼠在交配后10.5天(10.5 dpc.,days post coitum)时,能够检测到胎儿生殖腺中Sry基因的表达；原位杂交显示，11.5 dpc阶段的Sr
36、y基因转录只局限在生殖嵴部位，邻近的中肾组织没有转录。11.5-12.5 dpc期间，未分化生殖腺获得雌或雄性特征。在12.0-12.5 dpc时,也就是在Sry开始表达36 h后,作为睾丸分化的形态学标志的睾索开始形成.Sry在生殖腺体细胞中的表达,启动了雄性支持细胞(Sertoli cells)的分化过程,Sertoli细胞是睾丸体细胞中的一种主要类型,是睾丸后期分化必须的.Sertoli细胞的极化、在生殖细胞周围聚集以及再组织过程,将生殖腺分割成下面两个部分：由Sertoli细胞和生殖细胞组成的管状睾索部分和睾索间隙部分。生精小管的管周收缩细胞包围Sertoli细胞后,一起沉积到管状系统
37、边缘的基底层.睾丸的其他间质细胞,有分泌类固醇激素的Leydig细胞、成纤维细胞和XY生殖腺典型的脉管系统,Primordial germ cells(PGCs)in the mouse become demethylated early in development.Remethylation begins in prospermatogonia on E16 in male germ cells,and after birth in growing oocytes.,Methylation reprogramming in the germ line,Timing of X-inactiv
38、ation during development.,Oogenesis（出生前）,两性分化之前生殖腺有两套生殖管道 Wolffian ducts 中肾管(武氏管)黑色旁中肾管（缪氏管）Mullerian ducts 白色,9、fetal development,Whole-mount mouse embryos at representative stages of development,6.5 dpc7.5 dpc8.5 dpc9.5 dpc10.5 dpc12.5 dpc14.5 dpcnewborn,Fetus and mother(fetal environment),子宫环境的变化（food&stress,etc），可能是成年人慢性疾病的根源Stressed-out ewes give birth to lambs with high blood pressure.Pigs lacking adequate protein during gestation are born with fewer nephrons(肾单位).Couzin J,Science,2002,296:2167-9,10、Parturition&Lactating,It is important for clone,
