1、(发育生物学）III 生殖细胞发生与受精The germ cell development and fertilization How are the germ cells specified and determined?How are the primordial germ cells(PGCs)differentiated into eggs and sperm?(including meiosis)Fertilization and activation of the egg by the sperm that initiates the embryonic development.Q
2、uestions need to be addressed:The germ cell development and fertilization1 The germ cell fate determination and development of gonads1.1 Specification and determination of the primordial germ cells(PGCs)1.2 The germ cell migration and development of gonads2 Gametogenesis2.1 Spermatogenesis2.2 Oogene
3、sis3 Fertilization3.1 The recognition of egg and sperm3.2 The prevention of polyspermy3.3 The activation of egg metabolismThe germ cell development and fertilization1 The germ cell fate determination and development of gonads1.1 Specification and determination of the primordial germ cells(PGCs)1.2 T
4、he germ cell migration and development of gonads2 Gametogenesis2.1 Spermatogenesis2.2 Oogenesis3 Fertilization3.1 The recognition of egg and sperm3.2 The prevention of polyspermy3.3 The activation of egg metabolismSpecification and determination of the PGCsn Gametes in all sexually reproducing organ
5、isms arise from the primordial germ cells(PGCs).n In many instances(including nematodes,flies,and frogs),the PGCs are specified and determined autonomously by the cytoplasmic determinants in the early embryos that are comprised of specific proteins and mRNAs.These cytoplasmic determinants are collec
6、tively referred to as the germ plasm（生殖质生殖质）(The cell-autonomous manner)n In other instances(such as salamanders蝾螈蝾螈 and mammals),the PGCs are specified by cell induction via neighboring cells.(The non cell-autonomous manner)Specification and determination of the PGCsGerm plasm can be identified mor
7、phologically by the presence of conspicuous membrane-unbound organelles with an electron-dense granulofibrillar appearance called germ granulesPosterior granules(P granules)in C.elegansPolar granules(P granules)in DrosophilaGerminal granules in XenopusThe germ cell fate of C.elegans is determined at
8、 the 16-cell embryoP4 blastomere is the PGC Origin of the PGCs in DrosophilaThe PGCs known as pole cells become distinct at the posterior pole of the egg about 90 minutes after fertilization.The cytoplasm at the posterior pole is called pole plasm(极质极质)and is distinguished by large organelles,the po
9、lar granules.Origin of the PGCs in DrosophilaThe PGCs known as pole cells become distinct at the posterior pole of the egg about 90 minutes after fertilization.The cytoplasm at the posterior pole is called pole plasm(极质极质)and is distinguished by large organelles,the polar granules.The role of pole p
10、lasm in PGC determination:No germ cells develop if the posterior end of the egg is irradiated with ultraviolet light If pole plasm of an egg is transferred to the anterior pole of another embryo,the nuclei surrounded by the pole plasm at the anterior pole are specified as germ cellsThe pole plasm at
11、 the posterior pole is critical for determination of the PGCsn The pole plasm includes several components that are crucial for the PGC determination.Those are Oskar,Nanos,Vasa,mRNA of gcl(germ cell less)gene,mitochondrial ribosomal RNA(mtr RNA)etcn So far,it has been reported that several maternal g
12、ene(at least 8 genes)are functionally necessary and sufficient for the pole plasm formation,and the germ cell specification/determination Mutations in any of those genes,e.g oskar,result in the affected homozygous females being“grandchildless”.The mutant female does not produce functional pole plasm
13、 in her eggs,thus having offsprings(F1)who lack germ cells and therefore are sterile(no F2 offsprings).Expression of oskar alone is sufficient for ectopically specifying the germ cellsThe molecular mechanisms underlying the specification and determination of the PGCs in Drosophila The gene oskar is
14、sufficient to specify the germ cells in DrosophilaFunctionally conserved genes in Germ-Cell Development Origin of the PGCs in mammalsn There is no obvious germ plasm in mammals,and mammalian germ cells are not morphologically distinct during early development.Rather,germ cells are induced in gastrul
15、ating embryos n In mice,the germ cells form at the posterior region of the epiblast,at the junction of the extraembryonic ectoderm,epiblast,primitive streak,and allantois(尿囊尿囊).The mammalian PGCs appear to be induced in the early gastrulating embryosThe molecular mechanisms underlying the specificat
16、ion and determination of the PGCs in Mammalsn There is no evidence for germ plasm being involved in germ cell formation in the mouse or other mammals.Instead,germ cell specification in the mouse involves cell induction(cell-cell interactions).n In mice,At day 6.5 of embryonic development,BMP4/BMP8b
17、from the extraembryonic ectoderm give certain cells in this area the ability to produce germ cells.Germ cell specification in the mouse involves cell-cell interactionsFig 11.3 Germ-cell formation in the mouse.The precursors(white)of primordial germ cells(PGCs)and extra-embryonic mesoderm are induced
18、 in the proximal epiblast by signals from the extra-embryonic ectoderm that include BMP-4.During gastrulation,these cells move to the posterior end of the embryo above the primitive streak.Here they form a cluster in which the central cells becomes specified as PGCs and the peripheral cells as extra
19、-embryonic mesoderm.After their formation,the PGCs migrate to the gonads.PGCs:Fragilis(transmembrane protein),Stella and Oct-4BMP4 is required for the generation of primordial germ cells in the mouse embryosA:Wild type embryo;B:High magnification of part of A showing individual PGCs in the hindgut;C
20、:Embryos heterozygous for BMP4 mutant.There are fewer PGCs compared with the wild type;D:Homozygous embryo.PGCs are entirely absent in the hindgutA molecular marker of the PGCs:AP(alkaline phosphatase):The germ cell development and fertilization1 The germ cell fate determination and development of g
21、onads1.1 Specification and determination of the primordial germ cells(PGCs)1.2 The germ cell migration and development of gonads2 Gametogenesis2.1 Spermatogenesis2.2 Oogenesis3 Fertilization3.1 The recognition of egg and sperm3.2 The prevention of polyspermy3.3 The activation of egg metabolismn In m
22、any animals,germ cells develop at some distance from the gonads,and only later migrate to them,where they differentiate into eggs and sperm.n In the mouse gastrula,germ cells first become detectable at the posterior region of the epiblast(上胚层上胚层).They become incorporated into the hindgut and then mo
23、ve from gut tube into the genital ridge(生殖嵴生殖嵴).Germ cell migration and the underlying mechanisms Pathway for the migration of mammalian PGCsThe PGCs are shown by positive staining for high levels of alkaline phosphatase(AP)n During the migration,the PGCs have proliferated from an initial population
24、 of 10-100 cells to the 2500-5000 PGCs present in the gonads by day 12 mouse embryos.2 genes White spotting and Steel,are involved in controlling proliferation of migrating germ cells.White spotting codes for the cell surface receptor Kit(RTK),which is expressed in the migrating PGCs.Steel encodes a
25、 stem cell factor,a ligand for the RTK,which is produced by the cells lining the migration pathway.Mutations that inactivate either of White spotting and Steel cause a decrease in germ-cell number.Germ cell migration and the underlying mechanisms n In Drosophila,the PGCs move from the posterior pole
26、 to the gonads in a manner similar to that of mammalian germ cells.Drosophila is a model organism for understanding the mechanisms underlying the germ cell migration Migration of PGCs in the Drosophila embryo The PGCs are labelled by staining of Vasa,a specific marker for the germline cells(A-F)The
27、segmentation is shown by staining with antibodies to Engrailed proteins(D)n In Drosophila,the PGCs move from the posterior pole to the gonads in a manner similar to that of mammalian germ cells.n Genetic studies have found that a number of genes function in controlling the PGC migration.wunen gene w
28、hich is expressed in the endoderm(midgut)appears to be responsible for directing the migration of the PGCs from the endoderm to the mesoderm columbus and hedgehog,which are expressed in the mesodermal cells of the gonads,appear to be critical for the attracting the Drosophila PGCs to the gonads.Dros
29、ophila is a model organism for understanding the mechanisms underlying the germ cell migration columbus(clb)gene is necessary for PGCs to migrate to gonadsa-d:Vasa labels the PGCse,f:Vasa labels the PGCs(purple);Clift labels gonadal mesodermal cells(brown)In columbus mutant embryos,some PGCs fail to
30、 migrate from the endoderm(midgut)to the mesoderm,and instead remain associated with the endoderm(midgut)(a).In addition,many PGCs that do migrate to the mesoderm fail to associate with the gonadal mesoderm(e),and instead scatter widely in the embryo(c)Ectopic expression of columbus gene is sufficie
31、nt to attract PGCs to new locationsa:the embryo was immunostained with anti-beta-galactosidase to show the abdominal segments.b-e:the embryos were immunostained with anti-Vasa to show the PGCsThe Gal4/Uas binary system was employed to ectopically express columbus gene in specific tissues of Drosophi
32、la embryos including abdominal segments(b),epidermis(c),the nervous system(d)and the PGCs(e);a:embryos ectopically expressing UAS-lacZ in the abdominal segments.The germ cell development and fertilization1 The germ cell fate determination and development of gonads1.1 Specification and determination
33、of the primordial germ cells(PGCs)1.2 The germ cell migration and development of gonads2 Gametogenesis2.1 Spermatogenesis2.2 Oogenesis3 Fertilization3.1 The recognition of egg and sperm3.2 The prevention of polyspermy3.3 The activation of egg metabolismSpermatogenesis(I)n Spermatogenesis is the prod
34、uction of sperm from the primordial germ cells.Basically,there are 3 major phases for spermatogenesis in mammals:PGCSpermatocyteSpermatidn Once the vertebrate PGCs arrive at the genital ridge of a male embryo,they become incorporated into the sex cords(生殖索生殖索).The PGCs remain there until maturity,at
35、 which time the sex cords hollow out to form the seminiferous tubules(生精小管生精小管),and the epithelium of the tubules differentiates into the Sertoli cells(支持细胞支持细胞)n The initiation of spermatogenesis during puberty(青春期青春期)is probably regulated by the synthesis of BMP8b in the spermatogonia(精原干细胞精原干细胞),
36、the stem cell-like spermatogenic germ cells.Mice lacking BMP8b do not initiate spermatogenesis at pubertySpermatocyteSpermatidSpermDiagram of human male germ cell developmentThe transition between spermatogonia and spermatocytes At maturityDiagram of human male germ cell developmentThe transition be
37、tween spermatogonia and spermatocytesSertoli cellsGDNF At maturitySpermatogenesis(II)Levels of the glial cell line-derived neurotrophic factor(GDNF),which is secreted by the Sertoli cells,determine whether the dividing spermatogonia remain spermatogonia or enter the pathway to become spermatocytes.L
38、ow levels of GDNF favor the differentiation of the spermatogonia into spermatocytesHigh levels of GDNF favor self-renewal of the stem cells Mice heterozygous for GDNF mutant allele show depletion of stem cell reserves due to defective self-renewal of spermatogoniaGDNF+/-mice(A,B)at 5 weeks of ageWil
39、d type(D)and GDNF+/-(E)mice at 8 weeks of ageD,E:BrdU incorporation assayA,B:In younger GDNF+/-mice,part of seminiferous tubules are degenerated.In the degenerating tubules,spermatocytes and spermatids are visible,but spermatogonia have disappearedD,E:In older GDNF+/-mice,decreased expression of GDN
40、F often resulted in Sertoli cellonly seminiferous tubules without spermatogonia,and the cell proliferation rate was reduced,reflecting depletion of spermatogonia,due to defects in the self-renewal of spermatogonia.Mice overexpressing GDNF show accumulation of undifferentiated spermatogonia B:Large c
41、lusters of spermatogonia(spermatogonial marker,EE2)are evident in the transgenic gonadsE:The spermatogonia cell cluster gradually degenerate after puberty,resulting in tubular atrophyA,D:wild typeB,E:transgenic mice at 3 or 10 weeks of age respectivelyA-E:EE2 labels spermatogoniaOogenesis(I)n Oogene
42、sis differs from spermatogenesis in several ways.Whereas the gamete formed by spermatogenesis is essentially a motile nucleus,the gamete formed by oogenesis contains all materials needed to initiate and maintain metabolism and development.Therefore,oogenesis needs to build up a store of cytoplasmic
43、enzymes,mRNAs,organelles and metabolic substrates,as well as forming a haploid nucleusn The mechanisms of oogenesis vary among species more than those of spermatogenesis.In some species,such as sea urchins and frogs,the female routinely produces hundreds or thousands of eggs at a time,whereas in oth
44、er species,such as humans and most mammals,only a few eggs are produced during the lifetime of an individual.Overview of the Drosophila oogenesisOogenesis(II)n In the human embryo,oogonia(卵原细胞卵原细胞),the self-renewing germ-line stem cells divide rapidly from the second to the seventh month of gestatio
45、n to form roughly 7million germ cells.Since then,most oogonia die,while the remaining oogonia enter the first meiotic prophase.At this stage,the germ cells become primary oocytes.Those oocytes are arrested until the female matures.With the onset of purberty,groups of oocytes periodically resume meio
46、sis.n In the human female,the first part of meiosis begins in the embryo,and the signal to resume meiosis is not given until roughly 12 years later.In fact,some oocytes are maintained in meiotic prophase for nearly 50 yearsn Primary oocytes continue to die even after birth.Of millions of primary ooc
47、ytes present at birth,only 400 mature during a womans lifetime.Schematic representation of Xenopus oocyte maturationThe meiotic cell divisions are regulated by progesterone(孕酮孕酮)and fertilizationThe germ cell development and fertilization1 The germ cell fate determination and development of gonads1.
48、1 Specification and determination of the primordial germ cells(PGCs)1.2 The germ cell migration and development of gonads2 Gametogenesis2.1 Spermatogenesis2.2 Oogenesis3 Fertilization3.1 The recognition of egg and sperm3.2 The prevention of polyspermy3.3 The activation of egg metabolismFertilization
49、 Although the details of fertilization vary from species to species,it involves 3 major events:Contact and recognition between sperm and egg.This ensures that fertilization is species-specific.Regulation of sperm entry into the egg.Only one sperm can ultimately fertilize the egg.That is to prevent p
50、olyspermy which leads to abnormal development in most organisms.Activation of egg metabolism to initiate embryonic development.Fertilizationn The recognition of egg and spermn The prevention of polyspermyn The activation of egg metabolismThe plasma membrane on the head of the sperm contains various
