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Unformatted text preview: Aneuploidy – leading cause of miscarriage, congenital defects and mental retardation Congression • 10-25% of fertilized Congression failure human oocytes are BPA aneuploid • Risk factors include: maternal age, irradiation, smoking or drinking, oral BPA BPA contraceptives and fertility drugs • Suggested that environmental pollutants/ pesticides Congression Congression failure failure Knockout (gene targetting) a gene is replaced with a selectable marker or reporter gene 1 2 Neo 3 4 Mouse Genome “double cross over” Linearized Targeting Vector Knockout allele – Gene is replaced with a selectable marker or a reporter gene. Generate Chimeras - Breed to transmit the knockout allele Mate heterozygotes to determine the effects of the mutation 4 Neo 4 ZP3 Knockout disrupts zona pellucia formation 1:2:1 ZP3 +/- X ZP3 +/- ZP3 +/+ : ZP3+/- : ZP3 -/- ZP3 -/- X ZP3 +/- ZP3 +/- ZP3 -/- No zona No fertilization Female infertile What does this say about ZP3ʼs role in sperm binding/recognition? Production of a Transgenic Mouse – random integration of DNA Introduce human ZP3 (huZP3) in the mouse ZP3 knockout background Human ZP3 can substitute for mouse ZP3 in fertilization, but human sperm can not bind to the humanized ZP : Human sperm Mouse sperm ZP3 +/+ ZP3 +/+ Zp3 -/- & hZP3 (inset are control fertilizations) Do differences in post translational Modiﬁcation confer species speciﬁcity? Mouse and Human place distinct post translational modiﬁcations on ZP proteins. Human ZP3 is 64 kDA, while mouse is 83 kDa. Does the composition of the mouse and human zona pellucida differ? Recently a 4th ZP protein was identiﬁed in human and rat ZP. Does the supramolecular architecture involving more than one determinant of the ZP dictate speciﬁcity? Histological analysis of mouse ZP comprised of huZP3 looks “thinner” Injection of monoclonal antibody recognizing human ZP3 prevents fertilization Monocolonal Antibody (mAb) human ZP3 normal female Monocolonal Antibody (mAb) mouse ZP3 Injection of monoclonal antibody recognizing human ZP3 prevents fertilization mAb human ZP3 mAb mouse ZP3 hZP3 females normal female ICSI - Intra cytoplasmic Sperm Injection Immobilizing the spermʼs tail before picking up sperm head Injection of sperm into the egg Fertilized egg - two pronuclei www.advancedfertility.com Zygote Male and Female Pronuclei Egg cytoplasm: Factors for remodeling the sperm chromatin – protamines are replaced with histones. Factors needed for the ﬁrst cell division. Why is diploid not sufﬁcient for some mammalian embryos? http://atlasgeneticsoncology.org/Deep/GenomImprintID20032.html Cleavage - Division of fertilized egg (zygote) into blastomeres: smaller cells with no increase in mass. Sea urchin Wolpert 5.3 ,3.5, 3.10, 3.22, http://worms.zoology.wisc.edu/ dd2/echino/cleavage/intro.html X X nematode Xenopus Zebraﬁsh Mouse http://www.biologycorner.com/ worksheets/tiger/read6-2.html In mouse and human the early cleavage divisions are slow After the 2-cell stage, much of the maternal mRNA and proteins are degraded. The embryo begins to take over transcription and translation. ?? Do some factors persist – what is their role in early development?? 24 hrs Compaction of the morula creates distinct outer and inner environments morula compacted morula Slack 2.8 What factors regulate early cleavage divisions? In the ovary, FIGLIA activates transcription of genes needed in the egg for development of the embryo Enhancer Target Genes: CANNTG DNA Zona Pellucida ZP1, ZP2, ZP3 MATER SCMC SubIn humans Figlia +/- results cortical FILIA Figlia Maternal in premature ovarian failure FLOPED bHLH transcrption factor
Complex TLE6 (basic helix-loop-helix) Tissue speciﬁc http://stke.sciencemag.org/content/vol2005/issue284/cover.dtl What other maternal genes are activated by ﬁglia? Do these genes regulate embryo development? Approach: Use microarray to compare gene expression Prepare Microarray: Each well contains a unique gene sequence Wt Figia -/- http://www.genome.gov/10000533 Yellow – wt = mutant Green – wt >> mutant Red – mutant >> wt In the ovary, Figlia activates transcription of genes needed in the egg for development of the embryo Enhancer Target Genes: CANNTG DNA Zona Pellucida ZP1, ZP2, ZP3 MATER Maternal Antigen That Embryos Require Figlia bHLH transcrption factor (basic helix-loop-helix) http://stke.sciencemag.org/content/vol2005/issue284/cover.dtl Factor Located in FLOPED
Oocytes Permiting Embryonic Development Gene trap insertion in the Floped gene – disrupts the gene and incorporates a reporter gene (β-gal) Normal Floped allele Mutant Floped allele Mutant Floped fusion transcript FLOPED null (-/-) females are sterile despite normal ovary and egg morphology FLOPED -/X FLOPED +/+ X
No viable progeny Lack of FLOPED in mom leads to cleavage failure in the embryo FLOPED+/- Embryonic (E) day FLOPED -/- X FLOPED +/+ E 0.5 1st cell division often delayed and asymmetrical E 1.5 E 2.5 Assessing Embryo quality Cell number Cell regularity Degree of fragmentation Multinucleation Vacuoles Granularity Thickness of Zona Pellucida FLOPED protein accumulates in the egg and persists until the blastocyst stage – Maternally supplied mRNA! RT-PCR (RNA abundance) Western (Protein abundance) FLOPED is localized in the egg cortex overlapping f-actin – after cleavage it is absent from the cell junctions FLOPED localization is dynamic and can redistribute if cell contact is lost. Cadherins are Ca+2 dependent cell adhesion molecules http://php.med.unsw.edu.au/cellbiology/index.php?title=2010_Lecture_8 FLOPED remains localized in the outer cortex through the blastocyst stage, even though there is no mRNA synthesis! Use Co-Immunuoprecipitation to isolate a complex of proteins that interact with FLOPED. These proteins comprise the Subcortical Maternal Complex (SCMC). Compare co-immunoprecipitates from ﬂoped null and wild type (norm) ovaries Ovary/egg Cell lysate Mass Spec: PARTNERS MATER FLOPED Antibody α-FLOPED Protein A or G Sepharose TLE6 FILIA FLOPED SCMC Subcortical Maternal Complex Bands speciﬁc to the normal mice are possible FLOPED interacting proteins FLOPED, MATER, TLE6, and FILIA co-localize in a Subcortical Maternal Complex (SCMC) Localization of FLOPED depends on MATER Localization of Mater depends on FLOPED -/- -/- Localization of other SCMC proteins requires FLOPED and MATER How do mutations in the SCMC disrupt cleavage stage development? Subcortical Maternal Complex (SCMC) Does the SCMC regulate zygotic transcription? Nucleus Zygotic transcription signiﬁcantly reduced in Mater mutants ? Does inheritance of the SCMC regulate early cell fate decisions? Subcortical Maternal Complex (SCMC) What determines the fate of the blastomere? When is this decision made? Is this decision ﬁxed or reversible? When is the fate of a blastomere decided? hChr X – blue h Chr 21 - Pink Removing a blastomere for genetic testing. FISH – ﬂuorescent in situ hybridization Figure 21.10 and 21.4 Gilbert http://members.cox.net/amgough/Fanconi-genetics-PGD.htm What is the developmental potential of a blastomere? 11.38 Gilbert What do identical twins tell us about the potential of human blastomeres to develop? ...
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This note was uploaded on 09/29/2010 for the course BIO 325 taught by Professor Bernadette-holdener,g during the Fall '08 term at SUNY Stony Brook.
- Fall '08