Ultrastructure Studies Of Unfertilized Oocytes And Undivided Zygoytes And Arrested Preimplantation Embryos

There are some realities in vitro fertilization, such as failures at which oocytes fertilize and zygote divide and embryos cleavage. While how to explain them, then to analysis the reason is always a difficult problem. Recently, evaluation of oocyte and embryo is limited to the mophological observation by lighe microscope. There is little about Ultrastructure. Electron microscopy is used to studying the ultrastructure due to resolving power and intuition. Some interest has been generated in studying the relationship between oocyte and embryo and their ultrastructure . Ultrastructure by electron microscopy is: incomplete of the cortical reaction, oocyte immaturity, abnormal spindle, abnormal assembly of annulate lamellae. Other reports suggested the structure of the zona pellucida is abnormally dense ,which may be the cause of sperm attchment failure. Reports on arrested embryo is little due to ethic and law. Structure and mophology of oocytes and embryos is closely related to function. So far, about the ultrastructure of unfertilized oocytes, undivided zygotes and arrested embryos, there is no report in our country. Our research is to examine ultrastructure of unfertilized oocytes, undivided zygotes and arrested embryos by electron microscopic, and then explore the reason of failure at in vitro fertilization combined with mitochondrial structure and function. The purpose of this study is to provide the basic and clinical rational for IVF-ET and may be help to select the best clinically assisted fertilization method and the pattern of the fertilization, the morphological information for study on development of oocytes and embryos , and the basic rationale to perfect the system in vitro fertilization.Materials1. All the oocytes and embryos were recruited from Reproductive Medical Center in the First Affiliated Hospital of Zhengzhou University during a five-month period from April 2006 to februaty 2007. All couples included in this study gave written consent to experimentation on their excess or discarded oocytes or embryos.2. The oocytes and embryos were choiced: unfertilized mature(metaphase MII) human oocytes from conventional IVF after fertilization 48 h; unfertilized mature(metaphase MII) human oocytes from ICSI after fertilization 48 h; undivided zygotes from IVF or ICSI after fertilization 48 h; arrested embryos from IVF or ICSI after fertilization72 h.Methods1. We have gathered 105 oocytes and embryos, after single gametes handle with fixed,dehydrated,embedded, ultrashined and observed by transmit electron microscopy, only 15 appear the structure: 8 unferlized oocytes( 4 for IVF, 4 for ICSI), 3 undivided zygotes and 4 arrested embryos by TEM.2. Mitochondria membrane potential were determined by fluorescence microscope with stained with Jc-1.3. The methods of IVF-ET refer to the routine ways in our center.Results1,unfertilized metaphase MII from conventional IVF, three different structures1.1 The structure of the zona pellucida ZP is abnormally dense;The perivitelline space (PVS) is too narrow, but oocyte microvilli appear normal.The oocyte cortex lack other organelles(SER-T, SER-V) besides cortical granules(CG) and mitochondria.1.2 The loosely fibrillar structure of the zona pellucida(ZP)is normal, and contains unmerous cytomembranse derived from the feet of follicular cells.The perivitelline space(PVS) is normal, appearing as a narrow, light space between the oolemma and the inner surface of the ZP. The surface of the oocyte shows normal small microvilli.The oocyte cortex has mitochondria, SER-T, SER-V. However, there are no cortical granules(CG) beneath the oolemma.1.3 The normal sperm have penetrated the zona pellucida with intact acrosomal vesile(Av).2,unfertilized metaphase MII fromICSI, three different structure2.1 The structure of the zona pellucida(ZP) is loosely fibrillar;The perivitelline space(PVS) is normal, appearing as a narrow, light space between the oolemma and the inner surface of the ZP. The surface of the oocyte shows abnormal small microvilli.The oocytes cortex has a normal structure: beneath the oolemma there are one row of dense cortical granules. The oocyte cortex also ontains mitochondria, SER-T, SER-V, and SER-T, SER-V are always associated with mitochondria. Injected spermatozoon has lost the nuclear envelope. But chromatin is decondensed.and some is like chromosome named premature chromosome condensation(PCC);2.2 The structure of the zona pellucida(ZP) is loosely fibrillar;The perivitelline space(PVS) is normal, appearing as a narrow, light space between the oolemma and the inner surface of the ZP. The surface of the oocyte shows normal small microvilli.The oocyte cortex has an abnormal structure: cortex granules(CG), which dispersed in the oocytes cortex and subcotex besides those correctly beneath the oolemma; but mitochondrial and SER-T are scarce.2.3 The structure of the zona pellucida(ZP) is loosely fibrillar;The perivitelline space(PVS) is normal, appearing as a narrow, light space between the oolemma and the inner surface of the ZP. The surface of the oocyte shows abnormal small microvilli.The oocyte cortex contains mitochondria which mainly appear isolated, and the presence of Golgi complexes still forming cortical granules(CG), and the abnormal presence of very large SER-V , some containing denser fibrillar contents,3,undivided zygotesThe structure of the zona pellucida ZP is abnormally dense;Cortical granules(CG) contents appear free in the perivitelline space. The microvilli appear swollen and decreased in number.The oocyte cortex contained : mitochondria is main organelle, a few SER-V appeared not associated with mitochondria, and large clumps of annulate lamellae(AL);The oocyte subcortex has some of second lysosomes filled with droplets.4,arrested 2-cell embryoThe structure of the zona pellucida ZP is loose;The perivitelline space is more narrow between the blastomere and ZP than between the two blastomeres, and microvilli has decreased between the blastomere and ZP.Mitochondria a is the major organelles, showing isolated or associated with SER-T. Blastomere exhibite nucleolus precursor bodies, chromatin: enchromatin present low electron-density, distributing around the nulear, some are connected with nucleolus forming nucleolus-associated chromatin. whereas ,heterochromatin present high electron-density, distributing the centre of nulear.5.Structure of mitochondria in unfertilized MII, undivided zygotes, arrested embryos.Unfertilized MII: mitochondria is rounded with arched cristae and a dense matrix and which distributed evenly in the ooplasm.Undivided zygotes: mitochondria is oval dense, with blur cristae, and accumulation electron-dense material on matrix. They appeared to migrate the oocyte subcortex.Arrested embryos: mitochondria is oval, with transverse cristae. They were not around the pronuclei, but concentrated between the nuclear and the oocyte membrane.6. Function of mitochondria in unfertilized MII, undivided zygotes, arrested embryos.A total of 71 unfertilized oocytes including IVF and ICSI, which showed no patten of J-aggregate fluorescence, the cortex presenting green fluorescence; 15 undivided zygotes showed no patten of J-aggregate fluorescence, presenting green fluorescence filled with the cytoplast; A total of 24 arrested embryos showed no J-aggregate fluorescence, the cortex presenting green fluorescence.Conclusions:1. Organelles appear in abnormal position is related to oocytes cytoplasmic maturation, which lead to fail in fertilization. Failed fertilization in conventional IVF due to absense of acrosome reaction and abnormal dense of the zona pellucida. Absence of oocyte activation is related to failure of fertilization in ICSI.2. The presense of lipofuscin body and abnormal assembly of annulate lamellae is concered with fertilization arrest at the pronuclear stage of human zygotic development.3. Increase of heterochromatin distribution in blastmere is involved in embyros blocked at the 2-cell stage.4. Abnormal structure and dysfunction of mitochondria may be related to oocytes failed in fertilization and zygoty arrested at the pronuclear stage and embryos blocked at the 2-cell stage.