本文關(guān)鍵詞：人未受精、受精未分裂卵母細(xì)胞及早期發(fā)育阻滯胚胎的超微結(jié)構(gòu)研究　出處：《鄭州大學(xué)》2007年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 受精失敗卵母細(xì)胞 不分裂受精卵 發(fā)育阻滯胚胎 超微結(jié)構(gòu) 線(xiàn)粒體膜電位

【摘要】： 在體外受精-胚胎移植(In vitro fertilization-Embryo transfer，IVF-ET)中，常常有受精失敗、受精不分裂及胚胎發(fā)育阻滯等現(xiàn)象發(fā)生，如何解釋這些現(xiàn)象，進(jìn)而分析其發(fā)生原因，一直是輔助生殖醫(yī)學(xué)領(lǐng)域面臨的難題。目前對(duì)卵母細(xì)胞和胚胎質(zhì)量的評(píng)價(jià)僅限于光鏡下形態(tài)學(xué)觀察，對(duì)其內(nèi)部結(jié)構(gòu)的研究較少。電子顯微鏡直觀性和高分辨率的特點(diǎn)，使其成為觀察超微結(jié)構(gòu)的主要手段。國(guó)外報(bào)道透射電子顯微鏡觀察下，受精失敗及受精后未分裂的卵母細(xì)胞的超微結(jié)構(gòu)主要表現(xiàn)在卵母細(xì)胞透明帶結(jié)構(gòu)異常、皮質(zhì)反應(yīng)不全、卵胞漿不成熟、紡錘體異常、環(huán)紋片層異常聚集等方面；發(fā)育阻滯胚胎的超微結(jié)構(gòu)研究，由于受倫理及法律限制，報(bào)道較少。卵母細(xì)胞及胚胎的內(nèi)部結(jié)構(gòu)與形態(tài)及功能密切相關(guān)，對(duì)它們內(nèi)部結(jié)構(gòu)的研究將有助于更好的了解受精失敗、胚胎停滯發(fā)育的內(nèi)部原因。目前，關(guān)于透射電鏡研究人未受精、受精后未分裂的卵母細(xì)胞及發(fā)育阻滯胚胎的超微結(jié)構(gòu)，國(guó)內(nèi)尚未見(jiàn)報(bào)道。本研究即應(yīng)用透射電子顯微鏡觀察人未受精、受精未分裂卵母細(xì)胞、發(fā)育阻滯胚胎的超微結(jié)構(gòu)，結(jié)合線(xiàn)粒體形態(tài)特征與功能的改變分析卵母細(xì)胞受精失敗、受精后不分裂、胚胎發(fā)育阻滯的原因，旨在為基礎(chǔ)和臨床上的治療提供依據(jù)，協(xié)助患者選擇下一周期的最佳治療方法、受精方式；為人卵母細(xì)胞、胚胎發(fā)育的調(diào)控機(jī)理研究提供微觀形態(tài)學(xué)資料；為建立完善的體外培養(yǎng)體系提供理論依據(jù)。 研究對(duì)象 1．所有研究的卵母細(xì)胞和胚胎均來(lái)自鄭州大學(xué)第一附屬醫(yī)院生殖中心2006年4月～2007年1月間進(jìn)行的IVF-ET周期；所有用于研究的廢棄卵母細(xì)胞和胚胎均經(jīng)患者知情同意。 2．選擇常規(guī)IVF 48h后未受精的第二次減數(shù)分裂中期卵母細(xì)胞(metaphaseⅡ，MⅡ)；ICSI 48h后未受精的MⅡ；常規(guī)IVF或ICSI受精48h后未分裂的受精卵母細(xì)胞(合子)；常規(guī)IVF或ICSI受精后72h發(fā)育阻滯胚胎。 研究方法 1、將收集的受精失敗、受精未分裂卵母細(xì)胞、發(fā)育阻滯胚胎，進(jìn)行單細(xì)胞固定、脫水、包埋、超薄切片，透射電子顯微鏡下觀察，拍照； 2、將收集的受精失敗、受精未分裂卵母細(xì)胞、發(fā)育阻滯胚胎用Jc-1熒光染料染色后觀察其線(xiàn)粒體膜電位情況； 3、體外受精-胚胎移植方法按我中心常規(guī)方案進(jìn)行； 結(jié)果 1、常規(guī)IVF未受精卵母細(xì)胞：可見(jiàn)3種不同的超微結(jié)構(gòu) 1．1透明帶(ZP)為異常緊密狀結(jié)構(gòu)；卵周間隙(PVS)狹窄，微絨毛(mv)正常；卵皮質(zhì)內(nèi)含有卵膜下一層線(xiàn)形排列皮質(zhì)顆粒(CG)，線(xiàn)粒體(mt)為其主要的細(xì)胞器，但缺少滑面內(nèi)質(zhì)網(wǎng)(SER)集合管(T)、SER泡(V)等細(xì)胞器； 1．2透明帶(ZP)為正常的疏松狀結(jié)構(gòu)，內(nèi)含有大量卵泡細(xì)胞胞膜殘留物；卵周間隙(PVS)狹窄透亮，微絨毛(mv)正常；卵皮質(zhì)內(nèi)有線(xiàn)粒體(mt)、滑面內(nèi)質(zhì)網(wǎng)(SER)集合管(T)、SER泡(V)，滑面內(nèi)質(zhì)網(wǎng)(SER)集合管(T)、SER泡常與線(xiàn)粒體(mt)相連；但卵膜下未見(jiàn)皮質(zhì)顆粒(CG)；亞皮質(zhì)區(qū)精核(N)呈濃縮狀態(tài)，周?chē)匆?jiàn)線(xiàn)粒體(mt)、SER泡(V)等細(xì)胞器圍繞； 1．3透明帶內(nèi)精子頂體(AV)完整；卵皮質(zhì)和亞皮質(zhì)未切到。 2、ICSI未受精卵母細(xì)胞：可見(jiàn)3種不同的超微結(jié)構(gòu) 2．1透明帶(ZP)為正常的疏松狀結(jié)構(gòu)；卵周隙(PVS)狹窄透亮，微絨毛正常(mv)；卵皮質(zhì)內(nèi)含有卵膜下一層線(xiàn)形排列皮質(zhì)顆粒(CG)，線(xiàn)粒體(mt)、SER集合管(T)、SER泡(V)，SER集合管(T)、SER泡(V)常與線(xiàn)粒體相連；亞皮質(zhì)內(nèi)注射的精子已褪去核膜，部分染色質(zhì)(C)已去濃縮，有的染色質(zhì)聚集成染色體樣結(jié)構(gòu)； 2．2透明帶(ZP)為正常疏松狀結(jié)構(gòu)；卵周隙(PVS)狹窄透亮，微絨毛正常(mv)；卵皮質(zhì)內(nèi)皮質(zhì)顆粒分散存在，未在卵黃膜下排列，缺乏線(xiàn)粒體、滑面內(nèi)質(zhì)網(wǎng)集合管等細(xì)胞器； 2．3透明帶(ZP)為正常疏松結(jié)構(gòu)；卵周隙(PVS)正常，但微絨毛(mv)異常短小；皮質(zhì)區(qū)線(xiàn)粒體(mt)孤立存在，可見(jiàn)正在形成皮質(zhì)顆粒的高爾基復(fù)合體(G)，異常極大的SER大泡，有的SER大泡內(nèi)含有致密纖維； 3、受精未分裂卵母細(xì)胞 透明帶(ZP)結(jié)構(gòu)致密；卵周隙(PVS)內(nèi)可見(jiàn)皮質(zhì)顆粒排放的皮質(zhì)內(nèi)容物，微絨毛腫脹、數(shù)量少；卵皮質(zhì)內(nèi)線(xiàn)粒體(mt)為主要的細(xì)胞器，，孤立存在，不與滑面內(nèi)質(zhì)網(wǎng)(SER)泡相連；環(huán)文片層(AL)異常聚集，成叢分布于胞漿中；亞皮質(zhì)區(qū)有體積較大的許多小脂滴填滿(mǎn)的脂褐體；原核未切到； 4、發(fā)育阻滯胚胎 透明帶(ZP)變薄，結(jié)構(gòu)疏松狀；細(xì)胞與透明帶間的卵周隙(PVS)較細(xì)胞間窄，微絨毛數(shù)量減少，線(xiàn)粒體(mt)為主要的細(xì)胞器，或孤立，或與滑面內(nèi)質(zhì)網(wǎng)(SER)相連形成SER集合管(T)；完整的核膜，核內(nèi)可見(jiàn)數(shù)個(gè)深染的核仁前體(Nu)，染色質(zhì)呈顆粒狀，其中異染色質(zhì)(eu)電子密度較高，緊靠于核內(nèi)膜，數(shù)量多，部分與核仁前體連接；而常染色質(zhì)(he)電子密度較淺，靠核中心分布，數(shù)量少。 5．未受精卵母細(xì)胞、未分裂受精卵母細(xì)胞、發(fā)育阻滯胚胎的線(xiàn)粒體結(jié)構(gòu)特征 5．1未受精卵母細(xì)胞：數(shù)量較少，分散于整個(gè)胞質(zhì)，基質(zhì)密度高，少量似弓形的嵴； 5．2未分裂受精卵母細(xì)胞：嵴模糊，基質(zhì)中度密集，內(nèi)有電子沉積物，分布于亞皮質(zhì)； 5．3發(fā)育阻滯胚胎：橢圓形，基質(zhì)密度高，嵴呈管形，多集中分布于胞核與胞膜之間； 6、未受精卵母細(xì)胞、未分裂受精卵母細(xì)胞、發(fā)育阻滯胚胎的線(xiàn)粒體膜電位 共觀察了110個(gè)標(biāo)本，其中71個(gè)未受精卵母細(xì)胞Jc-1在卵皮質(zhì)區(qū)產(chǎn)生綠色熒光，15個(gè)未分裂受精卵母細(xì)胞Jc-1在整個(gè)胞漿產(chǎn)生綠色熒光，24個(gè)發(fā)育阻滯胚胎Jc-1在卵皮質(zhì)區(qū)產(chǎn)生綠色熒光。 結(jié)論 1．卵胞質(zhì)中細(xì)胞器異常分布導(dǎo)致卵母細(xì)胞胞質(zhì)不成熟影響其受精能力；透明帶結(jié)構(gòu)異常及誘導(dǎo)頂體反應(yīng)缺陷可導(dǎo)致精子穿透障礙，常規(guī)IVF受精失�。宦涯讣�(xì)胞激活失敗與ICSI受精失敗有關(guān)。 2．脂褐體的出現(xiàn)及環(huán)紋片層異常聚合與受精卵母細(xì)胞不分裂有關(guān)。 3．卵裂球核內(nèi)異染色質(zhì)增多與胚胎發(fā)育阻滯有關(guān)。 4．線(xiàn)粒體結(jié)構(gòu)改變、能量代謝降低與卵母細(xì)胞受精失敗、受精卵未分裂及胚胎發(fā)育阻滯有關(guān)。
[Abstract]:In in vitro fertilization and embryo transfer (In vitro fertilization-Embryo transfer, IVF-ET), often with fertilization failure, and embryo development block is not split fertilization, how to explain these phenomena, and then analyzes its causes, is always a difficult problem in the field of reproductive medicine. The evaluation of the secondary oocyte and embryo quality only limited to morphological observation under light microscope, to study the internal structure of the electronic microscope. Less intuitive and high resolution characteristics, make it become the main means to observe the ultrastructure of the foreign reports. Transmission electron microscope, the ultrastructure of unfertilized and undivided fertilized oocytes mainly in oocytes the cell structure of the zona pellucida abnormal cortical reaction is not complete, the egg cytoplasm of immature, spindle abnormalities, abnormal aggregation of annulate lamellae; ultrastructure of arrested embryos, Because of the ethical and legal constraints, reported less. Oocytes and embryo morphology and internal structure and function are closely related, will contribute to a better understanding of their internal structure of fertilization failure, the internal reason of embryonic stagnate development. At present, a transmission electron microscopy study of unfertilized, undivided fertilized egg the mother cell and the ultrastructure of arrested embryos, has not been reported. This study observed by transmission electron microscopy in unfertilized human oocytes, fertilization is not split, the ultrastructure of arrested embryos, with the grain line shape features and functions of the state change analysis of the failure of oocytes after fertilization, not split, reason embryo development block, in order to provide basis for the treatment and clinical basis, help patients to choose the best treatment method, the next cycle of insemination; for human oocytes, embryo development regulation The study of control mechanism provides micromorphological data and provides a theoretical basis for the establishment of a perfect culture system in vitro.
Research object
1. all the oocytes and embryos were from the IVF-ET cycle between April 2006 and January 2007 in the reproductive center of the First Affiliated Hospital of Zhengzhou University. All the oocytes and embryos used in the study were informed consent by the patients.
2., we chose the second meiotic metaphase oocytes (metaphase II, M II) which were not fertilized after routine IVF 48h, M 48h which was not fertilized after ICSI 48h, the unfertilized oocytes (zygote) after conventional IVF or ICSI fertilized 48h, and the development of embryos blocked by normal or ICSI fertilization.
research method
1, we will collect the fertilization failure, fertilize undivided oocytes, block embryos, develop single cell fixation, dehydration, embedding, ultrathin section, and observe by transmission electron microscope.
2, insemination failed, undivided oocytes were fertilized, and the developmental block embryos were stained with Jc-1 fluorescent dye to observe the mitochondrial membrane potential.
3, the method of in vitro fertilization and embryo transfer was carried out according to the routine plan of my center.
Result
1, conventional IVF unfertilized oocytes: 3 different ultrastructures
1.1 zona (ZP) abnormal close type structure; the perivitelline space (PVS) stenosis, microvilli (MV) normal; egg membrane under a layer of a linear array of cortical granules containing egg cortex (CG), mitochondria (MT) as the main organelles, but the lack of smooth endoplasmic reticulum (SER) tube set (T), SER (global V) and other organelles;
1.2 zona (ZP) is a loose structure normal, contains a large number of follicle cell membrane residues; perivitelline space (PVS) narrow translucent, microvilli (MV) in normal; egg cortex mitochondria, smooth endoplasmic reticulum (MT) (SER) (T), SER collection tube (V), global smooth endoplasmic reticulum (SER) collection tube (T), SER constant and global mitochondrial (MT) connected; but there was no oocyte cortical granules (CG); sub cortex sperm nucleus (N) was concentrated state, seen around the mitochondria (MT), SER (global V) and other organelles around;
1.3 the sperm acrosome (AV) in the zona pellucida is complete, and the egg cortex and subcortex are not cut.
2, ICSI unfertilized oocyte: 3 different ultrastructures
2.1 zona (ZP) is a loose structure normal; the perivitelline space (PVS) narrow translucent, normal microvilli (MV); egg membrane under a layer of a linear array of cortical granules containing egg cortex (CG), mitochondria (MT), SER (T), SER collection tube (V), SER Global the collecting duct (T), SER (global V) often associated with mitochondria; sub cortical injected sperm nuclear chromatin has faded, part (C) has to concentrate, some chromatin aggregation of chromosome structure;
2.2 transparent tape (ZP) for normal loose structure; the perivitelline space (PVS) narrow translucent, normal microvilli (MV); cortical granule egg cortex scattered, not arranged, lack of mitochondria in the vitelline membrane, endoplasmic reticulum organelles such as collecting tube;
2.3, the zona pellucida (ZP) is a normal loose structure. The PVS is normal, but the microvilli (MV) are very short. The mitochondria in the cortex (MT) are isolated. The Golgi complex (G) is forming the cortical granules, the abnormal SER bullae is very large, and some SER bubbles contain dense fibers.
3, fertilized undivided oocyte
Zona pellucida (ZP) structure is compact; the perivitelline space (PVS) contents in the cortex, cortical granule visible emission microvilli swelling, a small number of mitochondria; egg cortex (MT) is the main organelle, isolated, and smooth endoplasmic reticulum (SER) vesicles connected; ring paper sheets (AL) clumps of abnormal accumulation and distribution in cytoplasm; sub cortical areas have brown fat large and many small lipid droplets filled; prokaryotic not cut;
4, developmental retardation embryo
Zona pellucida (ZP) thin, loose structure; cell and the zona pellucida between the perivitelline space (PVS) is a narrow intercellular, reducing the number of microvilli, mitochondria (MT) as the main organelles, or isolated, or with the smooth endoplasmic reticulum (SER) connected to SER collection tube formation (T); complete the nuclear membrane, nucleus visible several hyperchromatic nucleolus precursor (Nu), granular chromatin, the heterochromatin (EU) with high electron density, close to the nuclear membrane, the number of parts, and nucleolar precursor connections; and euchromatin (he) shallow electron density, quantity distribution, rely on nuclear the center is less.
5. unfertilized oocytes, undivided fertilized oocytes, and mitochondrial structure characteristics of developmental retardation embryos
5.1 unfertilized oocyte: a small number, scattered in the whole cytoplasm, high matrix density, a small amount of arched ridge;
5.2 undivided fertilized oocytes: the crista is blurred, the matrix is moderately dense, and there is an electronic deposit in the subcortex.
5.3 the development of retarding embryos: oval, high density of matrix, tubular shaped ridge, mostly distributed between the nucleus and the membrane of the cell.
6, the unfertilized oocyte, undivided fertilized oocyte, and the mitochondrial membrane potential of developmental retardation embryos
A total of 110 specimens were observed, of which 71 of the unfertilized oocytes Jc-1 produced green fluorescence in the egg cortex, 15 of the unfertilized oocytes Jc-1 produced green fluorescence in the whole cytoplasm, 24 embryos blocked the development of embryos, and Jc-1 produced green fluorescence in the egg cortex.
conclusion
1., the abnormal distribution of organelles in oocytes causes the immature cytoplasm of oocytes to affect their fertilization ability. The abnormal structure of zona pellucida and the induction of acrosome reaction defects can lead to sperm penetration barrier, and failure of conventional IVF fertilization. The failure of oocyte activation is related to the failure of ICSI fertilization.
The appearance of 2. fat Brown bodies and the abnormal polymerization of the lamellar layer are related to the unsplit of fertilized oocytes.
3. blastomere nuclei heterochromatin increased associated with embryo development block.
4. the mitochondrial structure changes, the decrease of energy metabolism is related to the failure of oocyte fertilization, the unsplit of fertilized eggs and the retardation of embryonic development.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R321

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