人未受精、受精未分裂卵母细胞及早期发育阻滞胚胎的超微结构研究

发布时间：2018-01-14 05:32

本文关键词：人未受精、受精未分裂卵母细胞及早期发育阻滞胚胎的超微结构研究　出处：《郑州大学》2007年硕士论文　论文类型：学位论文

【摘要】： 在体外受精-胚胎移植(In vitro fertilization-Embryo transfer，IVF-ET)中，常常有受精失败、受精不分裂及胚胎发育阻滞等现象发生，如何解释这些现象，进而分析其发生原因，一直是辅助生殖医学领域面临的难题。目前对卵母细胞和胚胎质量的评价仅限于光镜下形态学观察，对其内部结构的研究较少。电子显微镜直观性和高分辨率的特点，使其成为观察超微结构的主要手段。国外报道透射电子显微镜观察下，受精失败及受精后未分裂的卵母细胞的超微结构主要表现在卵母细胞透明带结构异常、皮质反应不全、卵胞浆不成熟、纺锤体异常、环纹片层异常聚集等方面；发育阻滞胚胎的超微结构研究，由于受伦理及法律限制，报道较少。卵母细胞及胚胎的内部结构与形态及功能密切相关，对它们内部结构的研究将有助于更好的了解受精失败、胚胎停滞发育的内部原因。目前，关于透射电镜研究人未受精、受精后未分裂的卵母细胞及发育阻滞胚胎的超微结构，国内尚未见报道。本研究即应用透射电子显微镜观察人未受精、受精未分裂卵母细胞、发育阻滞胚胎的超微结构，结合线粒体形态特征与功能的改变分析卵母细胞受精失败、受精后不分裂、胚胎发育阻滞的原因，旨在为基础和临床上的治疗提供依据，协助患者选择下一周期的最佳治疗方法、受精方式；为人卵母细胞、胚胎发育的调控机理研究提供微观形态学资料；为建立完善的体外培养体系提供理论依据。研究对象 1．所有研究的卵母细胞和胚胎均来自郑州大学第一附属医院生殖中心2006年4月～2007年1月间进行的IVF-ET周期；所有用于研究的废弃卵母细胞和胚胎均经患者知情同意。 2．选择常规IVF 48h后未受精的第二次减数分裂中期卵母细胞(metaphaseⅡ，MⅡ)；ICSI 48h后未受精的MⅡ；常规IVF或ICSI受精48h后未分裂的受精卵母细胞(合子)；常规IVF或ICSI受精后72h发育阻滞胚胎。研究方法 1、将收集的受精失败、受精未分裂卵母细胞、发育阻滞胚胎，进行单细胞固定、脱水、包埋、超薄切片，透射电子显微镜下观察，拍照； 2、将收集的受精失败、受精未分裂卵母细胞、发育阻滞胚胎用Jc-1荧光染料染色后观察其线粒体膜电位情况； 3、体外受精-胚胎移植方法按我中心常规方案进行；结果 1、常规IVF未受精卵母细胞：可见3种不同的超微结构 1．1透明带(ZP)为异常紧密状结构；卵周间隙(PVS)狭窄，微绒毛(mv)正常；卵皮质内含有卵膜下一层线形排列皮质颗粒(CG)，线粒体(mt)为其主要的细胞器，但缺少滑面内质网(SER)集合管(T)、SER泡(V)等细胞器； 1．2透明带(ZP)为正常的疏松状结构，内含有大量卵泡细胞胞膜残留物；卵周间隙(PVS)狭窄透亮，微绒毛(mv)正常；卵皮质内有线粒体(mt)、滑面内质网(SER)集合管(T)、SER泡(V)，滑面内质网(SER)集合管(T)、SER泡常与线粒体(mt)相连；但卵膜下未见皮质颗粒(CG)；亚皮质区精核(N)呈浓缩状态，周围未见线粒体(mt)、SER泡(V)等细胞器围绕； 1．3透明带内精子顶体(AV)完整；卵皮质和亚皮质未切到。 2、ICSI未受精卵母细胞：可见3种不同的超微结构 2．1透明带(ZP)为正常的疏松状结构；卵周隙(PVS)狭窄透亮，微绒毛正常(mv)；卵皮质内含有卵膜下一层线形排列皮质颗粒(CG)，线粒体(mt)、SER集合管(T)、SER泡(V)，SER集合管(T)、SER泡(V)常与线粒体相连；亚皮质内注射的精子已褪去核膜，部分染色质(C)已去浓缩，有的染色质聚集成染色体样结构； 2．2透明带(ZP)为正常疏松状结构；卵周隙(PVS)狭窄透亮，微绒毛正常(mv)；卵皮质内皮质颗粒分散存在，未在卵黄膜下排列，缺乏线粒体、滑面内质网集合管等细胞器； 2．3透明带(ZP)为正常疏松结构；卵周隙(PVS)正常，但微绒毛(mv)异常短小；皮质区线粒体(mt)孤立存在，可见正在形成皮质颗粒的高尔基复合体(G)，异常极大的SER大泡，有的SER大泡内含有致密纤维； 3、受精未分裂卵母细胞透明带(ZP)结构致密；卵周隙(PVS)内可见皮质颗粒排放的皮质内容物，微绒毛肿胀、数量少；卵皮质内线粒体(mt)为主要的细胞器，，孤立存在，不与滑面内质网(SER)泡相连；环文片层(AL)异常聚集，成丛分布于胞浆中；亚皮质区有体积较大的许多小脂滴填满的脂褐体；原核未切到； 4、发育阻滞胚胎透明带(ZP)变薄，结构疏松状；细胞与透明带间的卵周隙(PVS)较细胞间窄，微绒毛数量减少，线粒体(mt)为主要的细胞器，或孤立，或与滑面内质网(SER)相连形成SER集合管(T)；完整的核膜，核内可见数个深染的核仁前体(Nu)，染色质呈颗粒状，其中异染色质(eu)电子密度较高，紧靠于核内膜，数量多，部分与核仁前体连接；而常染色质(he)电子密度较浅，靠核中心分布，数量少。 5．未受精卵母细胞、未分裂受精卵母细胞、发育阻滞胚胎的线粒体结构特征 5．1未受精卵母细胞：数量较少，分散于整个胞质，基质密度高，少量似弓形的嵴； 5．2未分裂受精卵母细胞：嵴模糊，基质中度密集，内有电子沉积物，分布于亚皮质； 5．3发育阻滞胚胎：椭圆形，基质密度高，嵴呈管形，多集中分布于胞核与胞膜之间； 6、未受精卵母细胞、未分裂受精卵母细胞、发育阻滞胚胎的线粒体膜电位共观察了110个标本，其中71个未受精卵母细胞Jc-1在卵皮质区产生绿色荧光，15个未分裂受精卵母细胞Jc-1在整个胞浆产生绿色荧光，24个发育阻滞胚胎Jc-1在卵皮质区产生绿色荧光。结论 1．卵胞质中细胞器异常分布导致卵母细胞胞质不成熟影响其受精能力；透明带结构异常及诱导顶体反应缺陷可导致精子穿透障碍，常规IVF受精失败；卵母细胞激活失败与ICSI受精失败有关。 2．脂褐体的出现及环纹片层异常聚合与受精卵母细胞不分裂有关。 3．卵裂球核内异染色质增多与胚胎发育阻滞有关。 4．线粒体结构改变、能量代谢降低与卵母细胞受精失败、受精卵未分裂及胚胎发育阻滞有关。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321

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