小鼠胚着床前线粒体的分布和超微结构变化

为了解小鼠着档前细胞中线粒体的分布和超微结构的变化规律，观察了２细胞胚，４细胞胚，８细胞胚，桑椹胚，早期囊胚和晚期囊胚，２细胞期和桑椹胚期，线粒体绕胞核集，在挤紧的８细胞胚中，线粒体在细胞接触面处的胞质边缘密集。囊胚期，滋养层细胞的线粒体在胞核周围较宽的区域中分布。     

溶酶体4次穿膜蛋白β在小鼠卵母细胞及早期胚卵中的表达

目的 观察溶酶体4次穿膜蛋白β(LAPTM4β)在小鼠卵母细胞及早期胚卵中的表达,分析新基因LAPTM4β在植入前胚卵发育中的作用,为研究植入前胚卵发育的基因调控提供实验依据.方法 从50只小鼠的子宫或输卵管中收集小鼠卵母细胞及从原核期到胚泡的早期胚卵共50枚.采用激光扫描共焦显微镜技术与免疫组织化学ABC法,观察小鼠卵母细胞及早期胚卵中LAPTM4β的表达;表达结果用Maticam2206图像分析系统进行半定量分析.结果激光扫描共焦显微镜下显示,小鼠卵母细胞和原核期胚卵细胞质的绿色荧光呈不均匀分布,近细胞膜处荧光信号强;2细胞期、4细胞期、8细胞期和桑椹胚卵裂球胞质中绿色荧光均呈局灶性分布,且靠近细胞膜处胞质中绿色荧光均较强,2细胞期中所见极体内也有绿色荧光信号;小鼠胚泡滋养层细胞及内细胞团中均可见中等强度的绿色荧光.免疫组织化学显示,在小鼠卵母细胞中可见LAPTM4β阳性反应颗粒呈浅棕色;原核期、2细胞期、4细胞期、8细胞期胚及桑椹胚,可见深棕色颗粒,且在卵裂球胞质中呈不均匀分布;胚泡的内细胞团及滋养层细胞中均可见棕色阳性反应颗粒.图像分析结果显示,卵母细胞与原核期胚相比,8细胞期与桑椹胚相比,桑椹胚与胚泡相比,LAPTM4β的表达量有显著差异(P<0.05).结论 LAPTM4β在正常小鼠卵母细胞及植入前各期胚卵中均有表达,且具有规律性;不同阶段早期胚卵中LAPTM4β的表达量均高于卵母细胞,推测IAPTM4β与小鼠早期胚卵的细胞分裂、增殖、分化及胚胎发育相关.     

GnRH mRNA and protein expression in human preimplantation embryos.

Gonadotrophin-releasing hormone (GnRH) regulates gonadotrophin biosynthesis and release in the anterior pituitary via specific receptors. Extrapituitary expression and action of GnRH have been demonstrated in several species. A possible role for GnRH in preimplantation embryonic development, endometrial preparation, and the implantation process has been previously suggested. Moreover, the presence of an immunoreactive GnRH in preimplantation embryos has been demonstrated in different species; however, there are no data for human embryos. We postulate that in humans GnRH may play a role in preimplantation embryonic development as well as in the implantation process. To examine this hypothesis, we assessed GnRH and GnRH-receptor mRNA and protein expression in human preimplantation embryos with three pronuclei. GnRH is expressed in peri-implantation human embryos at both the mRNA and protein level. GnRH-receptor mRNA is also present in the embryos studied. Immunohistochemical localization of GnRH showed intense staining in all the blastomeres at morula stage as well as in the trophectoderm and inner cell mass of the blastocysts. The results of the present study challenge the widely held view that GnRH has a predominantly central action, and suggests a pathway to describe a local role for the GnRH system in successful preimplantation embryonic development and implantation.     

Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases

To perform preimplantation genetic diagnosis for women carrying heteroplasmic mitochondrial DNA (mtDNA) mutations, it is necessary to ensure that the proportion of mutant mtDNA diagnosed in the biopsied cell gives an accurate indication of the mutant load in the remaining embryo. A heteroplasmic mouse model, carrying NZB and BALB mtDNA genotypes, was used to study the relative proportions of each mtDNA genotype in the ooplasm and first polar body of mature oocytes, and between blastomeres of early cleavage stage embryos. The levels of heteroplasmy varied widely in the gametes compared with the maternal genotype. However, the distribution of the two mtDNA genotypes was virtually identical between the ooplasm and polar body of a mature oocyte, and also between the blastomeres of each 2-, 4- and 6-8-cell embryo. Therefore, the level of heteroplasmy diagnosed from the polar body of an unfertilized oocyte or from a single blastomere of an embryo is representative of the level in the embryo as a whole. Reliable results were obtained from both polar bodies and blastomeres, but the efficiency of diagnosis was greater with blastomeres. We conclude that preimplantation genetic diagnosis is feasible for mtDNA diseases, although it should be approached with caution, as it is possible that transmission of some pathogenic mutations could behave in a different manner.     

Conceptus polarity and cell-zona adhesion during early cleavage (fertilized tubal egg to 8-cell stage) in the marsupial Sminthopsis macroura

This study outlines the ultrastructural changes that occur in Sminthopsis macroura tubal zygotes to the 8-cell stage in relation to observations of development in vitro, oocyte polarity and cell-zona adhesion. The extremely polarized mature oocytes and zygotes have nuclear material at one pole and accumulated vesicular bodies at the other. The first division is associated with extrusion of vesicular bodies and some cytoplasm as a membrane-bound yolk mass into the perivitelline space. Early cleavage is accompanied by the appearance of an extensive, highly structured extracellular matrix (ECM) comprised of amorphous substance, granules and filaments. At the 2- and 4-cell stage the decrease in density of the ECM in the vicinity of the blastomeres may facilitate cell-zona contact. At the 8-cell stage, discharge of vesicular bodies, which mostly appear to be empty, may contribute to the ECM by increasing the area of plasma membrane for synthesis of a hyaluronan-like ECM. As in other marsupials, the precedence of cell-zona adhesion over cell-cell contacts prevents morula formation. The earliest cell-zona contacts appear when microvilli contact the zona in the uterine zygote 12-16 h after uterine entry and continue at later stages. This early contact is possible because of the absence of a dense subzonal ECM in this species. Between late zygote and late 4-cell stage the cytoplasm also contains, beside a large amount of vesicular bodies, demarcated areas where smooth endoplasmic reticulum encloses mitochondria, vesicles, granular material and fibrillar arrays. The latter develop in the late zygote stage and are found outside demarcated areas as well, often closely surrounding large vesicles, probably helping vesicle extrusion. A putative germ plasm was identified at the 4-cell stage.     

Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells

Ova with two pronuclei were co-cultured with established human ampullary cell lines and various stages of preimplantation embryonic development were monitored by Nomarski optics and then assessed by transmission electron microscopy (TEM). Fifteen embryos ranging from the 2-cell stage to blastocyst hatching were examined for normal and abnormal features. Their ultrastructure was similar to that of embryos cultured in Whittingham's T6 medium, reported previously. Seven embryos were evidently morphologically normal and showed good organization of fine structure. Most cellular organelles underwent progressive changes during early development. There was evidence of enhanced embryonic genome activation at the 8-cell stage. Invariably, all embryos had few too many fragments, some internalized, which were later segregated into the blastocoele or found outside the trophoblast of the late morula and blastocysts. Six grossly 'normal' embryos assessed by Nomarski had multiple nuclei of various dimensions, which highlights the subjectivity of embryo assessment in the IVF laboratory. Incomplete incorporation of chromatin into nuclei and formation of micronuclei were evident in some blastomeres. The results are discussed in relation to early embryonic loss, prevalent in IVF. Significant events reported include the detection of centrioles at the 8-cell stage, cavitation of the early blastocyst and the initiation of blastocyst hatching visualized by TEM.     

Secreted proteins of the oviduct

During late follicular development and estrus, the mammalian oviduct undergoes specific physiological and biochemical modifications which contribute to an optimization of the microenvironment for fertilization and early cleavage-stage embryonic development. These changes appear to be hormonally regulated by ovarian steroids, most importantly, estrogen. The hundreds of macromolecules found within the oviductal lumen are contributed by selective serum transudation and active biosynthesis and secretion from nonciliated epithelial cells. Recent studies have indicated temporal and regional (infundibulum, ampulla and isthmus) differences in steady-state levels of specific mRNAs and in de novo protein synthesis and secretion by the oviduct. One protein synthesized de novo, the estrogen-dependent oviductal secretory glycoprotein (OSP), has been shown to be unique to the oviduct and is conserved across a number of mammalian species. This protein associates with the zona pellucida, perivitelline space and vitelline or blastomere membrane of ovulated eggs and preimplantation embryos. OSPs have been shown to enhance sperm binding and penetration in oocytes and may regulate development in early preimplantation embryos. Other regulatory molecules, protease inhibitors, growth factors, cytokines, binding proteins, enzymes and immunoglobulins have been identified in the oviductal microenvironment. The identification and potential roles for oviduct-secreted proteins will be reviewed and discussed. Current research focuses on continued identification and characterization of specific oviductal proteins and a determination of the molecular basis of their interactions with the oocyte, sperm or embryo.     

Geminin is essential for the development of preimplantation mouse embryos

Replication of DNA is strictly controlled to ensure that it occurs only once per cell cycle. Geminin has been thought to serve as a central mediator of this licensing mechanism by binding to and antagonizing the function of Cdt1 and thereby preventing re-replication during S and G2 phases. We have now generated mice deficient in geminin to elucidate the physiologic role of this protein during development. Lack of geminin was shown to result in preimplantation mortality. A delay in the development of homozygous mutant embryos was first apparent at the transition from the four- to eight-cell stages, concomitant with the disappearance of maternal geminin protein, and development was arrested at the eight-cell stage. The mutant embryos manifest morphological abnormalities such as dispersed blastomeres with nuclei that are irregular both in size and shape as well as impaired cell-cell adhesion. DNA replication occurs but mitosis was not detected in the mutant embryos. The abnormal blastomeres contain damaged DNA and undergo apoptosis, likely as a consequence of the deregulation of DNA replication. Our results suggest that geminin is essential for cooperative progression of the cell cycle through S phase to M phase during the preimplantation stage of mouse development.     

Variation in the dry mass of mouse embryos throughout the preimplantation period.

Information on the net balance of embryonic metabolism during the preimplantation period is provided by measurements of embryo dry mass. The dry mass of mouse embryos at 10 defined stages of preimplantation development was measured using the Vickers M86 scanning microinterferometer. The results demonstrate that there is an overall loss of dry mass from the unfertilized ovum (39.10 +/- 0.6 ng) to the late blastocyst stage (32.80 +/- 0.53 ng) with these differences being highly significant. However, there is a significant increase in embryonic dry mass from the 1-cell (36.88 +/- 0.34 ng) to 2-cell stage (40.48 +/- 0.40 ng). These results suggest that protein synthesis exceeds degradation during the 2-cell stage but that from the 4-cell to morula stage the reverse is true. In addition, the variation in dry mass between embryos at the same developmental stage is extremely small, suggesting that this may be a useful indicator of embryonic viability.     

From oocyte to 16-cell stage: cytoplasmic and cortical reorganizations that pattern the ascidian embryo.

The dorsoventral and anteroposterior axes of the ascidian embryo are defined before first cleavage by means of a series of reorganizations that reposition cytoplasmic and cortical domains established during oogenesis. These domains situated in the periphery of the oocyte contain developmental determinants and a population of maternal postplasmic/PEM RNAs. One of these RNAs (macho-1) is a determinant for the muscle cells of the tadpole embryo. Oocytes acquire a primary animal-vegetal (a-v) axis during meiotic maturation, when a subcortical mitochondria-rich domain (myoplasm) and a domain rich in cortical endoplasmic reticulum (cER) and maternal postplasmic/PEM RNAs (cER-mRNA domain) become polarized and asymmetrically enriched in the vegetal hemisphere. Fertilization at metaphase of meiosis I initiates a series of dramatic cytoplasmic and cortical reorganizations of the zygote, which occur in two major phases. The first major phase depends on sperm entry which triggers a calcium wave leading in turn to an actomyosin-driven contraction wave. The contraction concentrates the cER-mRNA domain and myoplasm in and around a vegetal/contraction pole. The precise localization of the vegetal/contraction pole depends on both the a-v axis and the location of sperm entry and prefigures the future site of gastrulation and dorsal side of the embryo. The second major phase of reorganization occurs between meiosis completion and first cleavage. Sperm aster microtubules and then cortical microfilaments cause the cER-mRNA domain and myoplasm to reposition toward the posterior of the zygote. The location of the posterior pole depends on the localization of the sperm centrosome/aster attained during the first major phase of reorganization. Both cER-mRNA and myoplasm domains localized in the posterior region are partitioned equally between the first two blastomeres and then asymmetrically over the next two cleavages. At the eight-cell stage the cER-mRNA domain compacts and gives rise to a macroscopic cortical structure called the Centrosome Attracting Body (CAB). The CAB is responsible for a series of unequal divisions in posterior-vegetal blastomeres, and the postplasmic/PEM RNAs it contains are involved in patterning the posterior region of the embryo. In this review, we discuss these multiple events and phases of reorganizations in detail and their relationship to physiological, cell cycle, and cytoskeletal events. We also examine the role of the reorganizations in localizing determinants, postplasmic/PEM RNAs, and PAR polarity proteins in the cortex. Finally, we summarize some of the remaining questions concerning polarization of the ascidian embryo and provide comparisons to a few other species. A large collection of films illustrating the reorganizations can be consulted by clicking on "Film archive: ascidian eggs and embryos" at http://biodev.obs-vlfr.fr/recherche/biomarcell/.     

Pulmonary Mycobacterium tuberculosis infection in leptin-deficient ob/ob mice

The development of active tuberculosis after infection with Mycobacterium tuberculosis is almost invariably caused by a persistent or transient state of relative immunodeficiency. Leptin, the product of the obese (ob) gene, is a pleiotropic protein produced mainly by adipocytes and is down-regulated during malnutrition and starvation, conditions closely connected with active tuberculosis. To investigate the role of leptin in tuberculosis, we intranasally infected wild-type (Wt) and leptin-deficient ob/ob mice with live virulent M. tuberculosis. Ob/ob mice displayed higher mycobacterial loads in the lungs after 5 and 10 weeks of infection, although the difference with Wt mice remained 1 log of M. tuberculosis colony forming unit. Nevertheless, ob/ob mice were less able to form well-shaped granuloma and lung lymphocyte numbers were reduced compared with Wt mice early during infection. In addition, ob/ob mice had a reduced capacity to produce the protective cytokine IFNgamma at the site of the infection early during infection and upon antigen-specific recall stimulation, and showed reduced delayed-type hypersensitivity reaction to intra-dermal tuberculin purified protein derivative. Leptin replacement restored the reduced IFNgamma response observed in ob/ob mice. Mortality did not differ between ob/ob and Wt mice. These data suggest that leptin plays a role in the early immune response to pulmonary tuberculosis.     

Leptin-dependent toll-like receptor expression and responsiveness in preadipocytes and adipocytes

Leptin, an adipokine mainly produced by adipocytes, has been well characterized with regard to its regulatory function on immune cells. Thus the question occurred of how adipocytes and preadipocytes interact with the immune system and whether or not this communication is regulated by leptin. With the present study we evaluated the Toll-like receptor (TLR) expression and TLR ligand-specific activation of murine preadipocytes and adipocytes in the presence [wild type (WT), 3T3L1] or absence of leptin (ob/ob) or leptin signaling (db/db). The ob/ob as well as db/db adipocytes and preadipocytes were characterized by a significant up-regulation of TLR1 to -9 expression when compared with WT cells. In WT preadipocytes the TLR responsiveness increased during maturation to adipocytes; however, stimulation of ob/ob and db/db cells resulted in a 10- to 20-fold higher interleukin-6 production. Signaling studies revealed, in addition to the increased TLR expression, alterations in the phosphoinositide 3 kinase signaling cascade in ob/ob and db/db cells as an explanation for this increased responsiveness. In conclusion, the present study indicates the expression and responsiveness of TLR1 to -9 in murine preadipocytes as well as adipocytes, both of which are strongly regulated by the adipokine leptin. In summary, these data further emphasize the role of fat tissue in the immune system.     

Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence

It has been suggested that mitochondrial DNA defects that effect metabolic capacity may be a proximal cause of failures in oocyte maturation, fertilization, or early embryonic development. Here, the distribution of mitochondria was examined by scanning laser confocal microscopy in living human pronuclear oocytes and cleavage stage embryos, followed either by measurements of the net ATP content of individual blastomeres or anti-tubulin immunofluorescence to determine the relationship between mitochondrial distribution and microtubular organization. The results indicate that specific patterns of perinuclear mitochondrial aggregation and microtubular organization are related, and that asymmetrical mitochondrial distributions at the pronuclear stage can result in some proportion of blastomeres with reduced mitochondrial inheritance and diminished ATP generating capacity. While the inability to divide appears to be a development consequence for an affected blastomere, for the embryo, reduced competence may occur during cleavage if several blastomeres inherit a mitochondrial complement inadequate to support normal cellular functions. The findings provide a possible epigenetic explanation for the variable developmental ability expressed within cohorts of morphologically normal early cleavage stage human embryos obtained by in-vitro fertilization.     

原癌基因产物Ras及c-erbB2在着床前小鼠胚胎中的表达

目的　观察小鼠着床前的胚胎细胞中原癌基因ras编码的蛋白产物 p2 1Ras及c erbB2的表达 ,并了解其对着床前的胚胎发育的可能作用。方法　采用免疫组织化学ABC法 ,检测着床前小鼠胚胎中p2 1Ras及c erbB2蛋白的表达。结果　小鼠着床前的胚胎细胞中 p2 1Ras的免疫反应呈阳性 ,阳性物质分布于胞质中 ,胚胎外透明带呈阴性反应 ;而在各期着床前的胚胎中均未检测到c erbB2蛋白的表达。结论　小鼠着床前的胚胎中 ,有原癌基因ras编码蛋白的表达 ,p2 1Ras是增殖和分化受体介导的信号传导途径中的重要组成部分 ,可能在小鼠着床前的胚胎发育过程中起重要的调节作用。C erbB2基因对于围着床期子宫的生长发育具有重要的作用 ;而在小鼠着床前的胚胎发育过程中似乎不起重要作用