Effects of visible light and room temperature on the ultrastructure of preimplantation rabbit embryos: a time course study

Summary In a time course study (4–20 h) rabbit early cleavage stages (day 1 p.c.) and compacted morulae (day 3 p.c.) were exposed to visible light or room temperature (23° C), respectively. An 8 h light exposure of day 1 embryos caused alterations in nuclear morphology (lobulated nuclei, loss of nucleolar diffentiation), an increased electron density of the cytoplasm, and cellular fragmentation leading to a considerable degeneration of blastomeres (central clustering of organelles, loss of cell surface differentiation) after a 20 h exposure. Room temperature exposure (compacted Day 3 morulae) led to decompaction and a cleavage delay after 8 h. After 10 h, arrested metaphases occurred in all examined morulae. Even after 20 h at 23° C, day 3 embryos were at the decompacted morula stage, and showed metaphase-arrested blastomeres. The general morphology of the blastomeres was unaffected at this temperature, except for vacuolated serand cis-side vesicles of the Golgi complex at 8, 12 and 20 h, respectively.     

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.     

人植入前胚胎细胞超薄电镜切片制作

目的改进人植入前胚胎细胞超薄切片的制备方法,观察不同时期人早期胚胎细胞超微结构的变化。方法选取我院生殖中心试管婴儿助孕技术后异常受精的3原核胚胎,于2~8细胞期进行固定,改进透射电镜的常规操作方法,使用3%~4%琼脂进行预包埋,制成超薄切片后在镜下观察植入前胚胎细胞凋亡的超微结构。结果使用本方法可以对少量胚胎进行超薄切片的制作。结论使用3%~4%琼脂预包埋的方法可以成功的制作人植入前胚胎细胞的超薄电镜切片。     

MATER蛋白在人类卵母细胞及着床前胚胎中的表达

目的探讨Mater在人卵发生和胚胎早期发育中的表达情况。方法收集体外受精助孕过程中未成熟的卵母细胞及不适合移植及冷冻保存的胚胎，采用免疫荧光联合激光共聚焦显微镜技术检测人类卵母细胞及着床前胚胎MATER蛋白的表达。结果16个未成熟卵母细胞15个MATER蛋白表达阳性；58个2—8细胞期人类早期胚胎中40个MATER蛋白检测阳性；5个囊胚和4个孵化出囊胚内细胞团均为阴性，滋养层细胞为弱阳性。结论MATER蛋白在人类卵母细胞中存在，并随着早期胚胎的发育其表达阳性率逐渐降低，至囊胚期只有滋养层细胞少量表达，这符合母性效应基因在哺乳动物中的表达模式。     

Ultrastructural changes of the early visceral yolk sac layer of mouse embryos after maternal injection of trypan blue

Summary The ultrastructural features of the early visceral yolk sac epithelium of normal mouse embryos on day 9 were compared to those whose mothers had received a single subcutaneous injection of 100 mg/kg trypan blue on day 8. The following results were obtained: In normal embryos the visceral yolk sac cells are predominantly characterized by numerous membrane bounded inclusions localized in the supranuclear cytoplasm. In embryos of animals treated with trypan blue, at about 12h after injection large single and only partly membrane bounded vacuoles are observed occupying most of the apical cytoplasm. Up to 24h after injection large cytoplasmic areas are seen which are in a stage of autodigestion possibly due to leakage of the vacuolar content. These alterations are exclusively limited to the visceral yolk sac epithelium whereas in the cells of the embryonic part, e.g. head process, no changes could be found. The observations are discussed in relation to the mechanism of the teratogenic activity of trypan blue.This investigation has kindly been supported by a grant of the Deutsche Forschungsgemeinschaft (Schl 166/1)     

Ultrastructural observations on cell necrosis during formation of the neural tube in mouse embryos

Summary Spontaneous cell death in the developing brain of 8.5–9 day old mouse embryos has been investigated with the electron microscope. Before closure of the neural tube, areas of cell death are found at the neuro-somatic junction. After closure of the neural tube degenerating cells are found in the dorsal midline of the prospective diencephalon. Ultrastructurally, cell degeneration is marked by chromatin condensation, increase in electron density of the structures in the cytoplasm and, in later stages, by a marked pycnosis of the dying cell. After fragmentation, the necrotic material as well as entire pycnotic cells are phagocytized and digested by cells of the neuroepithelium. An invasion of macrophages has not been observed. The significance of cell necrosis is discussed in relation to the normal formation of the neural tube and to the occurrence of certain induced malformations (exencephalies).     

韦-伯综合征相关印记基因在人类卵母细胞及植入前胚胎的正常表达

目的 检测韦-伯综合征(Beckwith-Wiedemann syndrome，BWS)相关印记基因在人类卵母细胞和植入前胚胎的正常表达，探讨辅助生殖技术(assisted reproductive technology，ART)和BWS的关系。方法应用嵌套式逆转录-聚合酶链反应技术检测印记基因P57KIP2、LIT1、TSSC3在人类卵母细胞及植入前胚胎的正常表达。结果 卵母细胞和各期植入前胚胎、囊胚泡中均存在P57KIP2表达。LIT1自8细胞胚胎开始表达，持续至囊胚泡期。TSSC3于卵母细胞及各期植入前胚胎中均未表达。结论 BWS相关印迹基因LIT1、P57KIP2表达于植入前胚胎，ART技术的体外干预可能影响其印迹基因的正常表达。     

Chromosome remodeling and differentiation of tetraploid embryos during preimplantation development

Although it is known that the tetraploid embryo contributes only to the placenta, the question of why tetraploid embryos differentiate into placenta remains unclear. To study the effect of electrofusion on the development of mouse tetraploid oocytes, mouse two‐cell embryos were fused and cultured in vitro in Chatot‐Ziomek‐Bavister medium. After electrofusion, two chromosome sets from the tetraploid blastomere were individually duplicated before nuclear fusion. At 8–10 hr after electrofusion, each chromosome set was condensing and the nuclear membrane was breaking down. Around 12–14 hr after electrofusion, the two chromosome sets had combined together and had reached the second mitotic metaphase, at this point with 8n sets of chromosomes. Interestingly, we discovered that expression of OCT4, an inner cell mass cells biomarker, is lost by the tetraploid expanded blastocysts, but that CDX2, a trophectoderm cells biomarker, is strongly expressed at this stage. This observation provides evidence clarifying why tetraploid embryos contribute only to trophectoderm. Developmental Dynamics 240:1660–1669, 2011. © 2011 Wiley‐Liss, Inc.     

Ultrastructure of early cleavage stages and preimplantation in the rabbit

Summary Fertilized rabbit ova were studied in the period from 25 to 144 hr after insemination. Eggs were recovered by flushing the uterine horns and oviducts with Hank's solution. The cells were morphologically alike in the 24 and 48 hr ova. At 661/2 hr blastomeres were differentiated into an inner mass of cells with dense cytoplasm and an outer trophoblastic layer with less dense cytoplasm. Otherwise no morphological differences were seen. Whether the 661/2 hr ova were morulas or blastocysts is discussed. The 96 hr ova were clearly blastocysts. Inner cells and trophoblastic cells at this stage bad the same cytoplasmic density. Mitochondria were increased in number and crystal-like figures were present for the first time. In the 120 and 144 hr ova the cytoplasm of the trophoblastic cells was denser than that of the inner cells. Trophoblastic cells were characterized by their density, crystal-like figures, elongated mitochondria with transverse cristae and many single ribosomes and they were interconnected with well developed junctional complexes. In a few cases a continuity seemed to exist between trophoblastic cells and inner cells. The latter were characterized by cytoplasm of less density than that of the trophoblastic cells, rounded mitochondria and fewer ribosomes. The fine structure of the crystal-like figures, their possible origin and differentiation of the mitochondria are discussed.This investigation was supported by the U.S. Public Health Service NIH International Postdoctoral Fellowship No. 4 FO5 TW 1365-02.     

Developmental and cytogenetic assessments of preimplantation embryos derived from in-vivo or in-vitro matured human oocytes

Aneuploidy is of great relevance to embryo selection, as it represents one of the important causes of implantation failure. Furthermore, immature oocytes, retrieved during gonadotrophin-stimulated IVF cycles, are generally discarded in clinics; whereas, there was no detectable comprehensive evidence on higher rates of aneuploidy based on maturity status on the day of oocyte retrieval. As well, the correlation between embryo morphology on aneuploidy remains unclear. The aim was to evaluate the developmental and genetic integrity of human preimplantation embryos from rescue in-vitro matured MII stage oocytes as well as in vivo matured oocytes. 541 rescue in-vitro matured oocytes as case as well as 659 in-vivo matured oocytes as control were used for the developmental assay. Finally, 121 cleaved embryos with good quality were analyzed by FISH technique for the detection of chromosomes X, Y, 13, 15, 16, 18, 21 and 22. The fertilization rates were 61.62% and 61.76% in case and control groups, respectively. Also, embryo formation rates of 89.1% vs. 92.2% were recorded for case and control groups, respectively. Good quality embryos on day 3 were 62.54% in case and 68.36% in control groups. There were insignificant differences in fertilization, embryo formation and quality between the groups. Total abnormality in 35 of the 60 embryos was 58.5% in case and 62.3% in control (p < 0.05). There were significant differences between aneuploidy rates of embryos using only sex chromosome preimplantation genetic screening (PGS) and sex chromosome in combination with autosomal chromosomes PGS in case (58.5% vs 28.3%, p = 0.000) and control groups (62.3% vs 21.3%; p = 0.000). The results demonstrated that a high proportion of good quality embryos were aneuploid in both patient groups with no obvious increase in aneuploidies as a result of rescue IVM application. Furthermore, the morphological characteristics of embryos do not completely consistent with chromosomal content. Despite the Rescue IVM is currently not a routine procedure in association with IVF, our finding suggested a viable option for young infertile women facing cancellation of their IVF treatment due to ovarian over-response or resistance factors as well as patients with low functional ovarian reserve considering good quality of embryos from rescue IVM-MII oocytes.     

Effect of the phenol antioxidant katavidan on autooxidation of microsomes exposed to visible light

A study is performed of the effect of the phenol antioxidant katavidan on autooxidation of microsomes from rat liver exposed to visible light. It is shown that katavidan in a concentration of 10⁻³ M inhibits but in concentrations of 10⁻⁵–10⁻⁷ M stimulates autooxidation of microsomes. No stimulation is observed under conditions of dark incubation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, № 10, pp. 393–394, October, 1994 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

Ultrastructure and protein transport of M cells in the rabbit cecal patch

Background: The gut-associated lymphoid tissue of the rabbit cecum includes a single lymphoid patch located close to the ileocecal orifice. Vimentin immunoreactivity, which can now be regarded as a marker for M cells in rabbits, has identified a subpopulation of epithelial cells as M cells in the domes of this patch. The aim of the present study was to demonstrate that these M cells are capable of antigen transport and to characterize their ultrastructure. Methods: M cells of the rabbit cecal lymphoid patch were studied by scanning, thin section, and freeze-fracture electron microscopy. The transcytosis across these M cells was investigated using horseradish peroxidase as a soluble tracer protein. Results: The M cells were concentrated at the flanks of the domes and had long, thick, branched microvilli, a well-developed terminal web, and a deep invagination of their apical membrane. Numerous small vesicles lay beneath the terminal web in close vicinity to the base of the invagination. These vesicles transported the luminally applied horseradish peroxidase through the M cells. In contrast to adjacent enterocytes, the glycocalyx of M cells was thin, stub-like, and had very few glycocalyceal bodies. Bacteria adhered to the surface of M cells and were also found in the apical invagination. Conclusions: The M cells of the rabbit cecal lymphoid patch differ from those of Peyer's patches of the small intestine in their ultrastructure and route of antigen transport. These differences might be related to the situations resulting from differences in the microbial populations at these locations. © 1995 Wiley-Liss, Inc.     

Effect of room temperature on percentage finger systolic blood pressure response to finger cooling

Percentage finger systolic blood pressure (%FSBP) in response to finger cooling is used to assess vascular components of the hand-arm vibration syndrome and the measurement method is under discussion for standardization. It has been suggested that measurement circumstances including room temperature may affect %FSBP. We investigated the effect of room temperature on %FSBP response to finger cooling in healthy subjects. Six healthy male subjects who were medical students volunteered for the study. Multi-channel plethysmograph was used for simultaneous multi-finger FSBP measurements. The examination room was kept at 21±1°C and 25±1°C, and the subjects were randomly assigned. Percentage finger systolic blood pressures for the index, middle, ring and little fingers at 15°C and 10°C cuff-water temperatures were calculated. Four-way analysis of variance was performed to determine the independent effect of subject, room temperature, finger and cuff-water temperature factors on %FSBP. The room temperature as an independent factor affecting %FSBP was statistically significant (P<0.01). From the results, it can be concluded that %FSBP response to finger cooling in healthy subjects may be affected by room temperature. Therefore, room temperature is expected to be controlled when assessing peripheral vascular components of the upper extremities using %FSBP response to finger cooling.     

Ultrastructure of the pinealocytes in rats exposed to light and radiation

Changes in pinealocytes (PC) were analysed using quantitative electron microscopy in 240 adult male rats from first minutes up to 180 days after their continuous exposure to bright light (CLE) for 48 hours, X-ray irradiation (XRI) or their combination (CE). After CLE early changes of PC included the reduction of rough endoplasmic reticulum (RER), Golgi complex and synaptic ribbons. At 24 hours and 10 days PC secretory activity was increased, while their ultrastructural organization was normalized by 30-180 days. 10 days after XRI degenerative changes were detected in PC that included dilation, fragmentation and vacuolization of RER cisterns, mitochondrial swelling, appearance of large vacuoles and osmiophilic inclusions, increase in lysosome content. Volume density of mitochondria and RER was lower, while that of Golgi complex was higher than in control. PC ultrastructure was restored 30-180 days after XRI. Following CE, the changes in PC ultrastructural organization were more significant at all time interval studied than after the action of single factors. The results obtained indicate that CLE increased the extent of postradiation changes in PC ultrastructural organization during the early time intervals after XRI and at the peak of radiation sickness development.     

Ultrastructure, protein synthesis and secretion of day-6 rabbit blastocysts cultured in a chemically defined, protein-free medium

Summary Day-6 rabbit blastocysts were cultured in Ham's F10 medium supplemented with polyvinylpyrrolidone as a macromolecular component, for 4 to 12 h. The integrity of the blastocyst cells was demonstrated by electron microscopy. Expansion and biosynthesis of proteins and of DNA were studied after culturing in the presence of ³⁵S-methionine and ³H-thymidine. Polyvinylpyrrolidone did not interfere with the subsequent protein analysis, which was performed by two dimensional gel electrophoresis followed by silver staining and fluorography. More than 600 labelled proteins were found in the blastocyst tissue, many of them were also present in the blastocyst fluid and in the blastocyst coverings. Several proteins seemed to be produced for incorporation into the blastocyst coverings; others, only detected in the culture medium, might have been synthesized for secretion into the environment.     

The cytogenetic analysis of human zygotes and preimplantation embryos

A review of cytogenetic studies on human zygotes and preimplantationembryos is presented. This survey documents the high incidenceof chromosomal abnormalities in embryos in vitro. Up to date,914 zygotes and embryos have been karyotyped. The rate of abnormalitiesis significantly higher in morphologically poor-quality embryosthan in good-quality embryos (86.6 and 36.6% respectively).In both groups, aneuploidy is the most frequently observed abnormality.In addition, various types of aberrations such as polyploidy,haploidy, mosaicism or fragmentation are also found. Among tripronucleatedzygotes, 81.9% display chromosomal abnormalities. Data suggestthat some parameters of IVF procedures might be responsiblefor the occurrence of some abnormalities.     

具有温度控制功能的复合光物理治疗系统

介绍了具有温度控制功能的复合光物理治疗系统的结构设计.系统以计算机为平台,采用宽谱辐射的卤钨灯作为辐照光源,将模糊控制算法应用于系统的温度控制中,实现对辐照表面温度的自动控制.通过对活体组织进行不同温度的控制实验证明,系统的控温精度均可达到允许误差±0.5℃范围以内,控温效果比较理想.     

Effects of evening bright light exposure on melatonin, body temperature and sleep

SUMMARY  Five male subjects were exposed to a single 2-h period of bright (2500 lux) or dim (<100 lux) light prior to sleep on two consecutive nights. The two conditions were repeated the following week in opposite order. Bright light significantly suppressed salivary melatonin and raised rectal temperature 0.3°C (which remained elevated during the first 1.5 h of sleep), without affecting tympanic temperature. Bright light also increased REM latency, NREM period length, EEG spectral power in low frequency, 0.75-8 Hz and sigma, 12–14 Hz (sleep spindle) bandwidths during the first hour of sleep, and power of all frequency bands (0.5–32 Hz) within the first NREMP. Potentiation of EEG slow wave activity (0.5-4.0 Hz) by bright light persisted through the end of the second NREMP. The enhanced low-frequency power and delayed REM sleep after bright light exposure could represent a circadian phase-shift and/or the effect of an elevated rectal temperature, possibly mediated by the suppression of melatonin.