Localization of proteasomes in human oocytes and preimplantation embryos

In the present study we describe the localization of proteasomes in human oocytes, apoptotic preimplantation embryos, and triploid preimplantation embryos by means of immunolabelling with the MCP21 monoclonal antibody detected by confocal microscopy. While in the oocytes proteasomes are scattered throughout the cytoplasm, in the pronuclear zygote they appear to concentrate at the periphery of the cytoplasm and do not enter the pronuclei. During early cleavage stages, proteasome immunolabelling is concentrated in the nuclei, while the examination of triploid blastocysts showed that proteasomes had a similar cellular distribution to somatic cell lines, i.e. in the nuclei but not in the nucleoli or the cytoplasm. It appears that the distribution of proteasomes dramatically changes during human preimplantation embryo development.     

Beta-N-acetylhexosaminidase activity in human oocytes and preimplantation embryos.

beta-N-acetylhexosaminidase is a lysosomal enzyme, which has two isoenzymes: beta-Hex A, a trimer consisting of one alpha-chain and two beta-chains (alpha beta 2) and beta-Hex B, a tetramer formed of four beta-chains (beta 2 beta 2). Genetic defects in the alpha-chain lead to Tay-Sachs disease, whereas mutations in the beta-chain gene lead to Sandhoff disease. In a previous study we developed a microassay for total beta-N-acetylhexosaminidase and used this for measuring activities in mouse oocytes and preimplantation embryos. In this study, to assess the feasibility of transferring this technique to the human for the purposes of preimplantation diagnosis for Tay-Sachs and Sandhoff disease, beta-Hex activity was assayed in human oocytes and embryos and in the medium in which they had been cultured. We showed that although the activity of beta-N-acetylhexosaminidase in human oocytes and embryos was > 500 times higher than in the mouse, it was not detectable in the culture medium and the activity in oocytes and embryos remained virtually constant throughout human preimplantation development, making it difficult to distinguish embryonic from maternal enzyme activity. In the absence of this distinction it would be inappropriate to use beta-N-acetylhexosaminidase activity for the purposes of preimplantation diagnosis of Sandhoff or Tay-Sachs disease. These experiments demonstrate that measuring the beta-N-acetylhexosaminidase activity in human embryos cannot be used at present for preimplantation diagnosis.     

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。     

Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos

Mitochondria play a vital role in the metabolism of energy-containing compounds in the oocyte cytoplasm to provide adenosine trisphosphate for fertilization and preimplantation embryo development. In this study, ratiometric confocal microscopy with the mitochondrion-specific membrane potential-sensitive fluorescence dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) was used to measure the activity of mitochondria in human oocytes and developing preimplantation embryos. Mitochondria in oocytes and embryos were characterized by distinct localized aggregation patterns. These patterns however did not determine localized regions of heterogeneity in mitochondrial activity. Mitochondrial activity was analysed during oocyte maturation and after fertilization. The activity of mitochondria in fresh metaphase II oocytes was negatively correlated with maternal age. This trend continued when the activity of developing embryos was analysed. Mitochondrial activity was strongly correlated with the rate of embryo development on day 3 after fertilization, but not on day 2. Partial regression analysis showed that the rate of cleavage of preimplantation embryos was more highly correlated with embryo mitochondrial activity than maternal age. These data suggest that the efficiency of mitochondrial respiration in oocytes and preimplantation embryos is closely correlated with the programmed rate of embryo development, and suggest that maternal age further influences this factor. The loss of mitochondrial activity in oocytes obtained from ageing couples may therefore contribute to lower embryo development and pregnancy rates observed during cycles of IVF.     

Fertilization and early embryology: Polypeptide profiles of human oocytes and preimplantation embryos

The polypeptides that direct fertilization and early developmentuntil activation of the embryonic genome occurs, at the 4–8cell stage in the human, are exclusively maternal in origin,and are either synthesized during oogenesis or translated laterfrom maternal mRNA. Using sodium dodecyl sulphate—polyacrylamidegel electrophoresis and silver stain, we have visualized andcompared the polypeptides present in different populations ofhuman oocytes and cleavage stage embryos obtained after superovulationand insemination in vitro. Two polypeptide patterns were resolved,differing in the region of mol. wt 69 kDa. The distributionof these patterns showed no correlation with the ability ofindividual oocytes to achieve fertilization and develop normallyto the 8-cell stage.     

Biogenic monoamines in mouse oocytes and preimplantation embryos

N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Moscow. Institute of Experimental Biology, Academy of Sciences of the Kazakh SSR, Alma-Ata. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 6, pp. 577–578, June, 1990.     

Expression of glucose transporter and glucose uptake in human oocytes and preimplantation embryos

The expression of glucose transporters 1, 2, 3 and 4 was evaluated in human oocytes and polyploid preimplantation embryos. Only glucose transporter 1 (GLUT-1) isoform was detected in oocytes and in 2-12-cell stage embryos. Glucose uptake was markedly increased in embryos as compared to oocytes (19.7 +/- 3.4 pmol/min/embryo and 2.3 +/- 0.3 pmol/min/oocyte), and GLUT-1 was inhibited by cytochalasin B. These results suggest that, although GLUT-1 is expressed in human oocytes and throughout preimplantation development, its function in mediating the rise in glucose uptake is triggered following fertilization.      

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。     

Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos

Pyruvate and glucose uptake by 73 indivudial human oocytes andpreimplantation embryos was measured non-invasively, using anultramicrofluorescence assay to analyse changes in substratelevels in microdroplets of culture medium. The uptake of bothsubstrates was measured over successive daily incubations betweendays 1(unfertilized oocytes)or 2 (‘spare’ embryoswhich were not transferred) and day 6 (dat 0= day of insemination).Under these conditions, 58% (25/43) of fertilized embryos withtwo pronuclei on day 1 developed to the blastocyst stage byday 6. The pyruvate uptake of these embryos increased from 28 to a maximum of 40 pmol/ embryo/h between days 2.5 and 4.5.Similarly, glucose uptake increased from 8 to 14 pmol/embryo/hbetween days 2.5 and 4.5, but then increased further to 24 pmol/embryo/hon day 5 at the blastocyst stage. The pyruvate uptake of fertilized4.5, but then increased further to 24 pmol/embryo/h on day 5at the blastocyst stage. The pyruvate uptake of fertilized embryoswhich arrested at cleavage stages was significantly lower thanfor those which developed to the blastocyst stage. Polyspermicand parthenogenetic embryos, and unfertilzed oocytes also hadlower pyruvate uptakes at later stages. The glucose uptake ofunfertilized oocytes and abnormal embryos never reached thelevel of fertilized embryos at the blastocyst stage on day 5.5.Non-invasive measurement of pyruvate uptake before embryo transfermay provide a valuable functional criterion for the selectionof viable embryos capable of developing to the blastocyst stage     

Non-invasive measurement of glucose and pyrovate uptake by individual human oocytes and preimplantation embryos

Pyruvate and glucose uptake by 73 individual human oocytes andpreimplantation embryos was measured non-invasively, using anultramicrofluorescence assay to analyse changes in substratelevels in mkrodroplets of culture medium. The uptake of bothsubstrates was measured over successive daily incubations betweendays 1 (unfertilized oocytes) or 2 (‘spare’ embryoswhich were not transferred) and day 6 (day 0 = day of insemination).Under these conditions, 58% (25/43) of fertilized embryos withtwo pronuclei on day 1 developed to the blastocyst stage byday 6. The pyruvate uptake of these embryos increased from –28to a maximum of 40 pmol/ embryo/h between days 2.5 and 4.5.Similarly, glucose uptake increased from 8 to 14 pmol/embryo/hbetween days 2.5 and 4.5, but then increased further to 24 pmol/embryo/hon day 5 at the blastocyst stage. The pyruvate uptake of fertilizedembryos which arrested at cleavage stages was significantlylower than for those which developed to the blastocyst stage.Polyspermk and parthenogenetic embryos, and unfertilized oocytesalso had lower pyruvate uptakes at later stages. The glucoseuptake of unfertilized oocytes and abnormal embryos never reachedthe level of fertilized embryos at the blastocyst stage on day5.5. Non-invasive measurement of pyruvate uptake before embryotransfer may provide a valuable functional criterion for theselection of viable embryos capable of developing to the blastocyststage.     

Calcium-binding proteins and calcium-release channels in human maturing oocytes,pronuclear zygotes and early preimplantation embryos

BACKGROUND: The study aim was to investigate the presence and localization of Ca2+-binding proteins and Ca2+-release receptor channels in human maturing oocytes, pronuclear zygotes and preimplantation embryos. METHODS: Immunocytochemical analysis, using specific antibodies against the proteins being studied, followed with confocal laser microscopy, was performed on human oocytes and embryos. RESULTS: Calreticulin and calsequestrin (the two major calcium storage proteins of somatic cells), two types of calcium release receptors, the inositol trisphosphate and ryanodine receptors (InsP(3)R-2, RyRs-1,2,3), and the molecular chaperone, calnexin, were identified in all investigated cell types. Calreticulin was predominant in the cell cortex and in the nuclear envelope, while calsequestrin was distributed throughout the entire cytoplasm. Generally, localization of the InsP(3)R-2 and RyRs was similar to that of calreticulin and calsequestrin respectively. Both types of receptor were enriched in the subplasmalemmal region of meiotic oocytes. In addition, the InsP(3)R was detected in the nuclear structures of oocytes and blastomeres. Calnexin distribution overlapped with that of calreticulin but appeared to be present in distinct subcompartments. CONCLUSIONS: Human oocytes and embryos express the calcium sequestration and release proteins in highly organized and developmentally regulated patterns. Fine-tuning of these proteins may play a crucial role in regulation of Ca2+ transience during oocyte maturation, fertilization and early embryo development.     

Positive outcome after preimplantation diagnosis of aneuploidy in human embryos.

usromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization (IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol concentrations on day +1, and average mature follicles. All these parameters were similar in test and control groups. Only embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (P < 0.05), and ongoing pregnancies and delivered babies increased (P < 0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation; (ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.     

Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes

Dual fluorescent in-situ hybridization (FISH) using X and Ychromosome specific probes has been used to identify the sexof human embryos for preimplantation diagnosis of X-linked disease.With a modified spreading method and directly labelled fluorescentDNA probes, we have examined the possibility of reducing thetime of the FISH procedure from 7 to 2 h. A total of 17 normallyfertilized human embryos were disaggregated and 98 intact blastomeresobtained. The spreading efficiency was 96% and FISH signalswere obtained from 97% of nuclei. In all cases, sibling blastomeresfrom the same embryo were the same sex. Mosaicism was observedin some embryos. Five cells which lysed during the dis–aggregationprocess were spread to determine whether FISH was possible inthese cells, but in all cases the morphology of the nuclei waspoor and multiple signals were observed so that reliable diagnosisof sex was not possible. The data reported here confirm thatby using an improved spreading method in combination with directlylabelled DNA probes, we have increased the efficiency and reducedthe time required for sexing embryos for preimplantation diagnosisof X-linked disease.     

Developmental competence of mouse embryos following zona drilling using a non-contact holmium: yttrium scandian gallium garnet (Ho: YSGG) laser system

The purpose of this study was to assess the efficacy of theholmium: yttrium scandian gallium garnet (Ho:YSGG) laser, operatingin a pipette-free, non-contact mode, to assist hatching andsustain normal embryonic development. Two-cell mouse embryoswere recovered and assigned to laser-assisted hatching (LAH)treatment or control human tubal fluid (HTF) culture with orwithout serum (HTF-s, HTF-o) or with late serum supplementation(HTF-o/s). The basic experimental apparatus for LAH consistedof a stationary 2.1 µm Ho: YSGG laser beam directed througha mechanical shutter into an input port of a Zeiss Axiomat invertedmicroscope. Fewer (P < 0.05) embryos developed to the blastocyststage in the HTF-s group (81%) than in the LAH (90%), HTF-o(94%) and HTF-o/s (92%) groups. The level of hatching was significantlyincreased (P < 0.01) after the LAH treatment (57%) comparedto HTF-o/s (32%), HTF-s (18%) or HTF-o (5%). Implantation rateswere not significantly impaired following the LAH treatment(21%). These data demonstrate that LAH using the Ho: YSGG laseris a simple, accurate and effective procedure for assisted hatching.     

The peptide nucleic acids as probes for chromosomal analysis: application to human oocytes, polar bodies and preimplantation embryos

Peptide nucleic acids (PNA) are synthetic DNA mimics based on an uncharged polyamide backbone, which hybridize with complementary DNA with high affinity and specificity. PNA have recently become recognized as efficient tools for in situ chromosomal identification. In the present study, this new approach has been tried on isolated human oocytes, polar bodies and blastomeres. Using centromeric PNA probes specific for chromosomes 1, 4, 9, 16, X and Y, we tested multicolour labelling PNA reaction on 27 oocytes and 23 blastomeres. Sequential PNA hybridization was performed on five oocytes and combined PNA and fluorescence in situ hybridization (FISH) reactions on two oocytes. Both the rates and the types of abnormalities observed are in agreement with results from previous FISH studies. This preliminary study indicates that PNA probes allow a reliable chromosomal analysis in isolated human oocytes and blastomeres and consequently might provide an interesting adjunct to FISH for diagnostic analysis.