Micromanipulation of cryopreserved embryos and cryopreservation of micromanipulated embryos in PGD

The possibility to employ cryopreservation in Preimplantation Genetic Diagnosis (PGD) should enlarge the opportunities for research and clinical activity. For these purposes, we tried three kinds of approaches on human abnormal embryos: (1) cryopreservation of biopsied embryos; (2) biopsy of thawed embryos; and (3) biopsy of embryos derived from thawed oocytes. Our preliminary results show that: (1) biopsy of thawed embryos is feasible and FISH analysis is possible on both survived and lysed cells; (2) Optimization of freezing/thawing procedures are necessary to obtain better survival rate after thawing of biopsied embryos; (3) Biopsy and FISH are feasible on embryos derived from thawed oocytes and they could be a good way to study the chromosomal arrangement of these poorly investigated embryos.     

Naegleria fowleri in chick embryos. Effects of embryo age and incubation temperature, and the infectivity of embryo-derived amebae for mice

Chick embryos were infected with Naegleria fowleri which was initially isolated from an ultimately fatal human case. Following inoculation of equivalent numbers of amebae on the chorioallantoic membrane, younger embryos died earlier than older embryos infected at the same time. Incubation of infected embryos at 32 degrees C prolonged survival only slightly in comparison with those at 37 degrees C. N. fowleri maintained for more than 25 serial passages in chick embryos retained infectivity for mice and the ability to convert to the biflagellate form in vitro.     

The hormonal activities associated with the cephalic and caudal regions of the cockerel pars distalis

The regional distribution of tropic hormone activities in the avian pars distalis was studied by transplanting the cephalic, middle, and caudal regions of the pars distalis of day-old cockerels onto the chorioallantoic membrane of 10-day-old partially decapitated (“hypophysectomized”) embryos. Bioassay was based on body growth (assessed by body weights and third toe lengths) and the growth and histological maturation of the adrenal and thyroid glands and the gonads. The experimental design consisted of normal embryos, hypophysectomized embryos, hypophysectomized embryos with one cephalic region graft, hypophysectomized embryos with one middle region graft, and hypophysectomized embryos with one caudal region graft. All embryos were examined at the end of 20 days of incubation.     

Short-term effects of altered shell conductance on oxygen uptake and hematological variables of late chicken embryos

The preceding report on the O2 uptake (MO2) of chicken embryos whose shell conductance (GO2) was altered from the beginning of incubation showed that the MO2 was decreased despite increased GO2 [Okuda, A. and H. Tazawa (1988) Respir. Physiol. 74: 187-198]. This was attributed to an excess water loss which reduced the growth of the embryos. The present study was designed to investigate the short-term effects of altered GO2, obviating the effect of excess water loss, on the MO2 and simultaneously on the hematological variables of embryos on days 16-17 and days 18-19 of incubation. The MO2 measured 5 h after increasing the GO2 was neither decreased nor increased significantly. The diffusing capacity of the chorio-allantoic membrane, which was estimated using the Bohr integration procedure, decreased as the GO2 was increased. When the GO2 was decreased, on the other hand, the decrease in MO2 was not so large as expected from the decrease in GO2, for both 16- and 18-day-old embryos. The effect of reduced GO2 on MO2 was more prominent in 18-day-old embryos than 16-day-old embryos. One-day-long hypoxia due to decreased GO2 induced erythropoiesis in 18-19-day embryos, but did not do so in 16-17-day embryos. The increase in hematocrit value of the latter group of embryos was attributed to an increase in cell volume due to concurrent hypercapnia.     

Apoptosis and its pathway in early post-implantation embryos of diabetic rats

It has been reported that diabetes-induced inappropriate apoptosis in embryos during neurulation may be one of the mechanisms leading to neural tube defects. We studied apoptosis and the apoptotic pathway occurring in early post-implantation period embryos of non-diabetic and streptozotocin (STZ)-induced diabetic rats. In quantitative RT-PCR, bax mRNA was constantly expressed to similar degree in embryos of non-diabetic and diabetic rats, while the expression of bcl-2 mRNA was significantly decreased in diabetic rat embryos compared to non-diabetic rat embryos. The increased number of terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells occurred selectively in the primitive brains of diabetic rat embryos compared to non-diabetic rat embryos. Immunohistochemical studies revealed that, in mirror sections, the staining of Bax and activated caspase-3 were observed in the TUNEL-positive cell area, but the expression of Bcl-2 in these apoptotic cells was generally too low to be detected. These results suggest that a Bax-regulated mitochondrial cytochrome c-mediated caspase-3 activation pathway might be involved in the diabetic embryopathy.     

Abnormal development and dye coupling produced by antisense RNA to gap junction protein in mouse preimplantation embryos.

Antisense RNA to the 27/32-kDa rat liver gap junction (GJ) protein was used to explore the role of GJs in preimplantation embryos. When all blastomeres of two- and four-cell embryos were injected with GJ antisense RNA, the percentage of embryos compacted at 60 hr of development was reduced to less than 20%, while 90% of uninjected embryos and 75% of embryos injected with an unrelated RNA were compacted. When most cells of compacted eight-cell embryos were injected with the GJ antisense RNA, 20% of the embryos were decompacted and only 5% had developed to the blastocyst stage at 90 hr, when blastulation had occurred in 90% of the control embryos. When antisense RNA was injected in one blastomere of four-cell embryos, 40% of the embryos presented a large cell that was not included in the compacted embryo at the time of compaction, and an additional 30% of the embryos had two smaller, excluded blastomeres. These excluded cells were identified as the injected cell with a rhodamine-conjugated dextran marker. To assess effects on junctional communication, one blastomere of some embryos was injected with Lucifer yellow, a GJ-penetrating dye, at various times after a blastomere was injected with antisense RNA. The dye was visible in the whole cell mass of control embryos, but it was excluded from a portion of experimental embryos when the delay between the RNA and the Lucifer yellow injections was 1 hr or longer.     

The inadequacy of existing theories on development of the proximal coronary arteries and their connexions with the arterial trunks

Coronary arterial development was studied in complete microseries of 20 human embryos and microseries of the hearts from 18 rat embryos. We never observed more than two coronary arterial orifices; these always originated from the facing aortic sinuses. In the human embryos these coronary orifices were variably identified between 16-19 mm crown-rump length, but were invariably present above 19 mm crown-rump length. In rat embryos, the orifices were variably identified at 13-17 mm and invariably present above 17 mm crown-rump length. In both human and rat embryos the left coronary orifice was observed significantly earlier. In all the embryos septation at arterial orifice level was complete. At the stages in which identification of the coronary orifices was variable, the proximal epicardial segments of the left and right coronary arteries could usually already be identified, in human as well as in rat embryos. On the other hand, a coronary orifice was never seen in the absence of a proximal coronary artery. At all stages studied (in human embryos from 10 mm crown-rump length and in rat embryos from 11 mm crown-rump length) vascular structures could be identified in the epicardial covering of the heart. The present theories on proximal coronary artery development are inadequate to explain either these data or the known possible congenital abnormalities of the coronary arteries. Our study offers a detailed chronology of development of these proximal coronary arteries and mostly supports dual coronary arterial development. The process by which the coronary orifices are brought into contact with the main coronary arteries still remains to be explained.     

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.     

The developmental potential of cryopreserved human embryos

Using rigorously matched non-frozen controls we have shown that cryopreservation does not alter the implantation potential of early cleavage stage (day 2) human embryos if no blastomere loss occurs. Thawed intact 4-cell embryos have a significantly higher implantation (fetal heart) rate (16.9%) than similar 2-cell embryos (7.2%). This difference is not due to blastomere number per se since increasing the cell number in frozen embryos by allowing an extended period in culture prior to freezing does not alter their intrinsic developmental potential. Blastomere loss, which occurred in almost half of all thawed embryos, is directly related to a reduction in developmental potential. We estimate that approximately 30% of the expected fresh embryo implantations are lost as a consequence of cryopreservation. Both preimplantation and peri-implantation losses may contribute to this outcome.     

Absence of increase of histidine decarboxylase activity in mast cell-deficient W/W mouse embryos before parturition.

The histidine decarboxylase (L-histidine carboxylase, EC 4.1.1.22) activity of whole W/W mouse embryos, which are devoid of mast cells, remained very low and did not show the rapid increase before birth (17-20 days of gestation) seen in wild-type +/+ embryos. During the same period, the histamine content also remained very low and no mast cells were detected in the W/W embryos, in the contrast to the large increase in both histamine content and number of mast cells in wild-type embryos. These findings imply that the histamine in embryos is largely derived from mast cells. In +/+ mice, histidine decarboxylase activity decreased rapidly soon after birth without concomitant decrease in histamine content or number of mast cells, suggesting that the enzyme activity in mast cells is regulated by some unknown mechanism.     

Manufacture of diploid/tetraploid chimeric mice.

Tetraploid mouse embryos were produced by cytochalasin B treatment. These embryos usually die before completion of embryonic development and are abnormal morphologically and physiologically. The tetraploid embryos can be rescued to develop to maturity by aggregating them with normal diploid embryos to produce diploid/tetraploid chimeric mice. The diploid/tetraploid chimeric embryos are frequently abnormal: the larger the proportion of tetraploid cells, the greater the abnormality. By karyotype analysis and by the use of appropriate pigment cell markers, we have demonstrated that two of our surviving chimeras are in fact diploid/tetraploid chimeras. One surviving chimera is retarded in growth and displays neurological abnormalities. The coat color chimerism suggests that this chimera is about 50% tetraploid. Another chimera with about 10% tetraploid pigment cells in the coat is only slightly retarded in growth and is a fertile male. Tetraploid cells are distributed in many, if not all, tissues of embryos but evidently are physiologically inadequate to support completely normal development and function in the absence of substantial numbers of normal diploid cells.     

Relation of early hemodynamic changes to final cardiac phenotype and survival after neural crest ablation in chick embryos

BACKGROUND. Microcinephotography was used to study a model of persistent truncus arteriosus created in chick embryos by ablation of premigratory neural crest destined for the third and fourth aortic arch arteries as well as the septum of the cardiac outflow tract. METHODS AND RESULTS. Twenty-five control embryos and 105 of 202 experimental embryos were filmed on day 3 of incubation and then reincubated. The remaining 97 experimental embryos were not filmed because of twisting of the embryos, but they were reincubated. There was no difference in either the survival rate (p greater than 0.23) from day 3 to day 11 of incubation or the incidence of persistent truncus arteriosus (p greater than 0.08) between the filmed and the nonfilmed embryos. Incomplete looping of the cardiac tube observed in experimental embryos during early cardiogenesis correlated with a right ventricular origin of the outflow vessels in the definitive heart. Hemodynamic measurements indicated that there was no difference in heart rate, ejection fraction, systolic and diastolic areas, stroke volume, and cardiac output between controls and the experimental group as a whole. However, embryos that did not survive to day 11 had decreased stroke volume (p less than 0.001) and cardiac output (p less than 0.001), whereas embryos that survived to day 11 with cardiac malformations had increased stroke volume and cardiac output in early embryogenesis. CONCLUSIONS. Increased stroke volume and cardiac output may be necessary factors for survival in embryos with cardiac dysmorphogenesis and probably are associated with dilation of the ventricular portion of the cardiac tube, which leads to malalignment of the outflow vessel or vessels.     

Variation during development in the response of chicken embryos to calcitriol administered via slow-release pellets.

Embryonic chickens were exposed to 0, 30, or 300 pg of calcitriol per day via slow-release pellets implanted adjacent to the chorioallantoic membrane. Pellets were placed in eggs on Days 10 and 15, and eggs were sampled on Days 12 and 17, respectively. The hormone induced high mortality among embryos receiving pellets on Day 10, but not among those whose treatment was begun on Day 15. Embryos receiving hormone were hypercalcemic and hypophosphatemic on both Day 12 and Day 17, but the concentration of magnesium in plasma was not affected. Size of embryos sampled on Day 12 was not affected by hormone treatment, but embryos sampled on Day 17 showed a dose-related reduction in size. Yolk-free carcasses of the embryos sampled on Day 17 also showed dose-dependent reductions in phosphorus and magnesium, but calcium content of carcasses on Day 17 was unaffected by treatment. These results indicate that both younger and older embryos respond to very small quantities of calcitriol administered via slow-release pellets. The absence of sustained hypercalcemia in earlier studies that used this protocol to dispense hormone was not caused by exposing older embryos to subthreshold quantities of calcitriol. The fact that reduced body size in late embryos receiving calcitriol was not accompanied by a reduction also in calcium content may mean that embryos deposit calcium in the carcass in an effort to deal with the extreme hypercalcemia induced by calcitriol.     

Essential role of glucose transporter GLUT3 for post-implantation embryonic development

Deletion of glucose transporter gene Slc2a3 (GLUT3) has previously been reported to result in embryonic lethality. Here, we define the exact time point of growth arrest and subsequent death of the embryo. Slc2a3(-/-) morulae and blastocysts developed normally, implanted in vivo, and formed egg-cylinder-stage embryos that appeared normal until day 6.0. At day 6.5, apoptosis was detected in the ectodermal cells of Slc2a3(-/-) embryos resulting in severe disorganization and growth retardation at day 7.5 and complete loss of embryos at day 12.5. GLUT3 was detected in placental cone, in the visceral ectoderm and in the mesoderm of 7.5-day-old wild-type embryos. Our data indicate that GLUT3 is essential for the development of early post-implanted embryos.     

Expression of imprinted genes in human preimplantation development

Imprinted gene expression in preimplantation development has been extensively studied in the mouse. Different imprinted genes vary in their time of onset of expression and also in the timing and tissue-specificity of mono-allelic expression. We have surveyed a range of imprinted genes for expression, and mono-allelic expression, in human development. Due to the scarcity of human embryos available for research, we first prepared amplified cDNA from replicate samples of human oocytes, four-cell, eight-cell and blastocyst stages. We then analysed these cDNAs for expression of a range of imprinted genes. Three of six genes analysed (SNRPN, PEG1 and UBE3A) are clearly expressed in preimplantation embryos. Expression was confirmed by direct analysis of embryos for these genes. For one of the expressed genes, SNRPN, we have shown that expression is mono-allelic from the paternal allele in human preimplantation embryos. This gene is also mono-allelically expressed in mouse preimplantation embryos. In our earlier work, we investigated the molecular mechanisms governing mono-allelic expression of the paternal allele of the Xist gene in preimplantation mouse embryos. We found that mono-allelic expression was correlated with differential methylation of Xist promoter sites in egg and sperm, and specific binding of a protein only to the methylated maternal (egg) allele. However, extension of these studies to the human showed that, unlike the mouse, XIST is expressed from both parental alleles in human preimplantation embryos. Since perturbation of imprinting is associated with disease and tumourigenesis, it is important to know the expression profiles of imprinted genes in human embryos and to monitor for normal imprinted gene expression with the introduction of new procedures in assisted conception.     

Use of slow-release pellets to administer calcitriol to avian embryos: effects on plasma calcium, magnesium, and phosphorus.

Calcitriol (1,25-dihydroxycholecalciferol) was administered to embryos of domestic fowl by slow-release pellets inserted into eggs on Day 10 of incubation. Mortality among embryos receiving 10 pg/day was no different from that of controls, but mortality among embryos receiving 100-1000 pg/day was elevated in later stages of incubation. Concentrations of calcium in plasma did not vary over the course of incubation among surviving embryos receiving carrier, but concentrations of magnesium and inorganic phosphorus declined with time. All doses of calcitriol elicited increases in plasma Ca and Mg on Day 12, and embryos may have responded also with a dose-dependent hypophosphatemia. The increases in plasma Ca and Mg were not sustained for the remainder of incubation, but the hypophosphatemia seemingly was of longer duration. This study demonstrates a new procedure for administering hormones to avian embryos and reveals that embryonic chickens respond in the expected manner to very small quantities of calcitriol.     

Conservation of methylation reprogramming in mammalian development: Aberrant reprogramming in cloned embryos

Mouse embryos undergo genome-wide methylation reprogramming by demethylation in early preimplantation development, followed by remethylation thereafter. Here we show that genome-wide reprogramming is conserved in several mammalian species and ask whether it also occurs in embryos cloned with the use of highly methylated somatic donor nuclei. Normal bovine, rat, and pig zygotes showed a demethylated paternal genome, suggesting active demethylation. In bovine embryos methylation was further reduced during cleavage up to the eight-cell stage, and this reduction in methylation was followed by de novo methylation by the 16-cell stage. In cloned one-cell embryos there was a reduction in methylation consistent with active demethylation, but no further demethylation occurred subsequently. Instead, de novo methylation and nuclear reorganization of methylation patterns resembling those of differentiated cells occurred precociously in many cloned embryos. Cloned, but not normal, morulae had highly methylated nuclei in all blastomeres that resembled those of the fibroblast donor cells. Our study shows that epigenetic reprogramming occurs aberrantly in most cloned embryos; incomplete reprogramming may contribute to the low efficiency of cloning.     

Genetic influence on dysmorphogenesis in embryos from different rat strains exposed to ethanol in vivo and in vitro

Background: The aim was to investigate the susceptibility of embryos from 2 rat strains (U and H) to a 48 hours ethanol exposure in early pregnancy, both in vivo and in vitro. Methods: The embryos were studied on gestational days 9 to 11. We used 1 ethanol dose in vivo (6 g/kg × 2), 3 different ethanol concentrations in vitro (88 mM, 132 mM, 176 mM) and also attempted to diminish the teratogenic effect in vitro by supplying the antioxidant N–acetylcysteine (NAC, 0.5 mM) to the culture medium. Results: The U embryos were more damaged by ethanol than the H embryos, both in vivo and in vitro. NAC addition diminished, but failed to completely normalize, the embryonic maldevelopment. Ethanol increased the Bax/Bcl‐2 ratio in the U embryos both in vivo and in vitro, but not in the H embryos. Furthermore, ethanol caused increased Caspase‐3 immunostaining in U embryos, but not in H embryos. Ethanol exposure in vivo did not alter CuZnSOD and MnSOD mRNA levels in U and H embryos. In vitro, however, the ethanol‐exposed U embryos increased their CuZnSOD and MnSOD mRNA levels, whereas the CuZnSOD mRNA was unchanged and MnSOD mRNA decreased in the H embryos, in neither strain did NAC exert any effect. The U embryos increased catalase gene expression in response to ethanol in vivo, but decreased catalase mRNA levels in vitro, changes normalized by NAC. The H embryos did not alter catalase mRNA levels in vivo, but increased gene expression in vitro, with no NAC effect. Ethanol affected the gene expression of the other ROS scavenging enzymes and the developmental genes studied – Bmp‐4, Ret, Shh, Pax‐6 – similarly in the 2 strains. Conclusions: The findings support a role for genetic predisposition, oxidative stress, and apoptosis in ethanol teratogenicity, and suggest that the teratogenic predisposition of the more susceptible U rats may reside, at least in part, in the regulation of the ROS scavenging enzymes in the U embryos.     

Experimental taphonomy shows the feasibility of fossil embryos

The recent discovery of apparent fossils of embryos contemporaneous with the earliest animal remains may provide vital insights into the metazoan radiation. However, although the putative fossil remains are similar to modern marine animal embryos or larvae, their simple geometric forms also resemble other organic and inorganic structures. The potential for fossilization of animals at such developmental stages and the taphonomic processes that might affect preservation before mineralization have not been examined. Here, we report experimental taphonomy of marine embryos and larvae similar in size and inferred cleavage mode to presumptive fossil embryos. Under conditions that prevent autolysis, embryos within the fertilization envelope can be preserved with good morphology for sufficiently long periods for mineralization to occur. The reported fossil record exhibits size bias, but we show that embryo size is unlikely to be a major factor in preservation. Under some conditions of death, fossilized remains will not accurately reflect the cell structure of the living organism. Although embryos within the fertilization envelope have high preservation potential, primary larvae have negligible preservation potential. Thus the paleo-embryological record may have strong biases on developmental stages preserved. Our data provide a predictive basis for interpreting the fossil record to unravel the evolution of ontogeny in the origin of metazoans.     

Repeated miscarriages in patients with antiphospholipid syndrome and subjected to in vitro fertilization: the importance of preimplantation genetic diagnosis

The aim of this study was to investigate two patients with antiphospholipid syndrome (APS) who suffered from multiple repeated pregnancy losses of probably genetically impaired embryos. Sera from these patients contained high levels of IgG antibodies against cardiolipin, IgG and IgM phosphatidyl inositol, IgG phosphatidyl L-serine, and IgG against anti-annexin V. The conventional treatment of APS was ineffective. Preimplantation genetic diagnosis (PGD) showed chromosomally impaired embryos. The course of pregnancies and deliveries were monitored due to the close collaboration of in vitro fertilization and PGD in early embryos. After the selection of normal embryos using PGD, and the treatment of APS, both patients became pregnant and delivered healthy babies. Without such selection, both women would probably have miscarried their embryos again.