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The ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes, cocultured with bovine cumulus/granulosa cells either in a serum-free medium (IVMD101) or in a serum-containing medium (TCM199+CS) was compared. Embryos up to the eight-cell stage had many cellular organelles and cytoplasmic components that were randomly distributed in the cytoplasm. Mitochondria were spherical or ovoid and had only a few peripheral cristae. There were no obvious differences in the ultrastructure between embryos developed in IVMD101 and TCM199+CS up to the eight-cell stage. However, conspicuous differences in the ultrastructural features between the embryos cultured in IVMD101 and TCM199+CS were observed at the morula and blastocyst stages. At the morula stage, embryos cultured in IVMD101 had cells containing elongated mitochondria, well-developed Golgi apparatus, lipid droplets, and large vesicles resembling lysosomes. The lysosome-like vesicles were partially filled with electron-dense materials and were frequently fused with lipid droplets. The blastomeres of morulae cultured in TCM199+CS contained numerous large lipid droplets and fewer lysosome-like vesicles than those cultured in IVMD101. In blastocysts cultured in IVMD101, lysosome-like vesicles were frequently observed in the trophoblast cells and lipid droplets were present in the cytoplasm of trophoblast and inner cell mass (ICM)-cells, but they were not abundant. On the other hand, the blastocysts developed in TCM199+CS contained fewer lysosome-like vesicles and large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM-cells. This study showed major differences in the ultrastructural features between the morulae and blastocysts from serum-free and serum-supplemented cultures, suggesting that the ultrastructural differences may reflect physiological characteristics of embryos.
Clonal production of loblolly pine ( Pinus taeda L.) through somatic embryogenesis has the potential to meet the increasing industrial demands for high-quality uniform raw materials. A major barrier to the commercialization of this technology is the low quality of the resulting embryos. Twenty-five newly initiated loblolly pine genotypes were followed through the process of liquid culture establishment, embryo maturation, germination, and retrieval from cryogenic storage. A maturation medium, capable of promoting the development of loblolly pine somatic embryos that can germinate, is presented that combines 1/2 P6 modified salts, 2% maltose, 13% polyethylene glycol 8000 (PEG), 5 mg/l abscisic acid (ABA), and 2.5 g/l Gelrite. A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also described. A set of somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting. Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed. The quality of the resulting embryos was examined and compared to that of zygotic embryos using such parameters as morphology, dry weight, germination performance, and gene expression. All of the observations that were made support the conclusion that even with the new maturation medium somatic embryos grow approximately only halfway through the normal sequence of development and then prematurely discontinue growth.
As part of a program to study the feasibility of using embryo transfer to control disease, initial experiments were undertaken to determine the virus susceptibility of early embryos. Two hundred and ninety-three preimplantation bovine embryos (16-cell to blastocyst stage) were exposed to either akabane virus (AV), bluetongue virus (BTV) or bovine viral diarrhea virus (BVDV). Two hundred and thirty-seven of these embryos were then cultured for 24-48 hours in order to determine whether the virus had any effect on embryonic development and to allow viral replication to occur. No infectious virus was isolated from any of the embryos and the in vitro development of virus exposed embryos proceeded normally. In addition, twenty-nine eggs/embryos isolated from donors that were seropositive to BVDV were found to be uninfected with this virus.
Bovine embryos, at the 16-cell to the blastocyst stage of development, were exposed to infectious bovine rhinotracheitis virus (IBRV) for either one or 24 hours. These embryos were then washed and incubated for 24 or 48 hours before being assayed for IBRV. Under these conditions, infectious virus at the level of 0-10(2.2) TCID(50)/ml was isolated from 57-64% of the embryos exposed to IBRV. Trypsin and IBRV-antiserum were found to be capable of removing and/or inactivating the IBRV from exposed embryos. Both the low level of the virus isolated from these embryos and the susceptibility of this virus to trypsin and antiserum suggests that IBRV attaches to the zona pellucida of embryos and cannot penetrate this structure to gain access to the embryonic cells. IBRV was found to have no effect on embryonic development in vitro . In addition, thirty-one eggs/embryos isolated from donors that were seropositive to IBRV were found to be uninfected with this virus.
This study was performed to investigate whether types and/or age of donor cells affect preimplantational embryo development and the incidence of apoptosis in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine fetal or adult ear fibroblasts were isolated, cultured in vitro and categorized into fresh or long-term cultured cells in terms of population doublings (PD): in fetal fibroblasts, <16 being considered fresh and >50 being long-term cultured; in adult ear fibroblasts, <16 being considered fresh and >30 being long-term cultured. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199, enucleated and reconstructed by SCNT. The reconstructed oocytes were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39 degrees C in a humidified atmosphere of 5% CO(2) air for 7 days. The early development of SCNT embryos was monitored under a microscope and the quality of blastocysts was assessed by differential counting of inner cell mass (ICM) and trophectoderm (TE) cells and by apoptosis detection in blastomeres using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. As results, types and/or age of donor cells did not affect the rate of blastocyst formation and the number of ICM and TE cells. However, a significant increase in apoptotic blastomeres was observed in SCNT embryos reconstructed with long-term cultured fetal or adult ear fibroblasts compared to those in SCNT embryos derived from fresh fetal or adult ear fibroblasts. In conclusion, these results indicated that the long-term culture of donor cells caused increased the incidence of apoptosis in bovine SCNT embryos but did not affect the developmental competence and the cell number of blastocysts.
The first experiment involved in vitro exposure of clean embryos to bluetongue virus (BTV) while three subsequent experiments involved the collection of embryos from BTV-infected donor ewes and their transfer to disease-free recipients. In Experiment I, 22 embryos/ova were exposed to BTV type 11 (BTV-11) for 1 h, washed 10 times in PBS and assayed in pairs for BTV. All 11 samples were positive for BTV in the 11-d-old embryonated chicken egg (ECE) assay system and 5/11 samples were positive in baby hamster kidney-21 (BHK-21) cells. In Experiment II, 5 donors were infected with BTV type 10 (BTV-10). All embryos were washed 10 times prior to assay or transfer. Thirty-three embryos/ova were assayed in groups of 2 or 3 and none yielded virus in ECE. Two BTV-seronegative recipients each received 6 embryos and a total of 3 lambs free of BTV antibodies were delivered. In Experiments III and IV, a total of 9 donors were infected with BTV-11. All embryos were washed 10 times prior to assay or transfer. Seventy-four embryos/ova were assayed in groups of 2 or 3 and none yielded virus in ECE, while for each experiment, 6 embryos were transferred into 2 BTV-seronegative recipients. The four recipients and their 3 lambs and 2 aborted fetuses were also seronegative for BTV.
Many chemicals that are not structurally related to estrogen have estrogen-like activity. In this study, we tried to apply a quail embryo culture system for assessing the in vivo effects of such chemicals on the development of quail embryos. Beta-estradiol induced feminization of the gonads of genetically male embryos, which was confirmed by the increase in the size of the left gonad and female-specific aromatase expression, while male-specific SOX9 expression was not affected. Nonylphenol, which has a weak estrogenic activity, reduced the viability and body weight of embryos. Simultaneously, several genetically male embryos were feminized in terms of gonadal size and aromatase expression. These results indicate that the avian embryo culture system was useful for evaluating endocrine disrupters.
Our previous studies have shown that the addition of 100 mircroM cysteamine to the in vitro maturation (IVM) medium increased the embryo development of prepubertal goat oocytes. The aim of the present study was to evaluate the effect of adding different concentrations of cysteamine to the IVM medium and to the in vitro embryo culture medium (IVC) on the embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue (BCB) test. Oocytes were exposed to BCB and classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) or oocytes without blue cytoplasm or growing oocytes (BCB-). In Experiment 1, oocytes were matured in a conventional IVM medium supplemented with 100 microM, 200 microM or 400 microM cysteamine. In Experiment 2, oocytes were matured with 400 microM cysteamine and following in vitro fertilization (IVF) were cultured in SOF medium supplemented with 50 microM and 100 microM cysteamine. In Experiment 1, BCB+ oocytes matured with 100 microM and 200 microM cysteamine showed higher normal fertilization and embryo development rates than BCB- oocytes. Oocytes matured with 400 microM cysteamine did not present these differences between BCB+ and BCB- oocytes. In Experiment 2, the addition of 50 microM and 100 microM cysteamine to culture medium did not affect the proportion of total embryos obtained from BCB+ oocytes (35.89% and 38.29%, respectively) but was significantly different in BCB- oocytes (34.23% and 29.04%, respectively, P < 0.05). In conclusion, the addition of 400 microM cysteamine to the IVM improved normal fertilization and embryo development of BCB- oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affected by the treatments.
The present experiments were designed to study the effects of Robertsonian translocations on the efficiency and kinetics of in vitro fertilization and early and advanced embryo development. Spermatozoa from bulls with rob(16;20), rob(1;29) and normal karyotype (A, B and C, respectively) were used. Oocytes were matured, fertilized and cultured by the standard protocol described previously. Twenty-four hours after fertilization, adequate numbers of oocytes were fixed, stained and examined. The development of embryos was evaluated on days 2 (D2), 7 (D7) and 8 (D8) after fertilization. The rate of normally fertilized oocytes was significantly lower (p < or = 0.01) for bull A than for bulls B and C. However, no significant differences in the kinetics of fertilization were found between bulls A, B and C. The D2 cleavage rate of embryos was significantly lower (p < or = 0.01) for bull A than for bulls B and C. Both D7 and D8 blastocyst rates for bull A or bull B were significantly lower (p < or = 0.01 or p < or = 0.05) than those for bull C. The percentages of both D7 advanced blastocysts and D8 expanded blastocysts were significantly lower (p < or = 0.01) for bulls A and B than for bull C. In conclusion, for rob(16;20), the efficiency of fertilization was strongly reduced; it resulted in low early and advanced embryo development. On the other hand, for the rob(1;29), neither fertilization nor early embryo development were affected and only advanced embryo development was decreased. But for both translocations, blastocyst formation was significantly delayed.
PURPOSE: To compare the frequency and the degree of mosaicism in human embryos from Robertsonian translocation (RT) t(13;14) carriers, with embryos from karyotypically normal IVF patients METHODS: FISH analysis of embryos from PGD cycles for RT t(13;14), with probes for chromosomes 13, 14, and 18 (Group I) and of embryos from karyotypically normal IVF patients with probes for chromosomes 13, 18, 21, X, and Y (Group II). RESULTS: The incidence of abnormal mosaic embryos was significantly higher in group I (38/51) as compared with group II (6/45) (chi2: P < 0.01). Furthermore, in group I the percentage of diploid cells per embryo was lower for chromosome 13 and 14 in comparison with 18, while in group II no differences were observed between the five chromosomes analyzed. CONCLUSIONS: RT induces a high frequency of mosaicism specifically for the chromosomes implicated in the translocation; the analysis by FISH of two blastomeres is strongly recommended for these patients
SMAD2 is a member of the transforming growth factor beta and activin-signaling pathway. To examine the role of Smad2 in postgastrulation development, we independently generated mice with a null mutation in this gene. Smad2-deficient embryos die around day 7.5 of gestation because of failure of gastrulation and failure to establish an anterior-posterior (A-P) axis. Expression of the homeobox gene Hex (the earliest known marker of the A-P polarity and the prospective head organizer) was found to be missing in Smad2-deficient embryos. Homozygous mutant embryos and embryonic stem cells formed mesoderm derivatives revealing that mesoderm induction is SMAD2 independent. In the presence of wild-type extraembryonic tissues, Smad2-deficient embryos developed beyond 7.5 and up to 10.5 days postcoitum, demonstrating a requirement for SMAD2 in extraembryonic tissues for the generation of an A-P axis and gastrulation. The rescued postgastrulation embryos showed malformation of head structures, abnormal embryo turning, and cyclopia. Our results show that Smad2 expression is required at several stages during embryogenesis.
We hypothesized that transforming growth factor beta1 (TGFbeta(1)) and its receptors play a role in the interaction between the preimplantation embryo and the reproductive tract. To investigate this hypothesis, TGFbeta 1 mRNA in mouse embryos was quantified by competitive reverse transcription-polymerase chain reaction using an RNA mimic. TGFbeta 1 was first detected in the unfertilized oocyte, disappeared after fertilization and was expressed again at the 2-cell stage (4410 +/- 1330 transcripts/embryo). Its expression increased gradually, peaked at the 8-cell stage (58 600 +/- 17 300 transcripts/embryo) and declined rapidly after the morula stage reaching a concentration of 1520 +/- 546 transcripts/embryo at the blastocyst stage. The mRNA levels of TGFbeta 1 at the 8-cell and morula stages were significantly higher than that at other cell stages (P < 0.05). The expression of TGF receptors in embryos and in the reproductive tract was also investigated. Both TGFbeta(1) type I (ALK-5) and type II TGFbeta receptors were detected in embryos from 1-cell to blastocyst stage by immunohistochemistry. Northern hybridization and immunohistochemistry showed a constant expression of both TGFbeta receptors in the oviduct from day 1 to day 4 of pregnancy, whilst in the uterus there was a marked increase in the expression of TGFbeta type I receptor on day 3. Expression of TGFbeta type II receptor in the uterus remained unaltered throughout the study period. This study has shown that preimplantation mouse embryos produce TGFbeta(1) and that both the embryos and the reproductive tract are responsive to TGFbeta(1) in the preimplantation period.
Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.
Differential screening of a cDNA library constructed using PCR amplification techniques from RNA isolated from the distal portion (embryonic ectoderm, mesoderm and visceral endoderm) of 7.5 days post coitum (dpc) mouse embryos led to the isolation of two cDNA clones expressed at higher levels in 7.5 dpc embryos than 12.5 dpc embryos. Nucleotide sequence analysis revealed that each of these clones was a different member of the family of facilitative glucose transporters (Glut genes). The differentially expressed cDNA clones represent mouse Glut-1 and Glut-3. Levels of the Glut-3 mRNA declined 14-fold between days 7.5 and 12.5 of gestation, and were under our limits of detection by 14.5 dpc. The levels of the Glut-1 mRNA declined about 3-fold between days 7.5 and 12.5 of gestation. Analysis of the expression of these genes by in situ hybridization revealed striking differences in transcript localization in early postimplantation mouse embryos. At 7.5 dpc, both transporters were expressed more strongly in extraembryonic tissues than in the embryo proper. While both transporters were expressed in the amnion and chorion, only Glut-1 was expressed in the ectoplacental cone. In the yolk sac, Glut-3 appeared to be expressed only in the endoderm while Glut-1, although expressed in both layers, was expressed more strongly in the mesoderm layer. Thus, the two transporters have relatively reciprocal sites of expression in the developing extraembryonic membranes. Expression of Glut-1 was fairly widespread in the embryo at 8.5 dpc, but by 10.5 dpc expression was down-regulated and was observed in the eye and the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)
Chondrodystrophic and normal 24-day old embryos obtained from matings of known heterozygotes (Ch/ch) of the Beltsville Small White turkey stock were compared for bone lengths and for bone weights as a percent of embryo weights. Bone and embryo weights were less in the chondrodystrophic embryos, and the bone weights were reduced proportionally more than embryo weights. The lengths of the humeri and tibiotarsi were shorter in chondrodystrophic embryos, and the tibiotarsi were shortened more than the humeri.
Suppression subtractive hybridization (SSH) cDNA plasmid libraries were constructed between gastrula embryos and tail bud embryos in gynogenetic gibel carp (Carassius auratus gibelio). 739 and 816 PCR positive clones were respectively selected to perform dot blot, and 72 dot blot positive clones and 98 dot blot positive clones were obtained from the SSH plasmid libraries specific for gastrula embryos and tail bud embryos. Sequencing analysis and database searches indicated that there were 19 known genes and 31 unknown/cDNA fragments in the sequenced 72 dot blot positive clones specific for gastrula embryos, and 52 known genes and 37 unknown cDNA fragments in the sequenced 98 dot blot positive clones specific for tail bud embryos. Moreover,specific expressions of partial genes were further confirmed by virtual Northern blots and RT-PCR. The screen of these differentially expressed genes will help us to understand the molecular mechanism in gibel carp embryogenesis.