Biomedical subjects

Y Heyman

Publications and source records attributed to Y Heyman.

At least 37 records · Page 2Linked to original sources

PubMed1999

Effect of lecithin on in vitro and in vivo survival of in vitro produced bovine blastocysts after cryopreservation.

The use of soybean lecithin in an glycerol-based solution for slow freezing of in vitro matured, fertilized and cultured (IVMFC) bovine embryos was examined. Embryos were developed in vitro in INRA Menezo's B2 medium supplemented with 10% fetal calf serum (FCS) on Vero cells monolayers. Day 7 blastocysts were frozen in a two-step protocol consisting of exposure to 5% glycerol and 9% glycerol containing 0.2 M sucrose in F1 medium + 20% FCS. Soybean lecithin was either added or not to the freezing solutions at a final concentration of 0.1% (w/v). In Experiment 1, blastocysts were equilibrated in cryoprotectant solutions without cooling. Cryoprotectant was diluted from embryos with 0.5 M and 0.2 M sucrose. The percentages of fully expanded and hatched blastocysts treated with or without lecithin after 24 and 48 h in culture were not significantly different (100 versus 100% and 93.3 versus 100%, respectively). In Experiment 2, the in vitro survival of frozen-thawed IVMFC blastocysts was compared when cryoprotectant solutions were either supplemented or not with lecithin. No significant effect of lecithin was found on the ability of frozen-thawed blastocysts to re-expand after 48 h in culture (65.6 and 54.2%, respectively). However, the post-thaw hatching rate of embryos cryopreserved in the presence of 0.1% lecithin was significantly higher after 72 h in culture (52 and 31.8%, respectively). In Experiment 3, the ability of frozen-thawed IVMFC blastocysts to establish pregnancy following single embryo transfer was determined. Transfers of 58 and 66 frozen-thawed embryos cryopreserved with or without lecithin resulted in 6 and 10 (10.3 and 15.1%, respectively) confirmed pregnancies at Day 60. Addition of lecithin to cryoprotectants did not improve the in vivo development rate of cryopreserved IVMFC bovine blastocysts.

Animals↗

PubMed1999

[Cloning: present and perspectives].

Human embryonic cells obtained through somatic cloning would allow selfgrafting for therapeutical purposes. Data available from animal research indicate that this issue should be considered with great care.

Cloning, Organism↗

PubMed1998

Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells.

Muscle and skin biopsies taken from bovine fetuses and young calves have been used as a source of donor nuclei for cloning experiments. After culture, cells were individually fused to enucleated matured oocytes and the resulting blastocysts obtained after 7 d of culture (3-8% depending on the cell type) were transferred to foster recipient heifers. Two calves, a female and a male, both originating from muscle cells were born, and four additional pregnancies have surpassed mild-term gestation. The pregnancies include one fetus established from a transgenic nucleus from a fetal skin cell, and another one resulting from a skin biopsy performed on a female calf. Our data demonstrate that nuclei from cultured bovine somatic cells obtained from differentiated tissues can be made multipotent.

Animals↗

PubMed1998

Prediction of transgene integration by noninvasive bioluminescent screening of microinjected bovine embryos.

Transgenesis in domestic species, as a research tool and in biotechnological applications, has been limited by the expense of producing transgenic offspring by standard microinjection techniques. A major factor is the inefficiency of maintaining large numbers of recipient females, when a high percentage of these carry nontransgenic fetuses. There are two approaches to reduce this cost, the fusion of transfected fetal fibroblasts with enucleated oocytes, and the screening of microinjected embryos for transgene integration in blastocysts, prior to transfer. Here, we develop a luminescent screening system to select transgenic bovine embryos. A transgene with scaffold attachment regions flanking the murine HSP70.1 promoter linked to firefly luciferase cDNA, was microinjected into pronuclei of in vitro produced zygotes. At the blastocyst stage, the transgene was induced by heat shock (45 degrees C, 15 min) and 4-6 h later, luciferase expression was analyzed by photon counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transgene integration. When nonluminescent blastocysts were transferred, transgene integration was never observed. Of 13 fetuses derived from luminescent blastocysts, 3 contained integrated transgenes that were functional in all tissues examined. Image analysis of the signal emitted by positive blastocysts revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuses derived from blastocysts that emitted light over more than 70% of their surface were transgenic. Thus, by selecting luminescent blastocysts on the basis of both signal intensity and distribution, the number of recipient females required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of transgenesis by microinjection through studies in which vector design and integration conditions are examined at the blastocyst stage.

Animals↗

PubMed1998

Nuclear transfer from sexed parent embryos in cattle: efficiency and birth of offspring.

The objectives of this study were to evaluate the efficiency and reliability of embryo sexing from isolated single blastomeres, and after nuclear transfer to examine the influence of the sex of donor embryos on development in vitro and in vivo up to calving. The sex of the donor embryo was determined by revealing a specific Y DNA sequence by PCR and electrophoresis after isolation of one, two, three, or more than five cells. The efficiency of sex determination was over 90% and reliability was 100% independent of the number of blastomeres used. In a second experiment, sex was determined from a single cell and the other cells were used for nuclear transfer. The effect of sex on in vitro development was studied in 386 male and 314 female reconstructed embryos derived from 19 male and 14 female parent embryos, respectively. Developmental competence in vitro of male and female constructs over 7 days was not statistically different (25.2 and 23.1% blastocysts on day 7, respectively; P > 0.05). After the transfer of predetermined male (n = 30) and female (n = 27) cloned embryos into recipient heifers, no effect of sex was observed on pregnancy rates at day 21, 35 and 90, or on calving rates (P > 0.05). These rates did not differ between single and twin transfer (P > 0.05). The sex of the calves born always corresponded to that determined from a single blastomere. These results show that sex can be determined accurately when using a single blastomere before nuclear transfer and that the sex of the parent embryo does not affect in vitro development or in vivo survival rates of cloned embryos.

Animals↗

PubMed1997

Beneficial effects of Vero cells for developing IVF bovine eggs in two different coculture systems.

A Vero cell line was used for coculture of bovine in vitro fertilized eggs up to blastocyst stage in comparison with bovine oviductal epithelial cells (BOEC) in two culture systems: monolayers or microdrops. Inseminated oocytes cocultured for 7 days with Vero cells in microdrops resulted in a significantly higher blastocyst rate compared to BOEC (29.5% vs 21.1%, respectively; P < 0.01). This difference was not significant in the monolayer coculture system although the blastocyst rate tended to be higher with Vero than with BOEC monolayers (27% vs 22.3%, respectively). Interestingly, the coefficient of variation between replicates was lower in both Vero cell groups than in BOEC groups indicating that Vero cells may help reduce variability. Medium conditioned by Vero cells partly supported embryo development compared to coculture itself (14.6% vs 26.5%, respectively; P < 0.01). Blastocysts developed on Vero cells contained significantly more cells (142 +/- 39) than those developed on BOEC (88.8 +/- 32.8, P < 0.001). Viability of blastocysts developed on Vero cells was evaluated by single transfer to 26 recipient heifers. Confirmed pregnancy rate after 3 months was 58%, demonstrating their high viability.

Animals↗

PubMed1996

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer.

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post coïtum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.

Journal Article↗

PubMed1996

Transcriptional activity and nucleolar ultrastructure of embryonic rabbit nuclei after transplantation to enucleated oocytes.

Changes in the level of transcriptional activity in 32-cell stage morula nuclei were studied after blastomere electrofusion to enucleated oocytes. Nuclear transplant recipients were pulse labelled with 3H-uridine during cultivation in vitro, embryos were then fixed and processed for autoradiography and electron microscopy. Transcriptional activity substantially decreased after 4.5 hr and was completely inhibited at last 15 hr after fusion. Transcription resumed thereafter in two-cell stage embryos and could be detected in both nuclei from 70% of the embryos analyzed. Transcription activity rapidly increased at the eight 16-cell stages, reaching the level typical for 32-cell stage nuclei used for the transfer. Changes in nucleolar ultrastructure after the nuclear transfer reflected the inhibition and subsequent reactivation of rRNA transcription. Nucleoli of 32-cell embryos had a typical structure of active nucleoli; many fibrillar centers surrounded and interconnected by threads of the dense fibrillar component and embedded in the granular component. Six hours following nuclear transplantation, these nucleoli underwent drastic changes including loss of granular material, collapse of nucleolar structure, and segregation of nucleolar components. Following the first cleavage, segregated fibrillar components of nucleoli manifested a complete inhibition of nucleolar transcription. Ribosomal RNA transcription was restored at the eight-cell stage and the sequence of ultrastructural changes was similar to that of the normal development. However, at the 32-cell stage, excessive extrusion of pre-ribosomal particles in the cytoplasm occurred, suggesting a possible alteration in regulating mechanisms of ribosome delivery. These results show that after fusion with enucleated metaphase II cytoplasm and subsequent activation, transcription is inhibited in donor embryonic nuclei and progressively increases again during cleavage; almost as in normal embryos. Migration of ribosomes into cytoplasm appears more intense in 32-cell stage reconstituted embryos but this does not seem to inhibit blastocyst building.

Animals↗

PubMed1995

Cellular evaluation of bovine nuclear transfer embryos developed in vitro.

Cloned blastocysts developed in vitro for 7 d had a mean number of cells (82.86 +/- 5.35) as evaluated by nuclei counting in serial optical sections using confocal microscopy, after staining with propidium iodide. This number was not significantly different from that of control IVF embryos cultured under the same conditions during the same period (mean = 88.89 +/- 7.53). Semi-thin sections revealed that most of the blastocysts had an inner cell mass (10/12) and a blastocoele. Under transmission electron microscopy, the trophectoderm appeared well differentiated as a polarized epithelium with apical microvilli and lateral junctions including desmosomes with bound intermediate filaments. The cytoplasm sometimes contained immature mitochondria or a large number of residual bodies. About half of the blastocysts examined had a large amount of cellular debris in the perivitelline space or inside the blastocoele cavity. The cloned blastocysts were also able to hatch in vitro by day 8 and SEM indicated a normal morphology of the trophectoderm cells with numerous apical microvilli. The high number of excluded or degenerating cells found in some embryos may partially explain early embryonic mortality that follows transfer. However, these observations do not give a clear explanation for the high incidence of fetal losses.

Animals↗

PubMed1995

Low developmental capacity of in vitro matured and fertilized oocytes from calves compared with that of cows.

The developmental competence of oocytes from 3-month-old calves was studied through in vitro maturation, fertilization and culture up to the blastocyst stage and by embryo transfer into a foster mother. Oocytes were recovered from antral follicles of calves after or without ovarian stimulation with exogenous FSH and their developmental potential was compared with that of oocytes recovered from cow ovaries. Fertilization and cleavage rates from calf oocytes did not differ significantly from those of cow oocytes. However, after 7 days of culture, the blastocyst formation rate was significantly lower for calves (9% and 11% for nontreated and treated animals, respectively) than for cows (over 20%). Transfer of blastocysts obtained from calf oocytes resulted in a lower pregnancy rate (1 of 23 recipients; 4%) than that achieved with cow oocytes (10 of 26; 38%). The recipient cow that was pregnant from calf embryos delivered a full-term live calf. These data show that some key regulative event that determines the ability to form blastocysts in cattle has not been fully achieved in oocytes from 3-month-old calves.

Aging↗

PubMed1994

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro.

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).

Journal Article↗

PubMed1993

Dynamic changes of gap junctions and cytoskeleton during in vitro culture of cattle oocyte cumulus complexes.

Changes in cell-to-cell contact and distribution of cytoskeletal components were investigated during in vitro culture of cattle oocyte cumulus complexes (OCC). Freeze-fracture analysis (FF), microinjections of the fluorescent dye Lucifer Yellow (LY), immunofluorescence, and ultrastructural immunocytochemistry were used. The cumulus cells (CC) remained in close contact via gap junctions (GJ) constituted of connexin43 (Cx43) during the entire culture time. Whereas the GJ decreased in diameter after 24 h of culture, their number was still substantially great at that time. The Cx43-positive GJ, localized between corona radiata cell projections and oolemma, disappeared after 6 h of culture. Concomitantly, the OCC lost the ability to transfer LY from cumulus to oocyte, and connexin32 (Cx32) became detectable in the oocytes. Both the changes in corona-oocyte coupling and cumulus expansion were preceded by the redistribution of F-actin in cytoplasm of CC. These data indicate that functional GJ linked the CC until the second meiotic arrest. However, the removal of Cx43-positive GJ interconnecting cytoplasmic projections of corona radiata cells with the oocyte was temporally correlated with germinal vesicle breakdown. The present results suggest the pivotal role of the cytoskeleton (F-actin) in cumulus expansion.

Actin Cytoskeleton↗

PubMed1993

Nuclear transfer in cattle: birth of cloned calves and estimation of blastomere totipotency in morulae used as a source of nuclei.

The birth of a clone of five bull calves, each produced by fusing a cell taken from the same young embryo with an oocyte whose own nucleus has been removed, opens the way to the use of these new animal models for research and animal selection. The embryonic cells came from a 31-cell morula. Our results indicate that most of the nuclei at this stage are totipotent which seems to no longer be the case for those taken from the cells of the inner cell mass at the blastocyst stage.

Animals↗

PubMed1990

The effect of sucrose and trehalose on viability of one- and two-cell rabbit embryos.

The effect of sucrose and trehalose on the viability of one- and two-cell rabbit embryos was investigated. A significant decrease in the viability of one- and two-cell embryos exposed for 30 min. at 20 degrees C was observed. At 38 degrees C none of the two-cell embryos in a sucrose solution survived after 30 min exposure, while approximately 50% of the embryos survived in a trehalose solution. The cleveage rate in culture of two-cell embryos exposed both to 2.0 M or 1.45 M trehalose was significantly lower in comparison with the control group. However the survival rate after transfer of two-cell embryos exposed to 1.45 M trehalose solution at 20 degrees C remained the same as that of the control group.

Journal Article↗

PubMed1989

Solvent effects on cytoskeletal organization and in-vivo survival after freezing of rabbit oocytes.

NBD-phallacidin revealed a polymerized actin distribution in the cortical region of the rabbit egg and along junctional feet. Staining with anti-alpha-tubulin antibody showed that the microtubule distribution was restricted to the barrel-shaped spindle. After cryoprotective treatment in the presence of propanediol, cortical polymerized actin was no longer visible within the egg and along junctional feet but filamentous actin was still present after treatment with dimethylsulphoxide. However, exposure to dimethylsulphoxide or propanediol led to the appearance of microtubules in the cytoplasm and to a disassembly of the spindle often associated with anomalies in chromosome position. Cytoplasmic microtubules formed by the action of propanediol were still present after freezing, thawing, and removal of the cryoprotectant, but after recovery of eggs in culture, they disappeared and barrel-shaped spindles were able to reform. When the effect of propanediol addition on in-vivo fertilization and development of frozen oocytes was examined, 39% (79/200) of frozen oocytes were fertilized and 9% (9/105) developed to normal fetuses, compared to 81% (38/47) and 32% (12/38) respectively for unfrozen control oocytes.

Actin Cytoskeleton↗

PubMed1988

[Timing of transplantation and success of pregnancy in mammals].

In domestic mammals, the optimum time for embryo transfer greatly depends upon the species and its embryo chronology development and the technique used. Three different models (rabbit, pig, bovine) have been studied to set up the importance of cycle synchrony between donor and recipient. In the rabbit, the best survival rates were obtained after tubal transfer to highly synchronous recipients even for frozen or cultured embryos. In the pig, there was no survival difference after embryo transfer on D2 (oviduct) or D6 (uterus) and an asynchrony up to 24 h was possible. In the bovine, embryo transfer is usually performed at the blastocyst stage directly into the uterus. A retrospective study over 2,000 recipients indicates that the highest pregnancy rates were obtained when the recipient was in heat 12 to 24 h before the donor.

Animals↗

PubMed1987

Transfer of frozen-thawed embryos in sheep.

Embryos collected from ewes six days after oestrus were frozen in straws using ethylene-glycol as a cryoprotectant. The efficiency of the simplified freezing and thawing procedure was assessed after transfer, which resulted in an overall survival rate of 58.3 per cent. Forty-two lambs were born from 72 frozen embryos which had been transferred without any attempt to evaluate them after the thawing and sucrose dilution process.

Animals↗

PubMed1987

Improvement of survival rate of frozen cattle blastocysts after transfer with trophoblastic vesicles.

An experiment was conducted to determine if the loss of viability due to deep freezing could be overcome by addition of trophoblastic tissue to the embryo at transfer time. Forty-nine recipient heifers in a cotransfer group each received one frozen blastocyst + two frozen trophoblastic vesicles. The confirmed pregnancy rates by Day 45, 60, and 90 were 73, 61, and 57%, respectively. In a control group of 53 recipients that received only a frozen blastocyst, pregnancy rates for the same periods were 43, 42, and 40%, respectively. The difference between groups was highly significant by Day 45. The addition of trophoblastic vesicles to frozen embryos contributed to luteal maintenance in recipients and likely magnified the intensity of embryonic signals resulting in improved pregnancy rates.

Journal Article↗