INDUCTION OF ECTODERMAL CELLS FROM VEGETALENDODERMAL BLASTOMERES OF AMPHIOXUS(BRANCHIOSTOMA BELCHERI TSINGTAUNESE) EMBRYOS BY THE CALCIUM IONOPHORE A23187

Timing of vegetal-endodermal cell determination in amphioxus embryos remains uncertain. We tentatively testal effects of A23187, the calcium ionophore, on the deveopment of vegetal blastomeres isolated at the 16-cell stage. It was found that when vegetal blastomres committed to endodermwere treated with A23187 prior to gastrulation, they were transformed into ectodermal cells as evidenced by the cell morphology and function characteristic of epidermis. Howver, the developmental fate of the sam blastomeres untreated or treated with DMSO at the same stage or of those treated with A23187 after gastrulation remained unchanged. Thus, vegetal-endodermal cells in amphioxus embryos are not irreversibly deermined before the gastrula stage, and artificial incarease in intracelluar Ca2+ concentration can induce transdetermination of the predetermined endodermal cells into ectodermal cells.

Production of early monozygotic twin bovine embryos in vitro by the blastomere separation and coculture technique

The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres（groups of at least 30 at the eight-cell stage） were separated into eight segments（to regard an eight-cell embryo as a tangerine, a blastomere as one segment） and one, two and four segments（blastomeres） were cultured respectively in microwells on the bottom of the four-well dish（Nunc, Denmark） with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics（cocultured with nothing, intact embryos, bovine cumulus cells（b CCs）, intact embryos ＆ b CCs） were applied to the group of four segments（blastomeres）. Finally, diameter and inner cell mass（ICM）：trophectoderm（TE） cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group（P〈0.05）. In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo ＆ b CCs（P〈0.05）. Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo（P〈0.05）, more important quality indicator of the blastocyst diameter and ICM：TE cell ratio was similar between our experimental group and the control group（P〉0.05）. Thus, these results suggest that combined with intact embryos ＆ b CCs coculture system, culturing four isolated segments（blastomeres） per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.

Human blastomere rotation in early cleavage embryos is not associated with reduced implantation: Evidence from time-lapse videography

Dear Editor,Time-lapse videography of human embryos allows for the easy visualization of the embryos without removing them from the protective environment of the incubator[1],the measurement of various morphokinetic(quantitative)parameters[2],and the identification of abnormalities of growth(qualitative parameters)such as direct cleavage[3],reverse cleavage[4]and intercellular contact of blastomeres[5].

Synchronous and Asynchronous Blastomere Cleavage at Cryopreservation: Effect on Subsequent Embryo Survival, Pregnancy and Live Birth Rates

Capsule: Although embryos with synchronous blastomere cleavage showed higher post-thaw survival rates, pregnancy rates did not differ. Thus, embryos with all cleavage patterns may be safely cryopreserved. Objective: To compare post-thaw embryo survival, pregnancy and live birth rates of embryos with synchronous vs asynchronous blastomere cleavage in frozen embryo transfer (FET) cycles. Design: Retrospective study. Setting: University-affiliated IVF unit. Patients: One thousand and sixty FET cycles performed from 2004-2006. Interventions: Cycles were divided into 3 groups: 1: cycles in which only embryos with synchronous blastomere cleavage were frozen;2: cycles in which only embryos with asynchronous blastomere cleavage were frozen;3: cycles in which both embryos with synchronous and asynchronous blastomere cleavage were frozen. Clinical and laboratory data were recorded and analyzed. Main Outcome Measures: Post-thaw embryo survival, morphologic grading, pregnancy and live birth rates. Results: A total of 1863 embryos were analyzed. Synchronous embryos had higher blastomere survival rates and morphological grading at thawing. Pregnancy and birth rates did not differ among groups. In a multivariant logistic regression analysis, a number of transferred embryos and embryo morphological grading at thawing were the only parameters that affected pregnancy and live birth rates. Conclusions: Embryos with both synchronous and asynchronous blastomere cleavage can be selected by classical embryo grading and safely cryopreserved.

Analysis for the dorsalization potency of the animal blastomeres of the 16 cell stage Xenopus embryo

The derso-ventral axis of Xenopus embryo is established after fertilization. Blastula stage blastomeres acquire different identities as they have inherited different maternal materials which distribute radial symmetrically along the animal vegetal aixs in the full-grown oocyte, and are rearranged by the cortical rotation triggered by fertilization. The vegetal blastomeres demonstrate the different dorsalization potencies in the previous transplantation experiments. The data of blastomere explanting and RT-PCR analyzing indicate that the dorsal ventral bias also exists among the animal blastomeres even during the early blastula stage.

In-vitro Developmental Potential of Single Blastomeres Derived from Day 3 Discarded Human Embryos

Objective To investigate whether isolated blastomeres of discarded human embryos could develop into blastocysts cultured in vitro without zona pellucida. Methods Total discarded 60 embryos, which were not suitable for transplanting or freezing, were collected from 21 patients. Of 60 embryos, 10, 8, 24, 12, and 4 embryos were at 2-, 3-, 4-, 5- and 6-cell stage, respectively. These embryos were split by 0.5% protease combined with mechanical method. The resulting single blastomere was cultured individually, evaluated daily and counted blastocyst development. Pluripotency of inner cell mass （ICM） of the blastocyst was analyzed by the expression of alkaline phosphatase（AKP）. Results A total of 229 single blastomeres were isolated from 60 embryos. Defining the day when the embryos were split as the first day （d 1）. The majority of the blastomere- derived embryos followed the normal pattern of development with compaction on d 3 and cavitationon d 4 and developed into small blastocysts on d 5. Rates of division, compaction, cavity and expansion of single blastomeres from 4-cell embryos were higher than those in other groups （P 〈O.05）, and AKP was expressed in ICM. Conclusion Some of the blastomeres from discarded human embryo are flexible and able to develop into blastocysts, the potential was related to their donor embryos closely. They seem to follow development pattern of their donor embryos. The blastomere- derived blastocysts have smaller size and fewer cells compared with regular in vitro cultured human embryos. However, AKP expression showed ICM cells with pluripotency. We believe that the value of single blastomere of discarded embryos will be further confirmed in the future.

Embryo quality and chromosomal abnormality in embryos from couples undergoing assisted reproductive technology using preimplantation genetic screening

Objective:To detect common chromosomal aneuploidy variations in embryos from couples undergoing assisted reproductive technology and preimplantation genetic screening and their possible associations with embryo quality.Methods:In this study,359 embryos from 62 couples were screened for chromosomes 13,21,18,X,and Y by fluorescence insitu hybridization.For biopsy of blastomere,a laser was used to remove a significantly smaller portion of the zona pellucida.One blastomere was gently biopsied by an aspiration pipette through the hole.After biopsy,the embryo was immediately returned to the embryo scope until transfer.Embryo integrity and blastocyst formation were assessed on day 5.Results:Totally,282 embryos from 62 couples were evaluated.The chromosomes were normal in 199(70.57%)embryos and abnormal in 83(29.43%)embryos.There was no significant association between the quality of embryos and numerical chromosomal abnormality(P=0.67).Conclusions:Embryo quality is not significantly correlated with its genetic status.Hence,the quality of embryos determined by morphological parameters is not an appropriate method for choosing embryos without these abnormalities.

Observation and analysis of morphology abnormalities in development of Oryzias melastigma embryos

Fish embryos are widely used as models in toxicology,drug development,and human disease research because of their high sensitivity,observability,and operability,providing the basis for an in-depth understanding of the embryogenesis.Increasing studies have indicated that birth defects are hereditary.In this study,we used Oryzias melastigma as a model to conduct a study of 185-day embryogenesis and observed self-induced non-pathological abnormal embryogenesis.O.melastigma experienced pre-puberty stage,adolescence stage,and senescence stage,and individuals produced up to 102 eggs per day.However,the fecundity was markedly reduced during the senescent stage.During the active egg and blastodisc stages,pseudo-fertilization and pseudo-blastocysts were observed.During cleavage at the 4-to 32-cell stages,we observed blastomeres separation or dislocation.Excessively separated blastomeres formed double blastoderms,eventually resulting in conjoined twins.During the blastula stage,we observed abnormally increased cell volume,narrowed and elongated blastocysts,and abnormally coated blastoderms.At the organogenesis stage,we observed abnormal numbers of Kupff er’s vesicles and conjoined twins.Abnormality in the location and number of oil droplets were observed in various development stages.Abnormal development was more commonly observed in fertilized eggs produced by broodstock in pre-puberty or senescence stages,which is probably related to the age of fish and the egg quality.This study can provide the materials for comparative analysis in toxicological and molecular studies of O.melastigma,and may provide evidence for other economic fish that produce sticky eggs.

Studies on Optimal Conditions of Two Freezing Methods for Preservation of Rabbit Embryos

Since the first report on the successful deep cryopreservation of mam-malian embryos in 1972,slow progressing cooling rate has been employ-ed in conventional embryos-freezing techniques;while more recent studieson freezing preimplantation embryos have focussed on the simplificationof cooling and thawing procedures and improvement of viability of embr-yos.Dimethylsulfoxide (DMSO)has been used as an effective cryoprotec-tant for freezing human and other mammalian embryos.It has been foundthat glycerol and other alcohols are effective to protect embryos fromcryoinjury