Faculty Bibliography

In human embryos, blastomeres differentiate into trophectoderm (TE) cells and inner cell mass (ICM) cells of blastocysts. Although morphologically indistinguishable, blastomeres at early cleavage stages are likely to undergo changes on a molecular level that make them destined to become ICM or TE cells. While the transcription factor Oct-4 might serve as a marker for totipotent ICM cells, human chorionic gonadotrophin might be used as the equivalent for TE cells. This study reports a reverse transcription-polymerase chain reaction procedure to assess human beta-HCG mRNA concentrations as well as ploidy in individual blastomeres from normally and abnormally fertilized human embryos. beta-HCG mRNA was detected in both euploid and aneuploid cells and in oocytes. Surprisingly, beta-LH mRNA was also detected in some euploid blastomeres. In regard to preimplantation genetic diagnosis, assessment of expression levels of beta-HCG and Oct-4 mRNA in individual biopsied cells might serve as a tool to identify embryogenic blastomeres in combination with testing for chromosome and single gene abnormalities

BACKGROUND: We assessed the maturational competence and the chromosomal pattern of mouse oocytes reconstructed by germinal vesicle (GV) transfer technique using nuclear and/or cytoplasmic components from cryopreserved GV stage oocytes. METHODS: From 657 GV oocytes (326 fresh and 331 frozen/thawed), four groups of reconstructed oocytes were obtained by micromanipulation and electrofusion: fresh GV-fresh cytoplast (FF), thawed GV-thawed cytoplast (TT), fresh GV-thawed cytoplast (FT), thawed GV-fresh cytoplast (TF). All reconstructed oocytes were cultured in vitro to metaphase II. RESULTS: Survival rate after manipulation and electrofusion, as well as progression to metaphase II, did not differ significantly among the four groups. Comparing reconstructed oocytes with fresh and thawed control pools, the only difference was a slightly but significantly higher maturation rate in the TT pool versus matched controls (P < 0.01). Cytogenetic analysis of 25 reconstructed oocytes showed the expected number of 20 chromosomes in 88% of them. CONCLUSIONS: We conclude that both nuclear and cytoplasmic components derived from cryopreserved immature oocytes are suitable for GV transfer procedure, and generate chromosomally normal oocytes able to progress to metaphase II in vitro. The possibility of using cryostored immature oocytes as a source of nuclei and cytoplasm could help in applying GV transfer procedure, both in research and clinical settings

Nuclear and pronuclear transfer procedures were used to assess the functional competence of the nucleus and cytoplasm of mouse germinal vesicle-stage oocytes denuded of granulosa cells and matured in vitro or in vivo before artificial activation using a sequential treatment of A23187 + cycloheximide. Following activation, in vitro-matured oocytes were 'fertilized' by inserting a male pronucleus (PN), cultured to the 2-cell stage, and then transferred to the oviducts of foster mothers. No live births were noted, whereas a 17% live birth rate was observed when in vivo-matured oocytes were used. The developmental competency of other zygotes was similarly assessed following the exchange of haploid PN of matured and activated eggs with the female PN of fertilized zygotes. When PN of oocytes subjected to maturation and activation in vitro were transferred, only 1 of 79 reconstructed zygotes developed to term. In contrast, the live birth rate was 21% (11 of 53) for zygotes reconstructed with PN from in vivo-matured oocytes. Moreover, a live birth rate of 23% (8 of 35) was observed for reconstructed zygotes with female PN from 'hybrid' oocytes created by transferring the metaphase II nuclei of in vitro-matured oocytes into enucleated, in vivo-matured oocytes before activation. Such results suggest that the nucleus of an in vitro-matured oocyte can support embryonic development, but only when it is activated in the proper ooplasmic milieu. The cellular factors creating this ooplasmic milieu appear to develop normally in vivo during follicle maturation to metaphase II, but they fail to do so when the oocytes are denuded of granulosa cells and cultured in vitro before the final stages of maturation. In parallel studies, male and female PN of in vivo-fertilized zygotes were inserted into oocytes that were activated and enucleated following either in vitro or in vivo maturation. Live birth rates were comparable at 19% (5 of 27) and 18% (9 of 49), respectively, suggesting that, regardless of the environment of the final stages of oocyte maturation, the resultant ooplasm is competent to support all aspects of embryonic development once activation and PN formation has been completed. Such findings only point further toward the importance of the condition of the ooplasmic milieu at the time of chemical activation. Whether a similar situation exists when eggs are activated following sperm penetration remains to be determined