Prostaglandin F2α Causes Fast Degenerative Changes in Ovulated Mouse Oocytes.

The effects of prostaglandin F2α on the cytoskeleton and membrane organelles of oocytes was investigated by culturing ovulated mouse oocytes in its presence (50 or 100 ng/ml) for 3 h. Tubulin, fibrillar actin, membranes and chromatin were visualized by specific antibodies, phalloidin, lipophilic dye DiOC6 and Hoechst 33342, respectively. Control oocytes were characterized by a meiotic spindle with chromosomes aligned at its equator, and a cortical layer of microfilaments with an actin cap. Intracellular membranes were localized mostly in the central region in metaphase I and in a broader volume, but still excluding the cell periphery, in metaphase II, and were slightly concentrated around the chromosomes. In oocytes treated with 50 ng/ml prostaglandin, cortical actin staining was diminished, the membrane distribution was clustered, and chromosomes showed signs of misalignment despite the apparently preserved spindle. In cells treated with 100 ng/ml prostaglandin, both the spindle and the actin cortex had degenerated or disappeared as microscopic objects. Metaphase plates were on average broader and more disorganized than in the 50 ng/ml group, and the distribution of membrane organelles had become uniform. These effects, to our knowledge observed for the first time, did not require presence of the cumulus during the incubation. They could be regarded as acceleration of the oocyte postovulatory aging, in which cytoskeletal deterioration seemed to have a leading role.

The status of the chromatin of human preimplantation embryos with good morphology.

The data about the relation and succession of blastomere fragmentation, cleavage rate and chromatin anomalies in preimplantation mammalian embryos are empirical and controversial at present. In this work we studied the proportion of nuclear fragmentation and condensation in 3-5-cell stage human embryos with no or minimal blastomere fragmentation (morphological class A and B, respectively) and the possibilities to perform FISH chromosomal analyses with them. We observed different stages of chromatin damage in blastomere nuclei corresponding to the steps of nuclear apoptotic changes well known in many cell types. The ploidity analysis of chromosomes 1, 5, 19 and X was determined as successful in embryos which had at least 2 out of 3, 3 out of 4 or 3 out of 5 normal nuclei with an equal number of FISH signals. There was no difference in the percentage of abnormal nuclei among the A- and B-class embryos. Tendencies noted by us suggest that the minimal blastomere fragmentation (up to 20% of perivitelline space) does not preclude the normal nuclear status allowing successful ploidy testing. The presence of condensed chromatin is a critical factor for interphase cytogenetic analysis of single early blastomeres.

Supernumerary human preembryos provide potential for preimplantation genetic diagnosis.

Preimplantation genetic diagnosis is an alternative to the classical prenatal diagnosis for couples undergoing in vitro fertilization. It allows very early embryo selection--before the intrauterine embryo transfer. Prior to clinical application of preimplantation diagnosis in the Infertility Treatment Centre "Technobioassistance", Sofia Medical Faculty, we have developed preimplantation diagnosis models of human spermatozoa and untransferred 2-8-cell human embryos obtained in vitro. Directly fluorescein isothiocyanate-labelled probes specific for the centromeric regions of chromosomes 1, 5, 19 and X (Boehringer Mannheim) were used. Eighty-six point three percent of fixed blastomeres with normal size and shape had unfragmented nuclei with dispersed interphase chromatin or mitotic chromosomes and all of them demonstrated successful hybridization. In cases with more than 75% of embryo cells suitable for analysis we were able to estimate the presence of mosaicism among the blastomeres.