Metaphase II karyoplast transfer from human in-vitro matured oocytes to enucleated mature oocytes.

Metaphase II karyoplast transfer is believed to be a useful method to rescue aged oocytes. This study attempted karyoplast transfer of in-vitro matured metaphase II (MII) oocytes, as a model of aged oocytes, into enucleated freshly ovulated metaphase II oocytes with visualization of their chromosomes under an inverted microscope. Recipient karyoplasts derived from immature oocytes were cultured in-vitro until first polar body extrusion. After 1-2 days culture, 52.1% extruded a polar body, 95.5% had PSC, aneuploidy was very low (4.5%) and none had structural aberrations. Donor oocytes were obtained from IVF or intracytoplasmic sperm injection (ICSI) patients. Chromosomes were easily confirmed in 92.3% and 95.0% of in-vivo and in-vitro matured oocytes respectively. Thirty-one karyoplasts were placed in the perivitelline space of enucleated donor oocytes, and 25 (80.6%) fused to form a reconstituted oocyte. Fertilization, cleavage and blastocyst formation rates following ICSI were 76.0%, 64.0% and 28.0% respectively for reconstructed oocytes and 59.2%, 48.0% and 3.1% respectively for control (in-vitro matured) oocytes. Chromosomal analysis of five embryos developed after karyoplast transfer and ICSI showed normal diploid sets of 46 chromosomes. In conclusion, this metaphase II karyoplast transfer technique can be applied to the solution of chromosomal abnormalities related to oocyte ageing.

Isolated spermatogonia protrude active pseudopodia in vitro.

When dispersed spermatogenic cells obtained by enzymatic digestion from prepuberal mice, adult male mice, nonazoospermic men and normospermic men were observed live using Normarski optics, it was found that, respectively, 47.4%, 1.4%, 5.1%, and 2.4% of them protruded active pseudopodia. These cells were 8 to 10 mum in diameter, had a high N/C ratio, and had one to two prominent nucleoli that were close to a distinct nuclear membrane. They showed low alkaline phosphatase activities and homogeneous nuclear immunoreactive patterns using gamma-H2AX, which suggests that they were spermatogonia.