Fertilization of bovine oocytes by microinjection of spermatozoa into the perivitelline space

THERIOGENOLOGY FERTILIZATION OF BOVINEOOCYTES BY MICROINJECTION OF SPERMATOZOA INTO THE PERIVITELLINE SPACE W. Heuwieser, X. Yang, S. Jiang and R. H. Foote Department of Animal Science Cornell University Ithaca, New York 14853-4801 USA The objective of this study was to evaluate the fertilization rate of in vitro matured oocytes (22 h incubation in TCM 199) following injection of spermatozoa (SPZ) into the perivitelline space (PVS). Cumulus cells were removed by hyaluronidase (1 mg/ml) and by pipetting. Frozen-thawed SPZ were treated either by heparin (100 Ag/ml; Trial 2) or by Ca ionophore A23187 (0.1 AM; Trial 3) or remained untreated. Oocytes were placed in a droplet of hyperosmotic (0.1 M) sucrose solution in PBS to enlarge the PVS. Before SPZ microinjection semen was diluted with TALP medium, suspended in 0.9% NaCl containing 10% polyvinyl pyrolidone (PVP), and transferred into a droplet of PVP near A motile SPZ was aspirated into the the oocyte-containing droplet. injection pipette, and the pipette was moved to the oocyte-containing droplet. The SPZ cell was injected into the PVS of an oocyte held by a holding pipette. As a control (Trial 1) a small amount of PVP without SPZ was injected into the PVS. Untreated SPZ were injected in Trials 4 (single) and 5 (multiple), and after injection of SPZ the oocytes with SPZ were treated with A23187 (0.1 AM; 5 min). All oocytes were washed twice, incubated in TCM 199 medium for 22 h at 39OC and stained with 1% aceto-orcein and examined for evidence of activation (i.e. SPZ decondensation, pronucleus formation). Results are in Table 1. Table 1. Trial 1 : 4 5

Fertilization of oocytes after injection of spermatozoa.

No. of oocytes 88 64 83

No. of sperm injected 0

;;

: 1 5

Treatment Sperm. Oocyte __ Heparin A23187 __ __

__ -A23187 A23187

Activation No. % :; 9b 7b 13c

I: 11 9 22

aybyc Values with different superscripts differ significantly (~~0.05). Successful injection of a single SPZ cell was verified in 92 to 98% of the oocytes. However, low rates of fertilization (9 to 22%) following SPZ microinjection into the PVS were obtained in this study. Results indicate that bovine oocytes are not sufficiently activated by the injection process of a motile SPZ injected into the PVS to complete meiosis. More distinctly decondensed SPZ were visible after multiple SPZ injection and treatment of the oocytes with SPZ by A23187 (pt0.05). It is noteworthy that only one SPZ per oocyte showed evidence of decondensation in Trial 5. In conclusion, microinjection of SPZ into oocytes is a useful technique to study spermatozoa-oocyte interactions; however fertilization rates were low.

JANUARY

1991VOL.35NO.l

213