Intra-cytoplasmic sperm injection in a marsupial

Here we report the first use of intra-cytoplasmic sperm injection (ICSI) in a marsupial, the tammar wallaby (Macropus eugenii), to achieve in vitro fertilization and cleavage. A single epididymal spermatozoon was injected into the cytoplasm of each mature oocyte collected from Graafian follicles or from the oviduct within hours of ovulation. The day after sperm injection, oocytes were assessed for the presence of pronuclei and polar body extrusion and in vitro development was monitored for up to 4 days. After ICSI, three of four (75%) follicular and four of eight (50%) tubal oocytes underwent cleavage. The cleavage pattern was similar to that previously reported for in vivo fertilized oocytes placed in culture, where development also halted at the 4- to 8-cell stage. One-third of injected oocytes completed the second cleavage division, but only a single embryo reached the 8-cell stage. The success of ICSI in the tammar wallaby provided an opportunity to examine the influence of the mucoid coat that is deposited around oocytes passing through the oviduct after fertilization. The presence of a mucoid coat in tubal oocytes did not prevent fertilization by ICSI and the oocytes cleaved in vitro to a similar stage as follicular oocytes lacking a mucoid coat. Cell-zona and cell-cell adhesion occurred in embryos from follicular oocytes, suggesting that the mucoid coat is not essential for these processes. However, blastomeres were more closely apposed in embryos from tubal oocytes and cell-cell adhesion was more pronounced, indicating that the mucoid coat may be involved in maintaining the integrity of the conceptus during cleavage.