The round spermatids of cynomolgus monkeys can be used as substitute gametes to support embryonic development at least to mid-gestation. This non-human primate is a suitable animal model for round spermatid conception in mammals, especially humans, and for biological and genetic characterization of events following ROSI.

To establish reproductive biological techniques in mammals, it is important to understand the growth environment of the embryo. Oviduct epithelial cells are in close proximity to the embryo during pre-implantation development. We, therefore, established an immortalized oviduct epithelial cell line from the cynomolgus monkey, evaluated the usefulness of these cells as feeder cells for embryo culture, and investigated the gene expression of several growth factors and cytokines in the cells. The immortalized cells were positive for the anti-cytokeratin antibody, as determined by immunocytochemistry, indicating that they are epithelial. They also expressed oviductin, which is specific to oviduct epithelial cells, glyceraldehyde-3-phosphate dehydrogenase (control), leukemia inhibitory factor, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor 1, transforming growth factor beta-2, and interleukin 4. Mouse embryo development was improved when the immortalized cells were used as feeder cells. This cell line is also useful for studying the factors secreted by oviduct epithelial cells.

Abstract:We have developed a new method for separating mouse eggs from other cells, such as cumulus cells, using centrifugation with Percoll. Solutions of 45, 22.5, 11.3, and 5.6% Percoll were tested. With the 22.5% solution, 99% of whole eggs obtained by in vitro fertilization were collected from the upper part of the Percoll solution, and 98% of 2-cell embryos collected from these eggs developed to the blastocyst stage. Offspring were obtained after transfer of collected embryos to female mice. The greatest advantage of this method is that undamaged eggs are separated from other cells in one simple operation, regardless of the number of eggs.

Sperm InjectionReproduction, Fertility and Development 307 60, 1657-1663]) 6-7 h after insemination and cultured for 65-66 h. Embryos which had cleaved by 72 h post insemination were further cultured 96 h in CDM-2 (Zhang M et al. 2003 Theriogenology 60, 1657-1663 containing 0.12 IU insulin mL −1 . Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7-8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There was no significant difference in cleavage rate or blastocyst rate between X and Y sperm treatments. These results indicate that embryos produced with Y sperm do not reach the blastocyst stage in significantly higher proportions than embryos produced with X sperm in this chemically defined medium + FAF-BSA. Apparently, this IVC system leads to a more synchronous development of male and female embryos than other methods of producing bovine embryos in vitro. The cleavage rate of bovine embryos is very low without activation of oocytes after intracytoplasmic sperm injection (ICSI), although both male and female pronuclei are formed. We previously reported that the stimulus due to the injected sperm alone was sufficient to lower the MPF activity of bovine oocytes after ICSI, and the activation treatment of oocytes with ethanol at 4 h after ICSI served to maintain the low levels of MPF activity until the next cell cycle started (Fujinami et al. 2004 J. Reprod. Dev. 50, 171-178). These results suggested that activation treatment is necessary to improve the embryonic development after bovine ICSI. In bovine fertilization, the sperm introduces the centrosome into the oocyte. The centrosome acts as the microtubule-organizing center and microtubules are organized within the oocyte. It is reported that the sperm aster is important for the normal fertilization process. Therefore, failure of sperm aster formation possibly causes the failure of cleavage following fertilization. To investigate the reason of the low cleavage rate after bovine ICSI without artificial activation treatment, we examined sperm aster formation and the microtubule organization in bovine oocytes with or without activation treatment after ICSI. Bull spermatozoa immobilized by piezopulse was injected into bovine oocytes matured in vitro. At 4 h after ICSI, oocytes were treated with 7% ethanol in TCM199 for 5 min for activation. Oocytes were fixed at 6 and 12 h after ICSI, and the microtubule organization was examined by using specific antibodies and immunofluorescence microscopy. The cleavage rate (51% vs. 15%) and the developmental rate to the blastocyst stage (13% vs. 3%) were increased by ethanol treatment after ICSI (with or without ethanol treatment, respectively, P < 0.05). In oocytes activated with ethanol after ICSI, both the sperm aster formation rate at 6 h and the microtubule organization rate at 12 h after ICSI were significantly higher than in oocytes without activation treatment (58%, 80% vs. 12%, 26%, P < 0.05). It was reported that t...