André Moens

The pluripotency of embryonic germ cells in the mouse suggests that mitotic bovine fetal germ cells might also be a source of pluripotent cells. To investigate the pluripotency of bovine oogonia, the development in vitro of bovine embryos reconstructed by fusing oogonia with enucleated oocytes was compared with that of embryos made similarly with either blastomeres or granulosa cells. The donor cells (fresh oogonia, cryopreserved oogonia, 16- to 32-cell-stage blastomeres, or granulosa cells) were fused to the enucleated oocytes electrically. The proportions of reconstructed embryos that had cleaved at 40 h after fusion using these types of donor cells were not significantly different (37%, 33%, 56%, and 31%, respectively; p > 0.05). However, the proportions of cleaved reconstructed embryos that developed to the blastocyst stage were 9%, 13%, 36%, and 3%, respectively, significantly higher (p < or = 0.05) with blastomeres than with the other three types of donor cells. After transfer of 3 morulae and 4 blastocysts made with oogonia into three recipient heifers, embryonic and extra-embryonic tissues developed in one animal. On recovery after 43 days gestation, this conceptus was shown to be genetically identical, at 11 microsatellite loci, to the fetus that had provided the oogonia. Cytological analysis of the embryos made with oogonia at 40-44 h after fusion and at the morula and blastocyst stages revealed that aberrant cytokinesis and nucleokinesis had given rise to multinucleated, anucleate, and polyploid cells in the reconstructed embryos. It is concluded that limited pluripotency of bovine oogonia has been demonstrated, warranting further study in this area.

Two prooxidant agents, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), a generator of free radicals in the culture medium, and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, were used to reinforce from the morula stage (day 5 post-insemination, p.i.) the oxidative stress encountered by bovine embryos in culture. Exposure to increasing concentrations of both prooxidants from the morula stage did not affect blastocyst formation but some blastocysts were found degenerated on day 8 in a dose-dependent manner (0, 0.001, 0.01, 0.1 mM AAPH gave respectively 0, 10%, 32%, 48% degeneration, while 0, 0.1, 0.2, 0.4 mM BSO led respectively to 0, 14%, 30%, 41% degeneration). Hatching rates and cell numbers of surviving blastocysts were not affected. Morulae and early blastocysts exposed from day 5 to day 6 p.i. appeared more resistant than expanded blastocysts (75-80% survival vs 20-65%; p < 0.05). Treatment with BSO significantly decreased the level of reduced glutathione in day 7 blastocysts (0.02 vs 0.42 pmol per embryo in the control) while AAPH had no effect (0.38 pmol per embryo). The proportion ofcells showing membrane lesions was increased in degenerated blastocysts from day 7.5 p.i. In AAPH-treated, but not in BSO-treated embryos, cell membrane permeabilisation seems to occur before blastocyst degeneration. DNA fragmentation evaluated by the TUNEL technique was increased in day 7 blastocysts by both prooxidants (2.8 +/- 0.4 in the control group vs 4.5 +/- 0.4 and 6.0 +/- 0.4 respectively in the AAPH- and BSO-treated groups). Addition of an inhibitor of caspase-3, DEVD-CHO, partially prevented DNA fragmentation, indicating that prooxidant treatment induced a caspase-dependent pathway of apoptosis.

Although toxic for early stages of embryo development, glucose is a physiological metabolic substrate at the morula and blastocyst stages. We evaluated the effect of adding 5.5 mM glucose from the morula stage on bovine blastocyst development and quality. In vitro matured and fertilised bovine oocytes were cultured in modified Synthetic Oviduct Fluid medium containing 5% fetal calf serum, but without added glucose, up to day 5 post-insemination (pi). Morulae were selected and further cultured in the presence or absence of 5.5 mM glucose. Blastocyst and hatched blastocyst rates were recorded. Oxygen, glucose and pyruvate uptakes as well as lactate release were evaluated. The quality of the resulting blastocysts was evaluated by the cell allocation to the inner cell mass (ICM) and trophectoderm (TE) and by the apoptotic index. Adding glucose increased the blastocyst rate at day 8 pi (80% vs 65%) but had no impact on hatching rate (25% vs 28%). A 22% decrease in oxygen uptake was observed in the presence of glucose, concomitant with an increase in lactate release, although no change was observed in pyruvate uptake. A slight decrease in blastocyst cell number was observed at day 7 in the presence of glucose while neither the ICM/TE cell ratio nor the apoptotic index were affected. In conclusion, adding 5.5 mM glucose from the morula stage has a limited impact on blastocyst rate and quality although important modifications were observed in embryo metabolism. It remains to be determined whether those modifications could influence embryo viability after transfer.