Effect of microinjection time during postfertilization S‐phase on bovine embryonic development

Abstract

Microinjection into bovine zygotes was performed to evaluate the effects of the timing of injection during the phase of DNA replication on the subsequent in vitro development of embryos and expression of injected chicken beta-actin promoter-lac Z gene construct. The period of DNA replication of bovine zygotes, determined by 3H-thymidine incorporation, begins between 12 hr and 13 hr postinsemination (hpi) of in vitro matured oocytes, reaches a maximum from 17 hpi to 19 hpi, and is complete by 21-22 hpi. Aphidicolin, an inhibitor of DNA polymerase alpha, was used to synchronize the pronuclei and the zygote population. Treatment with aphidicolin at 9-18 hpi arrested DNA replication without affecting formation of the pronuclei or embryo development. Cycloheximide, an inhibitor of protein synthesis, was used for nucleocytoplasmic resynchronization of the aphidicolin-treated zygotes. Microinjection was performed at 15 (early), 18 (mid), and 21 (late S phase) hpi. Embryonic development was affected following each of the three microinjection times. The development of zygotes injected at 18 hpi was significantly higher (P < 0.01) after 5 days of culture than those injected at 15 hpi or 21 hpi. Expression of the marker gene was observed in the higher stage of development (> 16 cells) only in the zygotes injected at 18 hpi. At the earlier stages of development, the proportions of embryos showing expression of the foreign gene were the same for all microinjection times. In aphidicolin- and cycloheximide-treated zygotes, expression of the marker gene followed the same curve as development, i.e., expression was low when injected early or late and higher (P < 0.005) when injected in the middle of zygotic S phase. The ability of the embryos to survive microinjection and to express the marker gene as a function of hpi seems to be influenced mostly in the cytoplasm processing stage rather than the pronuclei processing stage.

Onset of the First S-Phase Is Determined by a Paternal Effect During the G1-Phase in Bovine Zygotes1

“…This is not in accordance with Gagné et al [16], who observed similar development rates after aphidicolin treatments but mentioned only the morula rates. When oocytes were fertilized by high-fertility sperm, the timing of DNA replication after a short aphidicolin exposure mimics that of oocytes fertilized by low-fertility sperm without aphidicolin exposure.…”

“…The paternal influence on the onset of the S-phase was exerted simultaneously on both pronuclei and not only on the paternal pronucleus; this was not evidenced in the study of Eid et al [15]. Thereafter both pronuclei of a zygote had the same duration of DNA replication, in agreement with Gagné et al [16].…”

“…However, contrary to the situation in the mouse, the sperm used for fertilization influences the time of the onset after fertilization [15], and there is no evidence that one of the pronuclei enters the Sphase before the other [16]. Eid et al [15] showed that DNA replication began earlier and lasted longer in zygotes fertilized by sperm with high in vivo fertility, whereas the end of DNA replication occurred at the same moment regardless of the bull's fertility.…”

Transient Expression of a Translation Initiation Factor Is Conservatively Associated with Embryonic Gene Activation in Murine and Bovine Embryos1

“…Bovine embryos transfected with reporter genes at the 1-cell stage express detectable amounts of reporter gene protein only after 42 h of culture around the 4-to 8-cell stage or later. In those experiments, reporter protein was also detected in arrested 1-cell embryos at this time [72,73].…”

Oogenetic and zygotic gene expression directing early bovine embryogenesis: A review

“…Bovine embryos transfected with reporter genes at the 1-cell stage by pronuclear injection or retroviral transfer only express detectable amounts of reporter gene product following 42 hr of culture around the 4-to 8-cell stage or later, with product also detected in arrested 1-cell embryos at this time (Gagne et al, 1995;Kubisch et al, 1997). Temporal control of transcriptional permissiveness is also apparent in other species but appears to begin as early as the G2 phase of the first cell cycle in the mouse and rabbit (Vernet et al, 1992;Ram and Schultz, 1993;Christians et al, 1994;Nothias et al, 1996).…”