Electrically induced calcium elevation, activation, and parthenogenetic development of bovine oocytes

Abstract

The influence of electrical stimulation on the level of intracellular Ca2+ in bovine oocytes, as well as activation and extent of parthenogenetic development, was investigated. Mature oocytes were electrically stimulated at 29 hr of maturation, and intracellular Ca2+ concentration was determined with the Ca2+ indicator fura-2 dextran (fura-2 D). The Ca2+ response of oocytes to a given electrical pulse was variable. Oocytes responded with either no Ca2+ rise from baseline (approximately 12 nM), a short-duration Ca2+ rise (from 12 nM to 300 nM) that returned to baseline within 2 min of the pulse, or a long-duration Ca2+ rise (from 12 nM to 1,000-2,000 nM) that never returned to baseline during the 8 min period over which the oocytes were monitored. In these oocytes, Ca2+ level returned to baseline when oocytes were removed from 0.30 M mannitol and placed in an ionic medium. Increasing field strength or pulse duration tended to increase the proportion of oocytes displaying a Ca2+ rise, and at 1.0 kVcm-1 for 40 microseconds, all oocytes displayed a long-duration Ca2+ elevation. Direct transfer of oocytes from culture medium to mannitol also triggered a Ca2+ rise. Multiple stimulations, either electrical or by transferring to mannitol, produced multiple Ca2+ rises. This mannitol-induced Ca2+ rise could be inhibited by first washing the oocytes in medium containing equal parts of 0.30 M mannitol and phosphate buffered saline (PBS). The level of Ca2+ stimulation affected activation and development of oocytes. Insufficient, or, conversely, excessive Ca2+ stimulation impaired development. Optimum development was obtained with 1) three pulses of 0.2 kVcm-1 for 20 microseconds, each pulse 22 min apart, after direct transfer of oocytes from culture medium to mannitol (22% blastocysts) or 2) three pulses of 1.0 kVcm-1 for 20 microseconds after transfer of oocytes from culture medium to medium containing equal parts mannitol and PBS, then to mannitol (24% blastocysts). This procedure avoided induction of a Ca2+ rise prior to the pulse. The results indicate that the level of Ca2+ stimulation can be regulated by incubation conditions prior to the pulse and, to some extent, by field strength and pulse duration. The level of electrical stimulation influenced oocyte Ca2+ response, activation, and parthenogenetic development.

Use of a variable electrical pulsing sequence in rabbit oocyte activation

“…However, some factors more related to the oocyte itself may determine different oocyte susceptibility to those artificial stimuli [4,5,11,31]. In the present work, the existence of this expected intrinsic oocyte activation variability in similarly-aged post-hCG oocytes was demonstrated.…”

“…Three types of parthenogenetic eggs were produced [5,10,16,23]. The first type had one pronucleus plus first and second polar bodies and further Culture beyond this limit was not studied in this work since it had been investigated earlier [10].…”

“…During this period, the number of applied pulses and their characteristics will determine the final oocyte activation rate and type [5,23,26]. In fact, we have recently reported that 8 pulses or more applied over 90 min overstimulate the oocytes, resulting in retention of PB2 and the production of diploid oocytes [10].…”

“…Standard activating sequences consist of a pre-determined number of electrical stimuli which are applied to all oocytes [5,6,10,22,23]. However, some factors related to the intrinsic quality of the oocyte itself, such as the strain utilised and the post-ovulatory oocyte age at the time of activation, could lead to different oocyte susceptibility to those electrical pulse stimuli [4,5,11,31].…”

“…However, some factors related to the intrinsic quality of the oocyte itself, such as the strain utilised and the post-ovulatory oocyte age at the time of activation, could lead to different oocyte susceptibility to those electrical pulse stimuli [4,5,11,31]. In polytocous species, such as rabbits, certain time (approximately 2-6 h) is required to complete the ovulation [3,13,15,21] and so, in similarly-aged post-hCG oocytes the existence of intrinsic variability in sensitivity to stimuli would be expected.…”