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pHi oscillations during bovine oocyte maturation
Theriogenology
369
pHi OSCILLATIONS DURING BOVINE OOCYTE MATURATION L. Edwards 1, P. Batt, D. Williams & D.K. Gardner University of Melbourne, Melbourne, 3052, Australia The activity of maturation promoting factor (MPF) plays a key role in the regulation of the mitotic and meiotic cell cycles. Oscillations in MPF activity during the mitotic cell cycle have been correlated with periodic fluctuations in intracellular pH (pHi) in the Xenopus embryo. MPF activity was found to peak at metaphase I (12-14 h) and II (20-22 h) during maturation of the bovine oocyte (Wu et al., 1997 Biol. Reprod. 56:253; Lonergan et al., 1997 J. Reprod. Fertil. 109:355). Similarly there is a dramatic drop in MPF activity at the time of anaphase and telophase (16-18 h). Thus the aim of this study was to determine if pHi oscillated during maturation of the bovine oocyte. Abattoir cow ovaries were collected and oocytes aspirated from 2-5 mm ovarian follicles. Oocytes with at least 5 distinct layers of cumulus cells were selected and washed 3 times in TCM-199 supplemented with 25 mM HEPES. Groups of 20 oocytes were placed in 500 ~tl of maturation medium (TCM-199 supplemented with 25 mM NaHCO3, 10% FCS, 0.075 units/ml LH and 0.075 units/ml FSH) covered with paraffin oil, under 5% CO2 in air at 39°C. Culture time varied from 2 to 24 h. Ratio-metric measurements of pHi were performed using the pH-sensitive probe carboxy-seminapthorhodafluor-1 (SNARF-1) and confocal microscopy. After 2h pHi was 7.11 + 0.01 (n = 38) and remained constant for the first 8 h of the experimental time period. During the next 16 h the bovine oocyte expressed dramatic oscillations.in pHi as shown in figure 1. 7.25
7.20
7.15 e~
m
7.10
I
7.05
7.00
i 0
2.
. 4.
.6 . 8.
10
12
I4
1;
18 . . 20.
. 22
24
Time (h) Figure 1. Intracellular pH (pHi) of the bovine oocyte during maturation, n = at least 30 oocytes per time point, 2 replicates were performed. ***significantly different to base-line pHi (P < 0.001) (base-line pHi = 2 h measurement). Statistical differences were determined using ANOVA and Dunn's multiple comparison test. In this study we have observed that significant pHi oscillations occur and appeared to precede the reported changes in MPF activity by approximately 2 h. This finding indicates that pHi correlates with meiotic progression and MPF activity in the maturing bovine oocyte. This work was supported by the Australian Research Council.
369
pHi OSCILLATIONS DURING BOVINE OOCYTE MATURATION L. Edwards 1, P. Batt, D. Williams & D.K. Gardner University of Melbourne, Melbourne, 3052, Australia The activity of maturation promoting factor (MPF) plays a key role in the regulation of the mitotic and meiotic cell cycles. Oscillations in MPF activity during the mitotic cell cycle have been correlated with periodic fluctuations in intracellular pH (pHi) in the Xenopus embryo. MPF activity was found to peak at metaphase I (12-14 h) and II (20-22 h) during maturation of the bovine oocyte (Wu et al., 1997 Biol. Reprod. 56:253; Lonergan et al., 1997 J. Reprod. Fertil. 109:355). Similarly there is a dramatic drop in MPF activity at the time of anaphase and telophase (16-18 h). Thus the aim of this study was to determine if pHi oscillated during maturation of the bovine oocyte. Abattoir cow ovaries were collected and oocytes aspirated from 2-5 mm ovarian follicles. Oocytes with at least 5 distinct layers of cumulus cells were selected and washed 3 times in TCM-199 supplemented with 25 mM HEPES. Groups of 20 oocytes were placed in 500 ~tl of maturation medium (TCM-199 supplemented with 25 mM NaHCO3, 10% FCS, 0.075 units/ml LH and 0.075 units/ml FSH) covered with paraffin oil, under 5% CO2 in air at 39°C. Culture time varied from 2 to 24 h. Ratio-metric measurements of pHi were performed using the pH-sensitive probe carboxy-seminapthorhodafluor-1 (SNARF-1) and confocal microscopy. After 2h pHi was 7.11 + 0.01 (n = 38) and remained constant for the first 8 h of the experimental time period. During the next 16 h the bovine oocyte expressed dramatic oscillations.in pHi as shown in figure 1. 7.25
7.20
7.15 e~
m
7.10
I
7.05
7.00
i 0
2.
. 4.
.6 . 8.
10
12
I4
1;
18 . . 20.
. 22
24
Time (h) Figure 1. Intracellular pH (pHi) of the bovine oocyte during maturation, n = at least 30 oocytes per time point, 2 replicates were performed. ***significantly different to base-line pHi (P < 0.001) (base-line pHi = 2 h measurement). Statistical differences were determined using ANOVA and Dunn's multiple comparison test. In this study we have observed that significant pHi oscillations occur and appeared to precede the reported changes in MPF activity by approximately 2 h. This finding indicates that pHi correlates with meiotic progression and MPF activity in the maturing bovine oocyte. This work was supported by the Australian Research Council.