Maturation conditions, post-ovulatory age, medium pH, and ER stress affect [Ca2+]i oscillation patterns in mouse oocytes

Abstract

Insufficiency of oocyte activation impairs the subsequent embryo development in assisted reproductive technology (ART). Intracellular Ca²⁺ concentration ([Ca²⁺]i) oscillations switch the oocytes to resume the second meiosis and initiate embryonic development. However, the [Ca²⁺]i oscillation patterns in oocytes are poorly characterized. In this study, we investigated the effects of various factors, such as the oocytes age, pH, cumulus cells, in vitro or in vivo maturation, and ER stress on [Ca²⁺]i oscillation patterns and pronuclear formation after parthenogenetic activation of mouse oocytes. Our results showed that the oocytes released to the oviduct at 17 h post-human chorionic gonadotrophin (hCG) displayed a significantly stronger [Ca²⁺]i oscillation, including higher frequency, shorter cycle, and higher peak, compared with oocytes collected at earlier or later time points. [Ca²⁺]i oscillations in acidic conditions (pH 6.4 and 6.6) were significantly weaker than those in neutral and mildly alkaline conditions (pH from 6.8 to 7.6). In vitro-matured oocytes showed reduced frequency and peak of [Ca²⁺]i oscillations compared with those matured in vivo. In vitro-matured oocytes from the cumulus-oocyte complexes (COCs) showed a significantly higher frequency, shorter cycle, and higher peak compared with the denuded oocytes (DOs). Finally, endoplasmic reticulum stress (ER stress) severely affected the parameters of [Ca²⁺]i oscillations, including elongated cycles and lower frequency. The pronuclear (PN) rate of oocytes after parthenogenetic activation was correlated with [Ca²⁺]i oscillation pattern, decreasing with oocyte aging, cumulus removal, acidic pH, and increasing ER stress. These results provide fundamental but critical information for the mechanism of how these factors affect oocyte activation.

Keywords: Calcium oscillations; Endoplasmic reticulum stress; In vitro oocyte maturation techniques; Oocytes; Parthenogenesis.

Fig. 1

Intracellular free calcium ([Ca²⁺]i) oscillation patterns after parthenogenetic activation in oocytes collected at different times post-human chorionic gonadotropin (hCG) injection. a, b [Ca²⁺]i oscillation patterns at 13-21-h post-hCG. c Relative [Ca²⁺]i oscillation parameters including “frequency,” “cycle,” “maintain,” “move,” “peak,” and “Δpeak” for statistical analyses; each ratio of different groups was compared with the 17-h group. d Rates of pronucleus (PN) formation of oocytes collected at different post-hCG time points after parthenogenetic activation. N = 25 to 30 for each group. Data represent mean ± SD, and chi-square test and one-way ANOVA are used for statistical analysis. *P < 0.05 versus the 17-h group

Fig. 2

Intracellular free calcium ([Ca²⁺]i) oscillation patterns during parthenogenetic activation under different pH conditions at 17 h post-hCG. a, b [Ca²⁺]i oscillation patterns after parthenogenetic activation in gradient pH conditions from 6.4 to 7.6. c Relative [Ca2+]i oscillation parameters in gradient pH conditions in parthenogenetic activation medium. Each group was compared with the pH 7.2 group. The [Ca2+]i oscillation parameters, “frequency” and “cycle,” were significantly different between pH 7.2 and pH 6.4 or pH 6.6 groups. d Pronucleus (PN) rate in gradient pH conditions of the parthenogenetic activation system. PN ratios of pH 6.4 and pH 6.6 treatments were significantly lower compared with the other groups. N = 25 to 30 for each group. Data represent mean ± SD, and chi-square test and one-way ANOVA are used for statistical analysis. *P < 0.05 versus pH 7.2 group

Fig. 3

Intracellular free calcium ([Ca²⁺]i) oscillation patterns of in vivo oocytes and in vitro matured cumulus-denuded oocytes (DOs) and cumulus-enclosed oocyte complexes (COCs) in vitro-matured for 17 h. a, b [Ca²⁺]i oscillation patterns after parthenogenetic activation. c Relative [Ca²⁺]i oscillation parameters of oocytes matured in vivo or in vitro. The in vivo group was compared with the in vitro groups. d Pronucleus (PN) rate of in vivo and in vitro matured oocytes. N = 25 to 30 for each group. Data represent mean ± SD, and chi-square test and one-way ANOVA are used for statistical analysis. *P < 0.05 versus the control group

Fig. 4

Intracellular free calcium ([Ca²⁺]i) oscillation patterns after parthenogenetic activation of oocytes treated with the endoplasmic reticulum stress inducer tunicamycin (TM). a, b Oocytes were treated with tunicamycin (TM) for 1.5 h. The 0-μM group was the control group, and 2.5, 5, 10, 20, 30, and 50 μg/ml TM were respectively added to the M2 medium. c [Ca²⁺]i oscillation parameters in oocytes treated with gradient TM concentrations ranging from 0 to 50 μg/ml. d The pronucleus (PN) rate of oocytes with ER stress induced by TM. N = 25 to 30 for each group. Data represent mean ± SD, and chi-square test and one-way ANOVA are used for statistical analysis. *P < 0.05 versus the control group