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Postovulatory ageing modifies sperm-induced Ca2+ oscillations in mouse oocytes through a conditions-dependent, multi-pathway mechanism.

ABSTRACT: Postovulatory ageing of mammalian oocytes occurs between their ovulation and fertilization and has been shown to decrease their developmental capabilities. Aged oocytes display numerous abnormalities, including altered Ca2+ signalling. Fertilization-induced Ca2+ oscillations are essential for activation of the embryonic development, therefore maintaining proper Ca2+ homeostasis is crucial for the oocyte quality. In the present paper, we show that the mechanism underlying age-dependent alterations in the pattern of sperm-triggered Ca2+ oscillations is more complex and multifaceted than previously believed. Using time-lapse imaging accompanied by immunostaining and molecular analyses, we found that postovulatory ageing affects the amount of Ca2+ stored in the cell, expression of Ca2+ pump SERCA2, amount of available ATP and distribution of endoplasmic reticulum and mitochondria in a manner often strongly depending on ageing conditions (in vitro vs. in vivo). Importantly, those changes do not have to be caused by oxidative stress, usually linked with the ageing process, as they occur even if the amount of reactive oxygen species remains low. Instead, our results suggest that aberrations in Ca2+ signalling may be a synergistic result of ageing-related alterations of the cell cycle, cytoskeleton, and mitochondrial functionality.

SUBMITTER: Szpila M

PROVIDER: S-EPMC6694115 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Postovulatory ageing modifies sperm-induced Ca2+ oscillations in mouse oocytes through a conditions-dependent, multi-pathway mechanism.

Szpila Marcin M Walewska Agnieszka A Sabat-Pośpiech Dorota D Strączyńska Patrycja P Ishikawa Takao T Milewski Robert R Szczepańska Katarzyna K Ajduk Anna A

Scientific reports 20190814 1

Postovulatory ageing of mammalian oocytes occurs between their ovulation and fertilization and has been shown to decrease their developmental capabilities. Aged oocytes display numerous abnormalities, including altered Ca2+ signalling. Fertilization-induced Ca2+ oscillations are essential for activation of the embryonic development, therefore maintaining proper Ca2+ homeostasis is crucial for the oocyte quality. In the present paper, we show that the mechanism un ...[more]

PMID: 31413272

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Project description:BackgroundWhile calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated.ResultsPartial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation.ConclusionsInhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.

Project description:Postovulatory aging of the mammalian oocytes causes deterioration of oocytes through several factors including oxidative stress. Keeping that in mind, we aimed to investigate the potential of a well-known antioxidant, resveratrol (RV), to evaluate the adverse effects of postovulatory aging in porcine oocytes. After in vitro maturation (IVM), a group of (25–30) oocytes (in three replicates) were exposed to 0, 1, 2, and 4 μmol/L of RV, respectively. The results revealed that the first polar body (PB1) extrusion rate of the oocytes significantly increased when the RV concentration reached up to 2 μmol/L (p < 0.05). Considering optimum RV concentration of 2 μmol/L, the potential of RV was evaluated in oocytes aged for 24 and 48 h. We used fluorescence microscopy to detect the relative level of reactive oxygen species (ROS), while GHS contents were measured through the enzymatic method. Our results revealed that aged groups (24 h and 48 h) treated with RV (2 μmol/L) showed higher (p < 0.05) ROS fluorescence intensity than the control group, but lower (p < 0.05) than untreated aged groups. The GSH content in untreated aged groups (24 h and 48 h) was lower (p < 0.05) than RV-treated groups, but both groups showed higher levels than the control. Similarly, the relative expression of the genes involved in antioxidant activity (CAT, GPXGSH-Px, and SOD1) in RV-treated groups was lower (p < 0.05) as compared to the control group but higher than that of untreated aged groups. Moreover, the relative mRNA expression of caspase-3 and Bax in RV-treated groups was higher (p < 0.05) than the control group but lower than untreated groups. Furthermore, the expression of Bcl-2 in the RV-treated group was significantly lower than control but higher than untreated aged groups. Taken together, our findings revealed that the RV can increase the expression of antioxidant genes by decreasing the level of ROS, and its potent antiapoptotic effects resisted against the decline in mitochondrial membrane potential in aged oocytes.

Dataset Information

Postovulatory ageing modifies sperm-induced Ca2+ oscillations in mouse oocytes through a conditions-dependent, multi-pathway mechanism.

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Postovulatory ageing modifies sperm-induced Ca<sup>2+</sup> oscillations in mouse oocytes through a conditions-dependent, multi-pathway mechanism.

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