Dataset Information

Mitochondrial regulation of [Ca²⁺]i oscillations during cell cycle resumption of the second meiosis of oocyte.

ABSTRACT: Oocyte is arrested at metaphase of the second meiosis until fertilization switching on [Ca²⁺]i oscillations. Oocyte activation inefficiency is the most challenging problem for failed fertilization and embryonic development. Mitochondrial function and intracellular [Ca²⁺]i oscillations are two critical factors for the oocyte's developmental potential. We aimed to understand the possible correlation between mitochondrial function and [Ca²⁺]i oscillations in oocytes. To this end, mitochondrial uncoupler CCCP which damages mitochondrial function and two small molecule mitochondrial agonists, L-carnitine (LC) and BGP-15, were used to examine the regulation of [Ca²⁺]i by mitochondrial functions. With increasing CCCP concentrations, [Ca²⁺]i oscillations were gradually diminished and high concentrations of CCCP led to oocyte death. LC enhanced mitochondrial membrane potential and [Ca²⁺]i oscillations and even improved the damage induced by CCCP, however, BGP-15 had no beneficial effect on oocyte activation. We have found that mitochondrial function plays a vital role in the generation of [Ca²⁺]i oscillations in oocytes, and thus mitochondria may interact with the ER to generate [Ca²⁺]i oscillations during oocyte activation. Improvement of mitochondrial functions with small molecules can be expected to improve oocyte activation and embryonic development in infertile patients without invasive micromanipulation.

SUBMITTER: Wang F

PROVIDER: S-EPMC6132958 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Mitochondrial regulation of [Ca2+]i oscillations during cell cycle resumption of the second meiosis of oocyte.

Wang Feng F Yuan Rui-Ying RY Li Li L Meng Tie-Gang TG Fan Li-Hua LH Jing Ying Y Zhang Ren-Ren RR Li Yuna-Yuan YY Liang Qiu-Xia QX Dong Feng F Hou Yi Y Schatten Heide H Sun Qing-Yuan QY Ou Xiang-Hong XH

Cell cycle (Georgetown, Tex.) 20180723 12

Oocyte is arrested at metaphase of the second meiosis until fertilization switching on [Ca2+]i oscillations. Oocyte activation inefficiency is the most challenging problem for failed fertilization and embryonic development. Mitochondrial function and intracellular [Ca2+]i oscillations are two critical factors for the oocyte's developmental potential. We aimed to understand the possible correlation between mitochondrial function and [Ca2+]i oscillations in oocytes ...[more]

PMID: 29965788

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Project description:ObjectiveHepatic Ca2+ signaling has been identified as a crucial key factor in driving gluconeogenesis. The involvement of mitochondria in hormone-induced Ca2+ signaling and their contribution to metabolic activity remain, however, poorly understood. Moreover, the molecular mechanism governing the mitochondrial Ca2+ efflux signaling remains unresolved. This study investigates the role of the Na+/Ca2+ exchanger, NCLX, in modulating hepatic mitochondrial Ca2+ efflux, and examines its physiological significance in hormonal hepatic Ca2+ signaling, gluconeogenesis, and mitochondrial bioenergetics.MethodsPrimary mouse hepatocytes from both an AAV-mediated conditional hepatic-specific and a total mitochondrial Na+/Ca2+ exchanger, NCLX, knockout (KO) mouse models were employed for fluorescent monitoring of purinergic and glucagon/vasopressin-dependent mitochondrial and cytosolic hepatic Ca2+ responses in cultured hepatocytes. Isolated liver mitochondria and permeabilized primary hepatocytes were used to analyze the ion-dependence of Ca2+ efflux. Utilizing the conditional hepatic-specific NCLX KO model, the rate of gluconeogenesis was assessed by first monitoring glucose levels in fasted mice, and subsequently subjecting the mice to a pyruvate tolerance test while monitoring their blood glucose. Additionally, cultured primary hepatocytes from both genotypes were assessed in vitro for glucagon-dependent glucose production and cellular bioenergetics through glucose oxidase assay and Seahorse respirometry, respectively.ResultsAnalysis of Ca2+ responses in isolated liver mitochondria and cultured primary hepatocytes from NCLX KO versus WT mice showed that NCLX serves as the principal mechanism for mitochondrial calcium extrusion in hepatocytes. We then determined the role of NCLX in glucagon and vasopressin-induced Ca2+ oscillations. Consistent with previous studies, glucagon and vasopressin triggered Ca2+ oscillations in WT hepatocytes, however, the deletion of NCLX resulted in selective elimination of mitochondrial, but not cytosolic, Ca2+ oscillations, underscoring NCLX's pivotal role in mitochondrial Ca2+ regulation. Subsequent in vivo investigation for hepatic NCLX role in gluconeogenesis revealed that, as opposed to WT mice which maintained normoglycemic blood glucose levels when fasted, conditional hepatic-specific NCLX KO mice exhibited a faster drop in glucose levels, becoming hypoglycemic. Furthermore, KO mice showed deficient conversion of pyruvate to glucose when challenged under fasting conditions. Concurrent in vitro assessments showed impaired glucagon-dependent glucose production and compromised bioenergetics in KO hepatocytes, thereby underscoring NCLX's significant contribution to hepatic glucose metabolism.ConclusionsThe study findings demonstrate that NCLX acts as the primary Ca2+ efflux mechanism in hepatocytes. NCLX is indispensable for regulating hormone-induced mitochondrial Ca2+ oscillations, mitochondrial metabolism, and sustenance of hepatic gluconeogenesis.

Project description:Overweight or obese women seeking pregnancy is becoming increasingly common. Human maternal obesity gives rise to detrimental effects during reproduction. Emerging evidence has shown that these abnormities are likely attributed to oocyte quality. Oxidative stress induces poor oocyte conditions, but whether mitochondrial calcium homeostasis plays a key role in oocyte status remains unresolved. Here, we established a mitochondrial Ca2+ overload model in mouse oocytes. Knockdown gatekeepers of the mitochondrial Ca2+ uniporters Micu1 and Micu2 as well as the mitochondrial sodium calcium exchanger NCLX in oocytes both increased oocytes mitochondrial Ca2+ concentration. The overload of mitochondria Ca2+ in oocytes impaired mitochondrial function, leaded to oxidative stress, and changed protein kinase A (PKA) signaling associated gene expression as well as delayed meiotic resumption. Using this model, we aimed to determine the mechanism of delayed meiosis caused by mitochondrial Ca2+ overload, and whether oocyte-specific inhibition of mitochondrial Ca2+ influx could improve the reproductive abnormalities seen within obesity. Germinal vesicle breakdown stage (GVBD) and extrusion of first polar body (PB1) are two indicators of meiosis maturation. As expected, the percentage of oocytes that successfully progress to the germinal vesicle breakdown stage and extrude the first polar body during in vitro culture was increased significantly, and the expression of PKA signaling genes and mitochondrial function recovered after appropriate mitochondrial Ca2+ regulation. Additionally, some indicators of mitochondrial performance-such as adenosine triphosphate (ATP) and reactive oxygen species (ROS) levels and mitochondrial membrane potential-recovered to normal. These results suggest that the regulation of mitochondrial Ca2+ uptake in mouse oocytes has a significant role during oocyte maturation as well as PKA signaling and that proper mitochondrial Ca2+ reductions in obese oocytes can recover mitochondrial performance and improve obesity-associated oocyte quality.

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Dataset Information

Mitochondrial regulation of [Ca²⁺]i oscillations during cell cycle resumption of the second meiosis of oocyte.

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Mitochondrial regulation of [Ca<sup>2+</sup>]i oscillations during cell cycle resumption of the second meiosis of oocyte.

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