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PLCζ is the physiological trigger of the Ca²⁺ oscillations that induce embryogenesis in mammals but conception can occur in its absence.

ABSTRACT: Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca²⁺ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca²⁺ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca²⁺ oscillations in eggs. Here, we show that sperm derived from Plcz1^-/- male mice fail to trigger Ca²⁺ oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca²⁺ oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, Plcz1^-/- males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility.

SUBMITTER: Hachem A

PROVIDER: S-EPMC5592814 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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PLCζ is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence.

Hachem Alaa A Godwin Jonathan J Ruas Margarida M Lee Hoi Chang HC Ferrer Buitrago Minerva M Ardestani Goli G Bassett Andrew A Fox Sebastian S Navarrete Felipe F de Sutter Petra P Heindryckx Björn B Fissore Rafael R Parrington John J

Development (Cambridge, England) 20170710 16

Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca2+ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca2+ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ab ...[more]

PMID: 28694258

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Project description:BackgroundMotor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle.MethodsA transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed "mitoflash." Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation.ResultsWe found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity.ConclusionsThe loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.

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.

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PLCζ is the physiological trigger of the Ca²⁺ oscillations that induce embryogenesis in mammals but conception can occur in its absence.

Publications

PLCζ is the physiological trigger of the Ca<sup>2+</sup> oscillations that induce embryogenesis in mammals but conception can occur in its absence.

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