Figure 9 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
A possible mechanism underlying cytoprotective action of pinacidil and glibenclamide in human oocytes. Cartoon summarizing possible underlying mechanisms mediating pinacidil- (A) and glibenclamide-induced (B) cytoprotection in MII human oocytes based on the findings from the present study as well as findings from previous studies that have investigated KATP channels in oocytes and other cell types (Brady et al., 1996; Holmuhamedov et al., 1998; Jovanović et al., 1999; Jovanović and Jovanović, 2001; Du et al., 2010).
Source publication
Study question:
Could drugs targeting ATP-sensitive K(+) (KATP) channels prevent any spontaneous increase in intracellular Ca(2+) that may occur in human metaphase II (MII) oocytes under in vitro conditions?
Summary answer:
Pinacidil, a KATP channel opener, and glibenclamide, a KATP channel blocker, prevent a spontaneous increase in intracellula...
Citations
... The two repaglinide concentrations were selected from a previous study by Kalehoei & Azadbakht (2017). Dimethyl sulfoxide 0.1% (DMSO) was used to dissolve repaglinide as described by Fernandes et al. (2016). Sperm from asthenozoospermic patients was first double washed in sperm wash medium (3000 rpm for 5 minutes). ...
... According to these reports, drugs belonging to the class of sulfonylureas such as meglitinide analogs may have a beneficial role in modulating Ca 2+ homeostasis. Fernandes et al. (2016) showed that both inhibition and activation of K-ATP channels with drugs targeting K-ATP channels such as glibenclamide, 2, 4-dinitrophenol (DNP), and pinacidil are useful in maintaining Ca 2+ homeostasis in oocytes in in vitro culture. Our research team recently indicated that the application of repaglinide in IVM, IVF, and follicle growth culture medium significantly improved in vitro mice oocyte maturation, fertilization, embryo cleavage, and follicle growth rates, probably by elevating intracellular calcium concentrations (Kalehoei & Azadbakht, 2017;Awadh et al., 2019;2020). ...
Objective:
Human sperm motility and hyperactivation (HA) are induced by different factors such as intracellular calcium concentration. Repaglinide is an antidiabetic drug that, via the blocking of ATP-sensitive potassium channels (K-ATP channels), depolarization of the β-cell membrane, and opening of the voltage-gated calcium channels leads to an increase in intracellular calcium. The present study aimed to examine the effects of repaglinide on in vitro sperm motility parameters, viability, and DNA integrity in normozoospermic and asthenozoospermic men.
Methods:
Semen samples were collected from two groups of normozoospermic donors and asthenozoospermic patients. The samples were washed free of seminal plasma and then treated with medium alone (control) or with 100 nM and 1µM concentrations of repaglinide. After 1 h of incubation, percent sperm motility and hyperactivation were assessed; after 2 h of incubation, sperm viability and DNA fragmentation rate were evaluated by the Eosin-Y and acridine orange staining, respectively.
Results:
In both groups, repaglinide at a concentration of 100 nM and 1µM significantly improved percent sperm motility, hyperactivation, and vital sperms with normal DNA; in specimens from normozoospermic men, the 1µM concentration had a noticeable effect on progressive motility; in samples from asthenozoospermic men, the highest hyperactivation rate was seen at a concentration of 100 nM as compared with the 1µM concentration and controls (p<0.05).
Conclusions:
Our results suggest that repaglinide can improve sperm motility, hyperactivity, viability, and DNA integrity in both normozoospermic and asthenozoospermic men.
... Assisted reproductive technology (ART) including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) has witnessed huge advancements in recent years to treat patients with infertility (Fernandes et al. 2015). Despite the advancements in ART treatments, the success rates are still sub-optimal and 1 in 7 couples still experiences infertility globally (Calhaz-Jorge et al. 2016). ...
... Disruption of Ca 2+ homeostasis by metabolic stress resulted from in vitro ageing has been linked to certain cellular events that ultimately lead to cell death (Clapham 2007). Therefore, any changes to Ca 2+ dynamics in oocytes might lead to defective signalling events and eventually will lead to failed fertilization and abnormal embryo development (Fernandes et al. 2015). Recently, Fernandes et al. have examined the effects of in vitro stress on Ca 2+ homeostasis in human oocytes, and they found that intracellular Ca 2+ was increased in human oocytes when exposed to in vitro conditions for 2 h (Fernandes et al. 2015). ...
... Therefore, any changes to Ca 2+ dynamics in oocytes might lead to defective signalling events and eventually will lead to failed fertilization and abnormal embryo development (Fernandes et al. 2015). Recently, Fernandes et al. have examined the effects of in vitro stress on Ca 2+ homeostasis in human oocytes, and they found that intracellular Ca 2+ was increased in human oocytes when exposed to in vitro conditions for 2 h (Fernandes et al. 2015). To prevent this increase in intracellular Ca 2+ , drugs targeting the ATP sensitive potassium channels (K ATP ) were applied to oocytes to investigate their cytoprotecting effects. ...
... It has been shown that Cx 26 has a role in the continuation of meiosis and promoting the GV stage to mature oocytes in humans [59]. It is well known that the mammalian oocyte PM is mainly penetrable to K + , Cl -, and partly to Na + and also has the K ATP, Na + /H + , Na + /NH 4 + , Na + /HCO 3 -, Cl -/HCO 3 -, Cl -/OH -, H + /organic cation + , Na + /K + , K + /Mg 2+ exchanges, and Clunidirectional flux activities which have not still reported in human oocytes [60,67,78]. Studies on oocytes showed that hyperpolarized RMP in meiotic arrest and depolarized RMP in withdrawal from the arrested state occur by the actions of gonadotropins on the CCs. ...
Purpose
Oocytes and spermatozoa are electrogenic cells with the ability to respond to electrical stimuli and modulate their electrical properties accordingly. Determination of the ionic events during the gamete maturation helps to design suitable culture media for gametes in assisted reproductive technology (ART). The present systematic review focuses on the electrophysiology of human gametes during different stages of maturation and also during fertilization.
Materials and Methods
The reports published in the English language between January 2000 and July 2021 were extracted from various electronic scientific databases following the PRISMA checklist using specific MeSH keywords.
Results
Subsequent to the screening process with defined inclusion and exclusion criteria, 60 articles have been included in this review. Among them, 11 articles were directly related to the electrophysiology of human oocytes and 49 physiology department to the electrophysiology of human spermatozoa.
Conclusions
Gametes generate electrical currents by ionic exchange, particularly Na+, K+, Cl-, H+, Zn2+, Cu2+, Se2+, Mg2+, HCO3-, and Ca2+ through specific ion channels in different stages of gamete maturation. The ionic concentrations, pH, and other physicochemical variables are modulated during the gametogenesis, maturation, activation, and the fertilization process following gamete function and metabolism. The electrical properties of human gametes change during different stages of maturation. Although it is demonstrated that the electrical properties are significant regulators of cell signaling and are fundamental to gamete maturation and fertilization, their exact roles in these processes are still poorly understood. Further research is required to unveil the intricate electrophysiological processes of human gamete maturation.
... Age-related disorders of oocyte maturation have been suggested to be caused by DNA damage, and this process also indirectly involves the function of chromosome cohesion (Marangos et al., 2015). In other respects, the in vitro processes result in an increase in intracellular Ca 2þ in MII oocytes, and this increase implies cell stress in oocyte, which influences oocyte quality and, potentially, gene expression (Fernandes et al., 2016). At the same time, oocyte derived from older patients seem to be susceptible to various types of stress in vitro (Bittle et al., 2019). ...
Study question:
Is CDC26 a key factor in human oocyte aging?
Summary answer:
The lack of CDC26 disrupts the oocytes maturation process, leading to oocyte aging, but these defects could be partially rescued by overexpression of the CDC26 protein.
What is known already:
Age-related oocyte aging is the main cause of female fertility decline. In mammalian oocytes, aberrant meiosis can cause chromosomal abnormalities that might lead to infertility and developmental disorders. CDC26 participates in the meiosis process.
Study design, size, duration:
Differential gene expression in young and old women oocytes were screened by single-cell RNA-seq technology, and the functions of differentially genes were verified on mouse oocytes. Finally, transfection technology was used to evaluate the effect of a differentially expressed gene in rescuing human oocyte from aging.
Participants/materials, setting, methods:
Discarded human oocytes were collected for single-cell RNA-seq, q-PCR and immunocytochemical analyses to screen for and identify differential gene expression. Female KM mice oocytes were collected for IVM of oocytes, q-PCR and immunocytochemical analyses to delineate the relationships between oocyte aging and differential gene expression. Additionally, recombinant lentiviral vectors encoding CDC26 were transfected into the germinal vesicle oocytes of older women, to investigate the effects of the CDC26 gene expression on oocyte development.
Main results and the role of chance:
Many genes were found to be differentially expressed in the oocytes of young versus old patients via RNA-seq technology. CDC26 mRNA and protein levels in aged oocytes were severely decreased, when compared with the levels observed in young oocytes. Moreover, aged oocytes lacking CDC26 were more prone to aneuploidy. These defects in aged oocytes could be partially rescued by overexpression of the CDC26 protein.
Large scale data:
N/A.
Limitations, reasons for caution:
Our study delineated key steps in the oocyte aging process by identifying the key role of CDC26 in the progression of oocyte maturation. Future studies are required to address whether other signaling pathways play a role in regulating oocyte maturation via CDC26 and which genes are the direct molecular targets of CDC26.
Wider implications of the findings:
Our results using in vitro systems for both mouse and human oocyte maturation provide a proof of principle that CDC26 may represent a novel therapeutic approach against maternal aging-related spindle and chromosomal abnormalities.
Study funding/competing interest(s):
This work was supported by grants from the National Natural Science Foundation of China (81571442 and 81170571), the outstanding Talent Project of Shanghai Municipal Commission of Health (XBR2011067) and Clinical Research and Cultivation Project in Shanghai Municipal Hospitals (SHDC12019X32). The authors declare no conflict of interest.
... Research to date has not yet determined a major causative factor responsible for oocyte impairment in vitro (1). However, many studies have suggested that oocyte quality may be compromised by stimulation regimes, environmental and biological factors among others (1,2). Existing research recognises that oocyte ageing and oxidative stress are the major causes of in vitro oocyte impairment (1). ...
... Fertilization is one of the most critical events in the oocytes in different maturation stages (14). Recently Fernandes et al. have examined the effects of in vitro stress on Ca 2+ homeostasis in human oocytes, and they found that intracellular Ca 2+ was increased in human oocytes when exposed to in vitro conditions (2). To prevent this increase in intracellular Ca 2+ , drugs targeting the ATP sensitive potassium channels (K ATP ) were applied to oocytes to investigate their cytoprotecting effects. ...
... To prevent this increase in intracellular Ca 2+ , drugs targeting the ATP sensitive potassium channels (K ATP ) were applied to oocytes to investigate their cytoprotecting effects. Glibenclamide, a K ATP channel blocker has been shown to reduce and prevent intracellular Ca 2+ loading and thus provide cytoprotection in human oocytes (2). However, no studies have tested Ca 2+ and plasmalemmal membrane potential dynamics in mouse oocytes. ...
Objectives: Ca²⁺ is critical for normal oocyte activation and fertilization, and any alteration to the Ca²⁺ homeostasis may lead to failed fertilization or even cell death. It has been shown that intracellular Ca2+ is increased in bovine and human oocytes when cultured in vitro. Additionally, ATP sensitive potassium channels have been characterised recently in human and Xenopus oocytes Glibenclamide a KATP channel blocker was shown to protect human oocytes from Ca+2 overloading. None of these studies have been conducted in mouse oocytes to determine if they are a suitable alternative to human oocytes in the research setting. Thus, this research note aims to demonstrate if cryopreserved metaphase II (MII) mouse oocytes show similar Ca⁺² and plasmalemmal membrane potential dynamics to those in human oocytes. Also, to show if glibenclamide influences Ca⁺², and plasmalemmal membrane potential in cryopreserved metaphase II mouse oocytes.
Results: our data did not show an increase in intracellular Ca²⁺ in untreated cryopreserved mouse oocytes loaded with Fluo-3 AM dye. However, an increase in the plasmalemmal membrane potential was noticed (hyperpolarization). Glibenclamide has shown no significant effect on Ca+2, mitochondrial and plasmalemmal membrane potential.
... Assisted Reproductive Technology (ART) including In Vitro Fertilization (IVF) and Intracytoplasmic Sperm Injection (ICSI) has witnessed huge advancements in recent years to treat patients with infertility (1). ...
... Despite the advancements in ART treatments, the success rates are still sub-optimal and I in 7 couples still experience infertility globally (1). In ART procedures, maintenance and correct handling of oocytes are critical for successful embryo development and healthy live births Existing research recognises that postovulatory ageing and oxidative stress are the major contributors to the loss of oocyte competence in vitro (2,3). ...
... Disruption of Ca 2+ homeostasis by metabolic stress resulted from in vitro ageing has been linked to certain cellular events that ultimately lead to cell death (8). Therefore, any changes to Ca 2+ dynamics in oocytes might lead to defective signalling events and eventually will lead to failed fertilisation and abnormal embryo development (1). ...
Objectives: Ca²⁺ is critical for normal oocyte activation and fertilization, and any alteration to the Ca²⁺ homeostasis may lead to failed fertilization or even cell death. It has been shown that intracellular Ca2+ is increased in bovine and human oocytes when cultured in vitro. Additionally, ATP sensitive potassium channels have been characterised recently in human and Xenopus oocytes. Glibenclamide a KATP channel blocker was shown to protect human oocytes from Ca+2 overloading via inhibition of plasmalemmal KATP channels. This research note aims to demonstrate the effects of oxidative stress and in vitro ageing on the intracellular Ca⁺² and plasmalemmal membrane potential dynamics in cryopreserved metaphase II (MII) mouse oocytes. Also, this study aims to show if glibenclamide (a KATP channel blocker ) has a role in regulating intracellular Ca⁺² and plasmalemmal membrane potential through KATP channels in cryopreserved metaphase II mouse oocytes.
Results: our data did not show an increase in intracellular Ca²⁺ in untreated cryopreserved mouse oocytes loaded with Fluo-3 AM dye. However, an increase in the plasmalemmal membrane potential was noticed (hyperpolarization). Glibenclamide has shown no significant effect on Ca²⁺ and plasmalemmal membrane potential.
... It was found, in this study, that dietary Ca deficiency induced long-term accumulation of Ca 2þ in ovarian tissue despite plasma Ca being decreased by dietary Ca depletion (Chen et al., 2015). Intracellular Ca 2þ concentration is kept at low levels in quiescent oocytes, but transient elevations (oscillations) of intracellular Ca 2þ i are required for oocyte meiotic resumption (maturation) and fertilization in various animal species (Fernandes et al., 2009;Cheon et al., 2013). Once triggered by FSH or fertilization, cytoplasmic Ca 2þ re-enters the endoplasmic reticulum through the endoplasmic reticulum Ca 2þ ATPase pump, activates Ca/calmodulindependent kinase II and triggers meiotic resumption through degradation of the maturation promoting factor regulatory unit (cyclin B) (Nunes et al., 2015). ...
Ovarian follicle selection is a natural biological process in the pre-ovulatory hierarchy in birds that drives growing follicles to be selected within the ovulatory cycle. Follicle selection in birds is strictly regulated, involving signaling pathways mediated by dietary nutrients, gonadotrophic hormones and paracrine factors. This study aimed to test the hypothesis that dietary Ca may participate in regulating follicle selection in laying ducks through activating the signaling pathway of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/extracellular signal-regulated kinase (ERK), possibly mediated by gonadotrophic hormones. Female ducks at 22 weeks of age were initially fed one of two Ca-deficient diets (containing 1.8% or 0.38% Ca) or a Ca-adequate control diet (containing 3.6% Ca) for 67 days (depletion period), then all birds were fed the Ca-adequate diet for an additional 67 days (repletion period). Compared with the Ca-adequate control, ducks fed 0.38% Ca during the depletion period had significantly decreased (P < 0.05) numbers of hierarchical follicles and total ovarian weight, which were accompanied by reduced egg production. Plasma concentration of FSH was decreased by the diet containing 1.8% Ca but not by that containing 0.38%. The ovarian content of cAMP was increased with the two Ca-deficient diets, and phosphorylation of PKA and ERK1/2 was increased with 0.38% dietary Ca. Transcripts of ovarian estradiol receptor 2 and luteinizing hormone receptor (LHR) were reduced in the ducks fed the two Ca-deficient diets (P < 0.05), while those of the ovarian follicle stimulating hormone receptor (FSHR) were decreased in the ducks fed 0.38% Ca. The transcript abundance of ovary gap junction proteins, A1 and A4, was reduced with the Ca-deficient diets (P < 0.05). The down-regulation of gene expression of gap junction proteins and hormone receptors, the increased cAMP content and the suppressed hierarchical follicle numbers were reversed by repletion of dietary Ca. These results indicate that dietary Ca deficiency negatively affects follicle selection of laying ducks, independent of FSH, but probably by activating cAMP/PKA/ERK1/2 signaling pathway.
... It was found, in this study, that dietary Ca deficiency induced long-term accumulation of Ca 2þ in ovarian tissue despite plasma Ca being decreased by dietary Ca depletion (Chen et al., 2015). Intracellular Ca 2þ concentration is kept at low levels in quiescent oocytes, but transient elevations (oscillations) of intracellular Ca 2þ i are required for oocyte meiotic resumption (maturation) and fertilization in various animal species (Fernandes et al., 2009;Cheon et al., 2013). Once triggered by FSH or fertilization, cytoplasmic Ca 2þ re-enters the endoplasmic reticulum through the endoplasmic reticulum Ca 2þ ATPase pump, activates Ca/calmodulindependent kinase II and triggers meiotic resumption through degradation of the maturation promoting factor regulatory unit (cyclin B) (Nunes et al., 2015). ...
Ovarian follicle selection is a natural biological process in the pre-ovulatory hierarchy in birds that drives growing follicles to be selected within the ovulatory cycle. Follicle selection in birds is strictly regulated, involving signaling pathways mediated by dietary nutrients, gonadotrophic hormones and paracrine factors. This study aimed to test the hypothesis that dietary Ca may participate in regulating follicle selection in laying ducks through activating the signaling pathway of cyclic adenosine monophosphate ( cAMP )/protein kinase A ( PKA )/extracellular signal-regulated kinase ( ERK ), possibly mediated by gonadotrophic hormones. Female ducks at 22 weeks of age were initially fed one of two Ca-deficient diets (containing 1.8% or 0.38% Ca) or a Ca-adequate control diet (containing 3.6% Ca) for 67 days (depletion period), then all birds were fed the Ca-adequate diet for an additional 67 days (repletion period). Compared with the Ca-adequate control, ducks fed 0.38% Ca during the depletion period had significantly decreased ( P < 0.05) numbers of hierarchical follicles and total ovarian weight, which were accompanied by reduced egg production. Plasma concentration of FSH was decreased by the diet containing 1.8% Ca but not by that containing 0.38%. The ovarian content of cAMP was increased with the two Ca-deficient diets, and phosphorylation of PKA and ERK1/2 was increased with 0.38% dietary Ca. Transcripts of ovarian estradiol receptor 2 and luteinizing hormone receptor ( LHR ) were reduced in the ducks fed the two Ca-deficient diets ( P < 0.05), while those of the ovarian follicle stimulating hormone receptor ( FSHR ) were decreased in the ducks fed 0.38% Ca. The transcript abundance of ovary gap junction proteins, A1 and A4, was reduced with the Ca-deficient diets ( P < 0.05). The down-regulation of gene expression of gap junction proteins and hormone receptors, the increased cAMP content and the suppressed hierarchical follicle numbers were reversed by repletion of dietary Ca. These results indicate that dietary Ca deficiency negatively affects follicle selection of laying ducks, independent of FSH, but probably by activating cAMP/PKA/ERK1/2 signaling pathway.
... KATP channel inhibitory drugs such repaglinide by an effect on voltage-dependent Ca 2+ channel causes to depolarization of cells membrane and increasing of intracellular calcium. 37 Fernandes and his workers (16) when they study human MII oocyte indicated that human oocytes maintain Ca2+ homeostasis with difficulty when exposed to routine in vitro conditions, which could interfere with fertilization. Both inhibition and activation of KATP channels is useful for maintaining Ca 2+ homeostasis in oocytes under in vitro conditions. ...
The development of in vitro culture systems that result to preantral follicles growth and increasing of developmental competency of oocytes obtained from follicles has an important role in fertility preservation and assisted reproductive techniques. In this research, we evaluated the effect of repaglinide on in vitro growth and maturation of preantral follicles. Preantral follicles were isolated from 12-14 day-old female NMRI mice ovaries and cultured for 12 days cultured in α-MEM (Control), α-MEM supplemented with 1µM of repaglinide. Follicles examined for development on 1, 3, 6, 9, 12 days of culture. At the end of culture period after HCG administration in vitro oocyte maturation was assessed. Results showed that in vitro follicle growth, survival, density of granulosa cells and steroidogenic activity were higher than the control group (p less than 0.05). In vitro maturation rate in oocytes derived from follicles in the treatment group was higher than control group (p less than 0.05). Therefore the supplementation of the culture medium with repaglinide can improve the ovarian follicle survival, growth and subsequently in vitro oocyte maturation.
... Pinacidil is a nonselective opener of ATP-sensitive K + (KATP) channels, including the plasma membrane KATP (pmKATP) and mitochondria KATP (mitoKATP) channels. This agent has been shown to have significant cardioprotective and vasorelaxant effects due to shortening the action potential duration (APD) and hyperpolarizing the membrane potential during cardiac ischemia/reperfusion injury, hypertension, and arrhythmia (Spinelli et al. 1991;Carlsson et al. 1992;Fernandes et al. 2016). Pinacidil also abolishes early afterdepolarizations, delayed afterdepolarizations (DADs), and abnormal automaticity in dog and rabbit hearts (Spinelli et al. 1991;Carlsson et al. 1992). ...
Pinacidil, a nonselective ATP-sensitive K⁺ (KATP) channel opener, has cardioprotective effects for hypertension, ischemia/reperfusion injury, and arrhythmia. This agent abolishes early afterdepolarizations, delayed afterdepolarizations (DADs), and abnormal automaticity in canine cardiac ventricular myocytes. DADs are well known to be caused by the Na⁺/Ca²⁺ exchange current (INCX). In this study, we used the whole-cell patch-clamp technique and Fura-2/AM (Ca²⁺-indicator) method to investigate the effect of pinacidil on INCX in isolated guinea pig cardiac ventricular myocytes. In the patch-clamp study, pinacidil enhanced INCX in a concentration-dependent manner. The half-maximal effective concentration values were 23.5 and 23.0 μM for the Ca²⁺ entry (outward) and Ca²⁺ exit (inward) components of INCX, respectively. The pinacidil-induced INCX increase was blocked by L-NAME, a nitric oxide (NO) synthase inhibitor, by ODQ, a soluble guanylate cyclase inhibitor, and by KT5823, a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor, but not by N-2-mercaptopropyonyl glycine (MPG), a reactive oxygen species (ROS) scavenger. Glibenclamide, a nonselective KATP channel inhibitor, blocked the pinacidil-induced INCX increase, while 5-HD, a selective mitochondria KATP channel inhibitor, did not. In the Fura-2/AM study pinacidil also enhanced intracellular Ca²⁺ concentration, which was inhibited by L-NAME, ODQ, KT5823, and glibenclamide, but not by MPG and 5-HD. Sildenafil, a phosphodiesterase 5 inhibitor, increased further the pinacidil-induced INCX increase. Sodium nitroprusside, a NO donor, also increased INCX. In conclusion, pinacidil may stimulate cardiac Na⁺/Ca²⁺ exchanger (NCX1) by opening plasma membrane KATP channels and activating the NO/cGMP/PKG signaling pathway.
