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New Insight into Sperm Capacitation: A Novel Mechanism of 17?-Estradiol Signalling.

ABSTRACT: 17?-estradiol (estradiol) is a natural estrogen regulating reproduction including sperm and egg development, sperm maturation-called capacitation-and sperm?egg communication. High doses can increase germ cell apoptosis and decrease sperm count. Our aim was to answer the biological relevance of estradiol in sperm capacitation and its effect on motility and acrosome reaction to quantify its interaction with estrogen receptors and propose a model of estradiol action during capacitation using kinetic analysis. Estradiol increased protein tyrosine phosphorylation, elevated rate of spontaneous acrosome reaction, and altered motility parameters measured Hamilton-Thorne Computer Assisted Semen Analyzer (CASA) in capacitating sperm. To monitor time and concentration dependent binding dynamics of extracellular estradiol, high-performance liquid chromatography with tandem mass spectrometry was used to measure sperm response and data was subjected to kinetic analysis. The kinetic model of estradiol action during sperm maturation shows that estradiol adsorption onto a plasma membrane surface is controlled by Langmuir isotherm. After, when estradiol passes into the cytoplasm, it forms an unstable adduct with cytoplasmic receptors, which display a signalling autocatalytic pattern. This autocatalytic reaction suggests crosstalk between receptor and non-receptor pathways utilized by sperm prior to fertilization.

SUBMITTER: Bosakova T

PROVIDER: S-EPMC6321110 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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New Insight into Sperm Capacitation: A Novel Mechanism of 17β-Estradiol Signalling.

Bosakova Tereza T Tockstein Antonin A Sebkova Natasa N Simonik Ondrej O Adamusova Hana H Albrechtova Jana J Albrecht Tomas T Bosakova Zuzana Z Dvorakova-Hortova Katerina K

International journal of molecular sciences 20181212 12

17β-estradiol (estradiol) is a natural estrogen regulating reproduction including sperm and egg development, sperm maturation-called capacitation-and sperm⁻egg communication. High doses can increase germ cell apoptosis and decrease sperm count. Our aim was to answer the biological relevance of estradiol in sperm capacitation and its effect on motility and acrosome reaction to quantify its interaction with estrogen receptors and propose a model of estradiol action during capacitation using kineti ...[more]

PMID: 30545117

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Project description:BackgroundDue to the unique nature of spermatozoa, which are transcriptionally and translationally silent, the regulation of capacitation is based on the formation of posttranslational modifications of proteins (PTMs). However, the interactions between different types of PTMs during the capacitation remain unclear. Therefore, we aimed to unravel the PTM-based regulation of sperm capacitation by considering the relationship between tyrosine phosphorylation and reversible oxidative PTMs (oxPTMs), i.e., S-nitrosylation and S-glutathionylation. Since reversible oxPTMs may be closely related to peroxyredoxin (PRDX) activity, the second aim was to verify the role of PRDXs in the PTM-based regulation of capacitation.MethodsCryopreserved bull sperm were capacitated in vitro with or without PRDX inhibitor. Qualitative parameters of sperm and symptoms characteristic of capacitation were analyzed. Posttranslational protein modifications (S-nitrosylation, S-glutathionylation, tyrosine phosphorylation) were investigated at the cellular level (flow cytometry, fluorescence microscopy) and at the proteomic level (fluorescent gel-based proteomic approach).ResultsZona-pellucida binding proteins (ACRBP, SPAM1, ZAN, ZPBP1 and IZUMO4) were particularly rich in reversible oxPTMs. Moreover, numerous flagellar proteins were associated with all analyzed types of PTMs, which indicates that the direction of posttranslational modifications was integrated. Inhibition of PRDX activity during capacitation caused an increase in S-nitrosylation and S-glutathionylation and a decrease in tyrosine phosphorylation. Inhibition of PRDXs caused GAPDHS to undergo S-glutathionylation and the GSTO2 and SOD2 enzymes to undergo denitrosylation. Moreover, PRDX inhibition caused the AKAP proteins to be dephosphorylated.ConclusionsOur research provides evidence that crosstalk occurs between tyrosine phosphorylation and reversible oxPTMs during bull sperm capacitation. This study demonstrates that capacitation triggers S-nitrosylation and S-glutathionylation (and reverse reactions) of zona-pellucida binding proteins, which may be a new important mechanism that determines the interaction between sperms and oocytes. Moreover, TCA-related and flagellar proteins, which are particularly rich in PTMs, may play a key role in sperm capacitation. We propose that the deglutathionylation of ODFs and IZUMO4 proteins is a new hallmark of bull sperm capacitation. The obtained results indicate a relationship between PRDX activity and protein phosphorylation, S-glutathionylation and S-nitrosylation. The activity of PRDXs may be crucial for maintaining redox balance and for providing proper PKA-mediated protein phosphorylation during capacitation. Video Abstract.

Project description:Sperm capacitation is a complex and indispensable physiological process that spermatozoa must undergo in order to acquire fertilization capability. Spermatozoa from several mammalian species, including mice, exhibit a capacitation-associated plasma membrane hyperpolarization, which is necessary for the acrosome reaction to occur. Despite its importance, this hyperpolarization event has not been adequately examined in human sperm. In this report we used flow cytometry to show that a subpopulation of human sperm indeed undergo a plasma membrane hyperpolarization upon in vitro capacitation. This hyperpolarization correlated with two other well-characterized capacitation parameters, namely an increase in intracellular pH and Ca(2+) concentration, measured also by flow cytometry. We found that sperm membrane hyperpolarization was completely abolished in the presence of a high external K(+) concentration (60 mM), indicating the participation of K(+) channels. In order to identify, which of the potential K(+) channels were involved in this hyperpolarization, we used different K(+) channel inhibitors including charybdotoxin, slotoxin and iberiotoxin (which target Slo1) and clofilium (a more specific blocker for Slo3). All these K(+) channel antagonists inhibited membrane hyperpolarization to a similar extent, suggesting that both members of the Slo family may potentially participate. Two very recent papers recorded K(+) currents in human sperm electrophysiologically, with some contradictory results. In the present work, we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition, we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 μM). Considered altogether, our data indicate that Slo1 and Slo3 could share the preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the hyperpolarization event.

Project description:Study questionWhy are many sperm required for successful fertilization of oocytes in vitro, even though fertilization occurs in vivo when only a few sperm reach the oocyte?Summary answerCreatine produced in the ovary promotes efficient fertilization in vivo; however, in vitro, creatine is not contained in the in vitro fertilization (IVF) medium.What is known alreadyThe IVF medium enables capacitation of sperm. However, the IVF medium does not fully mimic the in vivo environment during fertilization. Consequently, fertilization in vitro is more inefficient than in the oviduct.Study design, size, durationFollicular and oviductal fluids were collected and then analyzed for creatine and glucose levels. To determine the physiological functions of creatine, the creatine antagonist 3-guanidinopropionic acid (GPA) was injected into hormonally primed mice. Using conventional IVF protocols, sperm were pre-incubated in IVF medium with creatine and then co-cultured with 10 ovulated cumulus-oocyte complexes (1-1000 per oocyte) in 50 μl medium droplets.Participants/materials, setting, methodsGlucose and creatine levels were measured using commercial enzymatic assay kits. The effect of creatine in vivo was assessed by mating experiments using mice treated with or without GPA just before ovulation. To assess the functions of sperm incubated in IVF medium containing creatine, we analyzed (1) the motility of sperm using computer-assisted sperm assay, (2) the capacitation level of sperm by western blot analyses, and (3) the condition of sperm acrosomes by peanut agglutinin lectin-FITC staining.Main results and the role of chanceOviductal creatine levels were significantly increased following ovulation. Injecting mice with GPA just before ovulation significantly reduced the number of fertilized oocytes. The addition of creatine to IVF medium enhanced sperm capacitation by increasing ATP levels. Successful fertilization was achieved with as few as five sperm/oocyte in the creatine group, and the number of fertilized oocytes was significantly higher than in the control without creatine (P < 0.01).Limitations, reasons for cautionIn the present study, a pharmacological approach, creatine antagonist (GPA) treatment, but not a knockout mouse model, was used to understand the role of creatine in vivo. The role of creatine in fertilization processes can only be shown in a mouse model.Wider implications of the findingsA modified IVF technique using creatine-containing medium was developed and shown to markedly improve fertilization with small numbers of sperm. This approach has the potential to be highly beneficial for human assisted reproductive technologies, especially for patients with a limited number of good quality sperm.Study funding/competing interest(s)This work was supported in part by JSPS KAKENHI Grant numbers JP24688028, JP16H05017 (to M.S.), and JP15J05331 (to T.U.), the Japan Agency for Medical Research and Development (AMED) (16gk0110015h0001 to M.S.), and National Institutes of Health (NIH-HD-076980 to J.S.R). The authors have nothing to disclose.

Dataset Information

New Insight into Sperm Capacitation: A Novel Mechanism of 17?-Estradiol Signalling.

Publications

New Insight into Sperm Capacitation: A Novel Mechanism of 17β-Estradiol Signalling.

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