RESUMO
Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.
Assuntos
Venenos Elapídicos/química , Peptídeos/química , Peptídeos/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Cromatografia por Troca Iônica , Dissulfetos/química , Egito , Venenos Elapídicos/farmacologia , Elapidae , Humanos , Macaca fascicularis , Masculino , Camundongos Endogâmicos , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Cauda do Espermatozoide/química , Cauda do Espermatozoide/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Espectrometria de Massas em TandemRESUMO
Male infertility is a global problem in modern society of which capacitating defects are a major cause. Previous studies have demonstrated that Ca2+ ionophore A23187 can make mouse sperm capable of fertilizing in vitro, which may aid in clinical treatment of capacitating defects. However, the detailed role and mechanism of Ca2+ in the capacitating process are still unclear especially how A23187 quickly renders sperm immotile and inhibits cAMP/PKA-mediated phosphorylation. We report that A23187 induces a Ca2+ flux in the mitochondria enriched sperm tail and excess Ca2+ inhibits key metabolic enzymes involved in acetyl-CoA biosynthesis, TCA cycle and electron transport chain pathways resulting in reduced ATP and overall energy production, however this flux does not destroy the structure of the sperm tail. Due to the decrease in ATP production, which is the main phosphate group donator and the power of sperm, the sperm is rendered immobile and PKA-mediated phosphorylation is inhibited. Our study proposed a possible mechanism through which A23187 reduces sperm motility and PKA-mediated phosphorylation from ATP generation, thus providing basic data for exploring the functional roles of Ca2+ in sperm in the future.
Assuntos
Trifosfato de Adenosina/biossíntese , Calcimicina/farmacologia , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ionóforos/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Acetilcoenzima A/biossíntese , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Capacitação Espermática/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/ultraestruturaRESUMO
Excessive fluoride (F) ingestion via drinking water interfered with spermatogenesis and lowered sperm quality of human and animals. However, it is still unclear why the effects of fluoride on sperm quality focus on mostly sperm motility rather than sperm count. The objective of this study is to investigate the potential relationship between alteration in the structure and function of sperm flagellum and fluoride exposure in the environment. 40 male mice were allocated to four groups which were treated with 0, 25, 50, 100 mg/L NaF deionized water, respectively, for 8 weeks continuously. The testicular morphology, ultra-structure of fibrous sheath and axoneme of sperm flagellum, and eleven key function genes Akap3, Akap4, Dnah1, Eno4, Cfap43, Cfap44, Hydin, Spef2, Spag6, Spag16, and Cfap69 were examined by histology, transmission electron microscopy, and real-time PCR methods respectively. The results displayed that fluoride damaged the typical "9 + 2â³ microtubule structure including fibrous sheathes and axoneme of sperm flagellum in testes of mice. Furthermore, the mRNA and protein expression levels of AKAP3 and AKAP4 related to fibrous sheathes formation, and CFAP43, CFAP44 and HYDIN in axoneme were down-regulated by fluoride exposure. Taken together, we revealed that fluoride altered the structures of the fibrous sheathes and axonemal in sperm flagellum via down-regulating the mRNA and protein expression levels of AKAP3, AKAP4, CFAP43, CFAP44, and HYDIN, which may be one of the reasons that fluoride lowered sperm quality and male reproductive function.
Assuntos
Fluoretos/toxicidade , Cauda do Espermatozoide/ultraestrutura , Testículo/metabolismo , Proteínas de Ancoragem à Quinase A , Animais , Dineínas , Poluentes Ambientais , Fluoretos/metabolismo , Humanos , Infertilidade Masculina , Masculino , Camundongos , Proteínas dos Microtúbulos , Fenótipo , RNA Mensageiro/metabolismo , Motilidade dos Espermatozoides , Cauda do Espermatozoide/efeitos dos fármacos , Espermatogênese , Espermatozoides/metabolismoRESUMO
Sperm are propelled by bending waves traveling along their flagellum. For steering in gradients of sensory cues, sperm adjust the flagellar waveform. Symmetric and asymmetric waveforms result in straight and curved swimming paths, respectively. Two mechanisms causing spatially asymmetric waveforms have been proposed: an average flagellar curvature and buckling. We image flagella of human sperm tethered with the head to a surface. The waveform is characterized by a fundamental beat frequency and its second harmonic. The superposition of harmonics breaks the beat symmetry temporally rather than spatially. As a result, sperm rotate around the tethering point. The rotation velocity is determined by the second-harmonic amplitude and phase. Stimulation with the female sex hormone progesterone enhances the second-harmonic contribution and, thereby, modulates sperm rotation. Higher beat frequency components exist in other flagellated cells; therefore, this steering mechanism might be widespread and could inspire the design of synthetic microswimmers.
Assuntos
Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/fisiologia , Espermatozoides/fisiologia , Fenômenos Biofísicos , Feminino , Humanos , Masculino , Modelos Biológicos , Periodicidade , Análise de Componente Principal , Progesterona/farmacologia , Progesterona/fisiologia , Rotação , Microscopia de Geração do Segundo Harmônico , Motilidade dos Espermatozoides/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Espermatozoides/efeitos dos fármacosRESUMO
STUDY QUESTION: Are there intracellular Ca2+ ([Ca2+]i) oscillations correlated with flagellar beating in human sperm? SUMMARY ANSWER: The results reveal statistically significant [Ca2+]i oscillations that are correlated with the human sperm flagellar beating frequency, when measured in three-dimensions (3D). WHAT IS KNOWN ALREADY: Fast [Ca2+]i oscillations that are correlated to the beating flagellar frequency of cells swimming in a restricted volume have been detected in hamster sperm. To date, such findings have not been confirmed in any other mammalian sperm species. An important question that has remained regarding these observations is whether the fast [Ca2+]i oscillations are real or might they be due to remaining defocusing effects of the Z component arising from the 3D beating of the flagella. STUDY DESIGN, SIZE, DURATION: Healthy donors whose semen samples fulfill the WHO criteria between the age of 18-28 were selected. Cells from at least six different donors were utilized for analysis. Approximately the same number of experimental and control cells were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Motile cells were obtained by the swim-up technique and were loaded with Fluo-4 (Ca2+ sensitive dye) or with Calcein (Ca2+ insensitive dye). Ni2+ was used as a non-specific plasma membrane Ca2+ channel blocker. Fluorescence data and flagella position were acquired in 3D. Each cell was recorded for up to 5.6 s within a depth of 16 microns with a high speed camera (coupled to an image intensifier) acquiring at a rate of 3000 frames per second, while an oscillating objective vibrated at 90 Hz via a piezoelectric device. From these samples, eight experimental and nine control sperm cells were analyzed in both 2D and 3D. MAIN RESULTS AND THE ROLE OF CHANCE: We have implemented a new system that allows [Ca2+]i measurements of the human sperm flagellum beating in 3D. These measurements reveal statistically significant [Ca2+]i oscillations that correlate with the flagellar beating frequency. These oscillations may arise from intracellular sources and/or Ca2+ transporters, as they were insensitive to external Ni2+, a non-specific plasma membrane Ca2+ channel blocker. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Analysis in 3D needs a very fast image acquisition rate to correctly sample a volume containing swimming sperm. This condition requires a very short exposure time per image making it necessary to use an image intensifier which also increases noise. The lengthy analysis time required to obtain reliable results limited the number of cells that could be analyzed. WIDER IMPLICATIONS OF THE FINDINGS: The possibility of recording flagellar [Ca2+]i oscillations described here may open a new avenue to better understand ciliary and flagellar beating that are fundamental for mucociliary clearance, oocyte transport, fertilization, cerebrospinal fluid pressure regulation and developmental left-right symmetry breaking in the embryonic node. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) (grants 253952 to G.C.; 156667 to F.M.M. and Fronteras 71 39908-Q to A.D. and Post-doctoral scholarships 366844 to P.H.-H. and 291028 to F.M.) and the Dirección General de Asuntos del Personal Académico of the Universidad Nacional Autónoma de México (DGAPA-UNAM) (grants CJIC/CTIC/4898/2016 to F.M. and IN205516 to A.D.). There are no conflicts of interest to declare.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Imageamento Tridimensional/métodos , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/fisiologia , Espermatozoides/fisiologia , Adolescente , Adulto , Compostos de Anilina/química , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Imageamento Tridimensional/instrumentação , Masculino , Níquel/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/ultraestrutura , Espermatozoides/efeitos dos fármacos , Espermatozoides/ultraestrutura , Gravação em Vídeo , Xantenos/químicaRESUMO
Male infertility due to abnormal spermatozoa has been reported in both animals and humans, but its pathogenic causes, including genetic abnormalities, remain largely unknown. On the other hand, contraceptive options for men are limited, and a specific, reversible and safe method of male contraception has been a long-standing quest in medicine. Some progress has recently been made in exploring the effects of spermatid-specifical genetic factors in controlling male fertility. A comprehensive search of PubMed for articles and reviews published in English before July 2016 was carried out using the search terms 'spermiogenesis failure', 'globozoospermia', 'spermatid-specific', 'acrosome', 'infertile', 'manchette', 'sperm connecting piece', 'sperm annulus', 'sperm ADAMs', 'flagellar abnormalities', 'sperm motility loss', 'sperm ion exchanger' and 'contraceptive targets'. Importantly, we have opted to focus on articles regarding spermatid-specific factors. Genetic studies to define the structure and physiology of sperm have shown that spermatozoa appear to be one of the most promising contraceptive targets. Here we summarize how these spermatid-specific factors regulate spermiogenesis and categorize them according to their localization and function from spermatid head to tail (e.g., acrosome, manchette, head-tail conjunction, annulus, principal piece of tail). In addition, we emphatically introduce small-molecule contraceptives, such as BRDT and PPP3CC/PPP3R2, which are currently being developed to target spermatogenic-specific proteins. We suggest that blocking the differentiation of haploid germ cells, which rarely affects early spermatogenic cell types and the testicular microenvironment, is a better choice than spermatogenic-specific proteins. The studies described here provide valuable information regarding the genetic and molecular defects causing male mouse infertility to improve our understanding of the importance of spermatid-specific factors in controlling fertility. Although a male contraceptive 'pill' is still many years away, research into the production of new small-molecule contraceptives targeting spermatid-specific proteins is the right avenue.
Assuntos
Anticoncepcionais/farmacologia , Fertilidade/fisiologia , Cabeça do Espermatozoide/fisiologia , Cauda do Espermatozoide/fisiologia , Espermátides/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Masculino , Cabeça do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Espermátides/efeitos dos fármacosRESUMO
It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.
Assuntos
Microtúbulos/fisiologia , Cauda do Espermatozoide/fisiologia , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/fisiologia , Animais , Axonema/efeitos dos fármacos , Axonema/fisiologia , Axonema/ultraestrutura , Cálcio/farmacologia , Bovinos , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Movimento (Física) , Elastase Pancreática/farmacologia , Ouriços-do-Mar , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/ultraestruturaRESUMO
Speract, a peptide from the egg jelly coat of certain sea urchin species, modulates sperm motility through a signaling pathway involving several ionic fluxes leading to pHi and [Ca²âº]i increases. [Ca²âº]i oscillations in the flagellum regulate its beating pattern modulating sperm swimming. Recent evidence showed the importance of pHi in controlling Ca²âº influx and chemotaxis. However, spatio-temporal characterization of the flagellar pHi increase triggered by speract, and its correlation to that of [Ca²âº]i is lacking. Here, we show for the first time in single sea urchin spermatozoa that the speract-induced flagellar pHi increase precedes and is independent of [Ca²âº]i increase. Our results support a leading role of pHi in modulating the Ca²âº signals that govern sperm swimming.
Assuntos
Sinalização do Cálcio , Citoplasma/metabolismo , Oligopeptídeos/metabolismo , Ouriços-do-Mar/fisiologia , Cauda do Espermatozoide/metabolismo , Espermatozoides/citologia , Álcalis/farmacologia , Cloreto de Amônio/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Cinética , Masculino , Análise de Célula Única , Cabeça do Espermatozoide/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismoRESUMO
Our previous studies revealed that in bank vole testicular cells octylphenol (OP) action is related either to its binding to estrogen receptors or increasing cAMP level that modulates spermatozoa motility and acrosome reaction (AR), (Kotula-Balak et al., 2011, 2014). To better understand the mechanisms underlying these changes, in the present study we aimed at evaluating the glycosylation pattern, calcium (Ca(2+)) level and ultrastructure of OP-treated vole spermatozoa. Glycans were recognized by lectins and localized for the first time on the surface of acrosomal cap and tail of vole spermatozoa. Expression and localization of glycans were determined histochemically and analyzed quantitatively. Following OP the expression of glycans markedly changed with concomitant increase of intracellular Ca(2+) concentration. Altered Ca(2+) signaling pathway and ultrastructural changes in sperm acrosome region were revealed by immunoenzymatic assay and electron microscope analysis together with hypo-osmotic swelling test, respectively. In detail, AR advancement reflected by the presence of small vesicles in the close vicinity to the sperm head was observed, while tail membrane integrity remained intact. Our study provides a new insight on OP direct effects on precocious AR of vole spermatozoa through modulation of the glycan expression and its distribution concomitantly with changes in Ca(2+) signaling pathway and acrosome ultrastructure in vitro.
Assuntos
Arvicolinae/fisiologia , Cálcio/metabolismo , Glicosilação/efeitos dos fármacos , Fenóis/farmacologia , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Animais , Membrana Celular , Técnicas In Vitro , Lectinas/metabolismo , Masculino , Pressão Osmótica , Cauda do Espermatozoide/efeitos dos fármacos , Espermatozoides/efeitos dos fármacosRESUMO
Most animal sperm are quiescent in the male reproductive tract and become activated after mixing with accessory secretions from the male and/or female reproductive tract. Sperm from the mosquito Culex quinquefasciatus initiate flagellar motility after mixing with male accessory gland components, and the sperm flagellum displays three distinct motility patterns over time: a low amplitude, a long wavelength form (Wave A), a double waveform consisting of two superimposed waveforms over the length of the flagellum (Wave B), and finally, a single helical waveform that propels the sperm at high velocity (Wave C). This flagellar behavior is replicated by treating quiescent sperm with trypsin. When exposed to either broad spectrum or tyrosine kinase inhibitors, sperm activated by accessory gland secretions exhibited motility through Wave B but were unable to progress to Wave C. The MEK1/2 inhibitor UO126 and the ERK1/2 inhibitor FR180204 each blocked the transition from Wave B to Wave C, indicating a role for MAPK activity in the control of waveform and, accordingly, progressive movement. Furthermore, a MAPK substrate antibody stained the flagellum of activated sperm. In the absence of extracellular Ca(2+), a small fraction of sperm swam backwards, whereas most could not be activated by either accessory glands or trypsin and were immotile. However, the phosphatase inhibitor okadaic acid in the absence of extracellular Ca(2+) induced all sperm to swim backwards with a flagellar waveform similar to Wave A. These results indicate that flagellar waveform generation and direction of motility are controlled by protein phosphorylation and Ca(2+) levels, respectively.
Assuntos
Cálcio/metabolismo , Culex/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/metabolismo , Animais , Cálcio/farmacologia , Culex/citologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Masculino , Fosforilação , Pirazóis/farmacologia , Piridazinas/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Fatores de TempoRESUMO
Mature and healthy male house rats, Rattus rattus (n= 160) were fed on bait (cracked wheat: powdered sugar, 98:2) containing different concentrations of triptolide (0.1, 0.05, 0.025 and 0%) for 7 and 14 days in no-choice and bi-choice feeding tests in the laboratory. The objective of the study was to record the antifertility affects of triptolide after 30 and 60 days of termination of treatment. Results revealed no significant effect of triptolide treatment on weights of testis, epididymis, seminal vesicles and prostate gland of rats. Overall, sperm motility, live sperm count, sperm density and sperm morphology in the cauda epididymal fluid were found to differ significantly (P≤ 0.05) between untreated and treated groups of rats. The major effect of triptolide on sperm morphology was in the form of sperm head tail separation, which was up to 56.0% in rats treated for 14 days in no-choice and autopsied after 30 days. A significant effect (P≤ 0.05) of triptolide treatment was observed on the histomorphology of the testis, which included a dose-dependent decrease in diameter of seminiferous tubules, thickness of germinal epithelium and numbers of various spermatogenic cells. Cell associations in the seminiferous epithelial cycle were poorly developed in rats ingesting medium (4.7-5.1 mg/100 g bw) and high doses (6.9-7.2 mg/100 g bw) of triptolide than rats ingesting low doses (1.8-2.3 mg/100 g bw) and untreated rats. The cell stages affected had not recovered fully within the 60 day period following triptolide withdrawal. The present study suggests the potential of triptolide in reproductive management of Rattus rattus.
Assuntos
Antiespermatogênicos/farmacologia , Diterpenos/farmacologia , Fertilidade/efeitos dos fármacos , Fenantrenos/farmacologia , Ratos/fisiologia , Controle de Roedores/métodos , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Antiespermatogênicos/administração & dosagem , Diterpenos/administração & dosagem , Relação Dose-Resposta a Droga , Epididimo/anatomia & histologia , Compostos de Epóxi/administração & dosagem , Compostos de Epóxi/farmacologia , Índia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fenantrenos/administração & dosagem , Próstata/anatomia & histologia , Glândulas Seminais/anatomia & histologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Espermatozoides/citologia , Testículo/anatomia & histologiaRESUMO
The function of Ca(2+) and cAMP in extruding doublet microtubules from sea urchin sperm axoneme and generating flagellar waves was investigated in order to clarify the regulatory mechanism of microtubule sliding and the formation mechanism of beating patterns of cilia and flagella. Almost all potentially asymmetric spermatozoa that were demembranated with Triton in the absence of Ca(2+) and reactivated with MgATP(2-) (Gibbons, B.H. and Gibbons, I.R. (1980). J. Cell Biol., 84: 13-27), beat with planar waves closely resembling those of the intact spermatozoa, whereas potentially symmetric spermatozoa, in which axonemal calmodulin was removed by detergent extraction in the presence of millimolar Ca(2+) (Brokaw, C.J. and Nagayama, S.M. (1985). J. Cell Biol., 100: 1875-1883), beat with three-dimensional waves if they were reactivated with low MgATP(2-). At a high MgATP(2-), almost all demembranated spermatozoa beat with planar waves. cAMP enhanced the three-dimensionality of the flagellar waves at a low Ca(2+). These changes in the flagellar waves were caused by different regulations of the microtubule sliding by calcium, cAMP, and MgATP(2-).
Assuntos
Cálcio/fisiologia , AMP Cíclico/fisiologia , Microtúbulos/fisiologia , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/fisiologia , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/fisiologia , Animais , Axonema/efeitos dos fármacos , Axonema/fisiologia , Cálcio/farmacologia , Membrana Celular/efeitos dos fármacos , AMP Cíclico/farmacologia , Detergentes/farmacologia , Hemicentrotus , Masculino , Modelos Animais , Octoxinol/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacosRESUMO
Flagellar and ciliary motility are driven by the activity of dynein, which produces microtubule sliding within the axonemes. Our goal is to understand how dynein motile activity is regulated to produce the characteristic oscillatory movement of flagella. Analysis of various parameters, such as frequency and shear angle in beating flagella, is important for understanding the time-dependent changes of microtubule sliding amounts along the flagellum. Demembranated flagella can be reactivated in a wide range of ATP concentrations (from 2 µM to several mM) and the beat frequency increases with an increase in ATP. By imposed vibration of a micropipette that caught a sperm head by suction, however, the oscillatory motion can be modulated so as to synchronize to the vibration frequency over a range of 20-70Hz at 2mM ATP. The time-averaged sliding velocity calculated as a product of shear angle and vibration frequency decreases when the imposed frequency is below the undriven flagellar beat frequency, but at higher imposed frequencies, it remains constant. In addition to the role of ATP, the mechanical force of bending is involved in the activation of dynein. In elastase-treated axonemes, bending-dependent regulation of microtubule sliding is achieved. This chapter provides an overview of several approaches, using sea urchin sperm flagella, to studying the measurements in the regulation of dynein activity with or without mechanical force.
Assuntos
Trifosfato de Adenosina/metabolismo , Dineínas do Axonema/metabolismo , Axonema/metabolismo , Ouriços-do-Mar/fisiologia , Cauda do Espermatozoide/metabolismo , Animais , Axonema/química , Axonema/efeitos dos fármacos , Fenômenos Biomecânicos , Movimento Celular/efeitos dos fármacos , Masculino , Elastase Pancreática/farmacologia , Ouriços-do-Mar/efeitos dos fármacos , Cabeça do Espermatozoide/química , Cabeça do Espermatozoide/efeitos dos fármacos , Cabeça do Espermatozoide/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/química , Cauda do Espermatozoide/efeitos dos fármacos , Tripsina/farmacologia , VibraçãoRESUMO
Successful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin-like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore-forming toxin, we conclude that rupture of the erythrocyte membrane is blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforin-like protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.
Assuntos
Membrana Eritrocítica/parasitologia , Células Germinativas/metabolismo , Perforina/metabolismo , Plasmodium berghei/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Eritrócitos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Animais , Fenótipo , Plasmodium berghei/efeitos dos fármacos , Saponinas/farmacologia , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/fisiologia , Cauda do Espermatozoide/ultraestruturaRESUMO
The aim of this study was to investigate the impact of a Chinese herbal decoction on the intracellular calcium (Ca2+) concentration of sperm and the expression of the cation channel 1 of sperm (CatSper1), which is a calcium-channel protein specific to sperm tail, in a murine model of asthenospermia induced with cyclophosphamide. After 34 days of intragastric administration of Chinese herb decoction to the murine model used, routine analyses of the mouse sperm were conducted, the intracellular Ca2+ concentration of the sperm tails was measured using flow cytometry, and the expression of CatSper1 protein was detected using reverse transcriptionpolymerase chain reaction (RT-PCR). Sperm concentration, percentage of grade A and B sperm (i.e., sperm activity) and percentage of grade A, B and C sperm (i.e., overall sperm motility) of the model group mice (MG) were markedly lower compared to the control murine group (CG) (one-way ANOVA, P<0.05). Subsequent to treatment, sperm concentration, percentage of sperm activity and overall sperm motility of the large dose of herbal medicine group murine (LG) were markedly increased compared to MG mice (P<0.05). Intracellular Ca2+ concentration in MG mice was markedly lower compared to CG mice (P<0.05). However, following therapy, a significant increase was observed in the intracellular Ca2+ concentration in LG mice as compared to MG mice (P<0.05). In addition, the expression of CatSper1 in LG mice was significantly higher compared to MG mice (P<0.05), while no statistically significant difference was observed for the CG mice. Intraperitoneal injection of cyclophosphamide reduced sperm concentration, percentage of sperm activity and overall sperm motility, intracellular Ca2+ concentration and CatSper1 expression. Large doses of this Chinese herbal decoction increased sperm intracellular Ca2+ concentration, sperm concentration, and percentages of sperm activity and overall sperm motility by upregulating CatSper1 expression. The findings of this study have demonstrated a therapeutic effect of this decoction on asthenospermia.
Assuntos
Canais de Cálcio/genética , Cálcio/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/metabolismo , Animais , Masculino , Camundongos , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/genética , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologiaRESUMO
The hypo-osmotic (HOS) test has been used in other species as an indicator of the fertilising capacity of spermatozoa. The aims of this study were to assess the response of domestic cat spermatozoa to the hypo-osmotic test, to determine the type of solution, concentration and time of incubation needed to obtain a maximum percentage of swelling, to correlate the selected combination with the percentages of progressive motility and to evaluate whether dilution of the ejaculate alters the results. Incubation for 30 and 45 min in solutions of fructose and of citrate of 50 and 100 mOsmol kg⻹ was evaluated. The highest percentage of swelling was obtained using the 50 mOsmol kg⻹ solution, and no significant differences were observed between the times of exposure to the solutions. A positive correlation was observed between the percentage of individual progressive motility and the percentage of sperm swelling in a 50 mOsmol kg⻹ fructose solution, with no significant differences being observed between raw and diluted semen samples. The results of this study suggest that the HOS test could be useful for evaluating membrane function in domestic cat spermatozoa, both in raw semen and in samples diluted in the EZ Mixin® commercial extender, and thus could be incorporated into routine semen evaluation protocols.
Assuntos
Análise do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Gatos , Citratos/farmacologia , Frutose/farmacologia , Soluções Hipotônicas , Masculino , Concentração Osmolar , Sêmen/fisiologia , Citrato de Sódio , Motilidade dos Espermatozoides , Cauda do Espermatozoide/efeitos dos fármacos , Espermatozoides/ultraestruturaRESUMO
We screened the gene that encodes tetratricopeptide repeat domain 29 (Ttc29) in the maturing rat testis. Gene expression was determined by Northern blotting of 7-week-old rat testes, and a strong signal was detected close to the 18S rRNA band in addition to two weak high-molecular-weight signals. In situ hybridization revealed that Ttc29 was expressed primarily in the spermatocytes. We evaluated the effect of gonadotropin on Ttc29 expression using hypophysectomized rats. The pituitary was removed from 3-week-old rats, gonadotropin was injected at 5 weeks, and Ttc29 expression was determined at 7 weeks. Although testicular development and hyperplasia of interstitial cells were observed following chorionic gonadotropin treatment after hypophysectomy, Ttc29 expression was upregulated by treatment with follicle-stimulating hormone. Ttc29 encodes axonemal dynein, a component of sperm flagella. Taken together, these data indicate that axonemal dynein expression starts in the spermatocytes and is regulated by follicle-stimulating hormone.
Assuntos
Dineínas/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Intersticiais do Testículo/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Animais , Gonadotropina Coriônica/farmacologia , Dineínas/metabolismo , Hormônio Foliculoestimulante/farmacologia , Hipofisectomia , Hibridização In Situ , Células Intersticiais do Testículo/citologia , Masculino , Hipófise/fisiologia , Hipófise/cirurgia , Ratos , Ratos Sprague-Dawley , Cauda do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/fisiologia , Espermatócitos/citologia , Espermatogênese/efeitos dos fármacosRESUMO
Sperm chemotaxis has an important role in fertilization. Most of our knowledge regarding this phenomenon comes from studies in organisms whose fertilization occurs externally, like sea urchins. Sea urchin spermatozoa respond to sperm-activating peptides, which diffuse from the egg jelly coat and interact with their receptor in the flagellum, triggering several physiological responses: changes in membrane potential, intracellular pH, cyclic nucleotide levels, and intracellular Ca2+ concentration ([Ca2+]). In particular, flagellar [Ca2+] has been shown to oscillate. These [Ca2+] oscillations are correlated with changes in the flagellar shape and so with the regulation of the sperm swimming paths. In this study, we demonstrate, from a mathematical modeling perspective, that the reported speract-activated signaling pathway in Strongylocentrotus purpuratus (speract being a sperm-activating peptide specific to this species) has the necessary elements to replicate the reported [Ca2+] oscillations. We further investigate which elements of this signaling pathway constitute the core oscillator.
Assuntos
Relógios Biológicos , Oligopeptídeos/metabolismo , Transdução de Sinais , Cauda do Espermatozoide/metabolismo , Strongylocentrotus purpuratus/metabolismo , Animais , Relógios Biológicos/efeitos dos fármacos , Simulação por Computador , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Modelos Biológicos , Ácido Niflúmico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Cauda do Espermatozoide/efeitos dos fármacos , Processos Estocásticos , Strongylocentrotus purpuratus/efeitos dos fármacosRESUMO
Progesterone is a physiological agonist for mammalian sperm, modulating its flagellar movement and facilitating the acrosome reaction. To study the initial action of progesterone, we developed a caged analog with a photosensitive group: nitrophenylethanediol, at position 20. Using this compound combined with stroboscopic illumination, we performed Ca(2)(+) imaging of human spermatozoa and analyzed the effects of progesterone on the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)) of beating flagella for the first time. We observed a transient [Ca(2)(+)](i) increase in the head and the flagellum upon photolysis of the caged progesterone and an increase in flagellar curvature. Detailed kinetic analysis revealed that progesterone elicits an increase in the [Ca(2)(+)](i) immediately in the flagellum (mid-piece and principal piece), thereafter in the head with a short time lag. This observation is different from the progesterone-induced Ca(2)(+) mobilization in mouse spermatozoa, where the Ca(2)(+) rise initiates at the base of the sperm head. Our finding is mostly consistent with the recent discovery that progesterone activates CatSper channels in human spermatozoa, but not in mouse spermatozoa.
Assuntos
Cálcio/análise , Progesterona/análogos & derivados , Progesterona/farmacologia , Cauda do Espermatozoide/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Canais de Cálcio/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Masculino , Nitrobenzenos/química , Fotólise , Progesterona/química , Espectrometria de Fluorescência , Cabeça do Espermatozoide/química , Cabeça do Espermatozoide/efeitos dos fármacos , Cauda do Espermatozoide/química , Cauda do Espermatozoide/fisiologia , Espermatozoides/química , Espermatozoides/fisiologiaRESUMO
Triton X-100-extracted mouse sperm treated with 0.1 mM ATP and 1.0 mM Ca(2+) exhibit an extremely coiled configuration that has been previously described as a curlicue. Sperm in the curlicue configuration exhibit a monotonically curved flagellum where the shear angle of the flagellum can reach a value as high as 14 radians at the flagellar tip. We utilized this strong reaction to Ca(2+) to elucidate the mechanism of the calcium response. The disintegration of the axoneme was facilitated by the use of an extraction procedure that removed the mitochondrial sheath without eliminating the calcium response. The order of emergence of the doublet microtubule outer dense fiber complexes was observed in the presence and absence of added Ca(2+). The identity of the emergent elements was confirmed by transmission electron microscopy. Ca(2+) altered the order of emergence of internal axoneme elements to favor the appearance of the elements of the 9-1-2 side of the axoneme. These elements are propelled baseward by the action of dyneins on doublets 1 and 2. It was also possible to establish that the motive force for maintaining the curlicue configuration is dynein-based. The curlicues were relaxed by inhibition with 50 µM NaVO(3) and were reestablished by disinhibiting the vanadate with 2.5 mM catechol.