Calpain inhibition prevents flotillin re-ordering and Src family activation during capacitation

Maldonado-García, Deneb; Salgado‐Lucio, Monica L.; Roa-Espitia, Ana Lilia; Reyes-Miguel, Tania; Hernández‐González, Enrique O.

doi:10.1007/s00441-017-2591-2

Cited by 7 publications

(12 citation statements)

References 89 publications

(109 reference statements)

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“…Spermatozoa were capacitated (60 minutes) in the absence and presence of NSC23766 (100 μM). In other studies, we have defined the different fluorescent patterns observed when spermatozoa are stained with CTC: pattern F for noncapacitated spermatozoa, pattern B for capacitated spermatozoa, and AR pattern for sperm that have experienced AR . These patterns are shown in Figure B.…”

Section: Resultsmentioning

confidence: 99%

“…In other studies, we have defined the different fluorescent patterns observed when spermatozoa are stained with CTC: pattern F for noncapacitated spermatozoa, pattern B for capacitated spermatozoa, and AR pattern for sperm that have experienced AR. 12,23 These patterns are shown in Figure 2B. After 60 minutes of capacitation, patterns B and AR increased significantly in comparison with noncapacitated spermatozoa (P = .001 and P = .002, respectively; Figure 2B).…”

Section: Rac1 Is Involved In Capacitation and Armentioning

confidence: 89%

“…These assays were performed following the procedure described by Maldonado‐Garcia et al Noncapacitated and capacitated spermatozoa, treated or no with NSC23766 during 1 hour, were fixed with a mixture of formaldehyde (3% vol/vol) and glutaraldehyde (0.2% vol/vol) diluted in phosphate‐buffered saline (PBS). Spermatozoa were pelleted by centrifugation (600 g for 3 minutes), resuspended and incubated in NH 4 Cl (50 mM) for 10 minutes, rinsed twice with PBS, and once with bidistilled water.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence images were registered using an Olympus BX5 fluorescence microscope. CTC staining patterns, 100 spermatozoa per sample were classified as Maldonado‐Garciía et al: ‘F,’ the acrosome region displays a faint fluorescence, feature of spermatozoa noncapacitated and with acrosome‐intact; “B,” a band along the equatorial segment and the acrosomal region exhibit a bright fluorescence, attribute of capacitated, and with acrosome‐intact spermatozoa; and “AR,” fluorescence in the postacrosomal region is distinctive of physiologically capacitated acrosome‐reacted spermatozoa. The status of the acrosomal cap on each spermatozoon was verified under phase‐contrast illumination.…”

Section: Methodsmentioning

confidence: 99%

“…We followed the procedure characterized by Ward and Storey 25 and adapted for guinea pig spermatozoa. 23 In a light-shielded container were mixed 45 μL of spermatozoa and the same volume of CTC solution (750 mM CTC diluted in 130 mM NaCl, 5 mM cysteine, and 20 mM Tris, at a pH of 7.8) and placed in a water bath at 37°C for 20 seconds. Then, the spermatozoa were fixed, adding glutaraldehyde (0.75 µL, final concentration 0.1%) diluted in Tris (0.5 mM, pH 7.4) at room temperature.…”

Section: Ctc Fluorescence Proceduresmentioning

confidence: 99%

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Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Ramírez‐Ramírez

Salgado‐Lucio²,

Roa-Espitia³

et al. 2019

J of Cellular Biochemistry

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Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras‐related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1‐GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem‐like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin‐1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F‐actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.

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Section: Resultsmentioning

confidence: 99%

Section: Rac1 Is Involved In Capacitation and Armentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Ctc Fluorescence Proceduresmentioning

confidence: 99%

See 3 more Smart Citations

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Ramírez‐Ramírez

Salgado‐Lucio²,

Roa-Espitia³

et al. 2019

J of Cellular Biochemistry

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Perfluorooctane sulfonate and perfluorooctanoic acid induce plasma membrane dysfunction in boar spermatozoa during in vitro capacitation

Ortiz-Sánchez

Roa-Espitia

Fierro

et al. 2022

Reproductive Toxicology

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Variation of sperm quality and circular RNA content in men exposed to environmental contamination with heavy metals in ‘Land of Fires’, Italy

Grazia Mele,

Chioccarelli,

Diano

et al. 2024

Human Reproduction

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STUDY QUESTION Can illegal discharge of toxic waste into the environment induce a new condition of morpho-epigenetic pathozoospermia in normozoospermic young men? SUMMARY ANSWER Toxic environmental contaminants promote the onset of a new pathozoospermic condition in young normozoospermic men, consisting of morpho-functional defects and a sperm increase of low-quality circular RNA (circRNA) cargo, tightly linked to contaminant bioaccumulation in seminal plasma. WHAT IS KNOWN ALREADY Epidemiological findings have reported several reproductive anomalies depending on exposure to contaminants discharged into the environment, such as germ cell apoptosis, steroidogenesis defects, oxidative stress induction, blood–testis barrier dysfunctions, and poor sperm quality onset. In this scenario, a vast geographical area located in Campania, Italy, called the ‘Land of Fires’, has been associated with an excessive illegal discharge of toxic waste into the environment, negatively impacting human health, including male reproductive functions. STUDY DESIGN, SIZE, DURATION Semen samples were obtained from healthy normozoospermic men divided into two experimental groups, consisting of men living in the ‘Land of Fires’ (LF; n = 80) or not (CTRL; n = 80), with age ranging from 25 to 40 years. The study was carried out following World Health Organization guidelines. PARTICIPANTS/MATERIALS, SETTING, METHODS Quality parameters of semen from CTRL- and LF-normozoospermic men were evaluated by computer-assisted semen analysis; high-quality spermatozoa from CTRL and LF groups (n = 80 for each experimental group) were obtained using a 80–40% discontinuous centrifugation gradient. Seminal plasma was collected following centrifugation and used for the dosage of chemical elements, dioxins and steroid hormones by liquid chromatography with tandem mass spectrometry. Sperm morpho-functional investigations (cellular morphology, acrosome maturation, IZUMO1 fertility marker analysis, plasma membrane lipid state, oxidative stress) were assessed on the purified high-quality spermatozoa fraction by immunochemistry/immunofluorescence and western blot analyses. Sperm circRNA cargo was evaluated by quantitative RT-PCR, and the physical interaction among circRNAs and fused in sarcoma (FUS) protein was detected using an RNA-binding protein immunoprecipitation assay. Protein immunoprecipitation experiments were carried out to demonstrate FUS/p-300 protein interaction in sperm cells. Lastly, in vitro lead (Pb) treatment of high-quality spermatozoa collected from normozoospermic controls was used to investigate a correlation between Pb accumulation and onset of the morpho-epigenetic pathozoospermic phenotype. MAIN RESULTS AND THE ROLE OF CHANCE Several morphological defects were identified in LF-spermatozoa, including: a significant increase (P < 0.05 versus CTRL) in the percentage of spermatozoa characterized by structural defects in sperm head and tail; and a high percentage (P < 0.01) of peanut agglutinin and IZUMO1 null signal cells. In agreement with these data, abnormal steroid hormone levels in LF seminal plasma suggest a premature acrosome reaction onset in LF-spermatozoa. The abnormal immunofluorescence signals of plasma membrane cholesterol complexes/lipid rafts organization (Filipin III and Flotillin-1) and of oxidative stress markers [3-nitrotyrosine and 3-nitrotyrosine and 4-hydroxy-2-nonenal] observed in LF-spermatozoa and associated with a sperm motility reduction (P < 0.01), demonstrated an affected membrane fluidity, potentially impacting sperm motility. Bioaccumulation of heavy metals and dioxins occurring in LF seminal plasma and a direct correlation between Pb and deregulated circRNAs related to high- and low-sperm quality was also revealed. In molecular terms, we demonstrated that Pb bioaccumulation promoted FUS hyperacetylation via physical interaction with p-300 and, in turn, its shuttling from sperm head to tail, significantly enhancing (P < 0.01 versus CTRL) the endogenous backsplicing of sperm low-quality circRNAs in LF-spermatozoa. LIMITATIONS, REASONS FOR CAUTION Participants were interviewed to better understand their area of origin, their eating habits as well as their lifestyles, however any information incorrectly communicated or voluntarily omitted that could potentially compromise experimental group determination cannot be excluded. A possible association between seminal Pb content and other heavy metals in modulating sperm quality should be explored further. Future investigations will be performed in order to identify potential synergistic or anti-synergistic effects of heavy metals on male reproduction. WIDER IMPLICATIONS OF THE FINDINGS Our study provides new findings regarding the effects of environmental contaminants on male reproduction, highlighting how a sperm phenotype classified as normozoospermic may potentially not match with a healthy morpho-functional and epigenetic one. Overall, our results improve the knowledge to allow a proper assessment of sperm quality through circRNAs as biomarkers to select spermatozoa with high morpho-epigenetic quality to use for ART. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by ‘Convenzione Azienda Sanitaria Locale (ASL) Caserta, Regione Campania’ (ASL CE Prot. N. 1217885/DIR. GE). The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.

show abstract

Calpain inhibition prevents flotillin re-ordering and Src family activation during capacitation

Cited by 7 publications

References 89 publications

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Perfluorooctane sulfonate and perfluorooctanoic acid induce plasma membrane dysfunction in boar spermatozoa during in vitro capacitation

Variation of sperm quality and circular RNA content in men exposed to environmental contamination with heavy metals in ‘Land of Fires’, Italy

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