Seminal Plasma Proteins Revert the Cold-Shock Damage on Ram Sperm Membrane1

Barrios, Beatriz; Pérez‐Pé, Rosaura; Gallego, Margarita; Tato, A.; Osada, Jesús; Muiño‐Blanco, T.; Cebrián‐Pérez, J. A.

doi:10.1095/biolreprod63.5.1531

Cited by 195 publications

(158 citation statements)

References 49 publications

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“…Furthermore, proteins from DRMs and non-DRMs are lost after capacitation and the acrosome reaction. These results are consistent with previous reports that describe the release of sperm membrane proteins during capacitation of bull [70], boar [71,72], rabbit [73] or ram [74] spermatozoa.…”

Section: Discussionsupporting

confidence: 94%

Remodelling of Lipid Rafts during In vitro Capacitation and Acrosome Reaction of Ram Spermatozoa

Colás¹

2013

Biochem Anal Biochem

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Background: Lipid rafts are often known as Detergent-Resistant Microdomains (DRMs). We report for the first time the presence of two lipid raft markers, caveolin-1 and ganglioside GM1, on the ram sperm surface, and the effect of in vitro capacitation and acrosome reaction on these marker distributions, the protein content and lipid composition of DRM and non-DRM fractions. Methods:Caveolin-1 and ganglioside GM1 were evidenced by immunocytochemical and fluorescence analysis, respectively. DRM and non-DRM fractions were separated by an OptiPrep TM density gradient. Cholesterol by fluorometry, GM1 by peroxidase reaction, protein content by spectrophotometry, and fatty acid profiling by gas chromatography were determined.Results: Caveolin-1 was evidenced at the acrosome of 59.2 ± 4.3% fresh spermatozoa, and the proportion of stained cells increased (P<0.05) after capacitation. GM1 was detected at the post-acrosome and tail of all spermatozoa, and no change was found after capacitation. Cholesterol and GM1 were distributed all along the gradient, with a peak in DRM fractions. A higher proportion (P<0.001) of saturated fatty acids was found in DRM fractions, confirmed by the unsaturation index and a higher lipid/protein ratio. In vitro capacitation induced a decrease in the content of saturated fatty acids in both DRM (P<0.001) and non-DRM (P<0.01) fractions. Polyunsaturated fatty acids increased in DRMs after the acrosome reaction. All treatments resulted in lower content of cholesterol and proteins in DRM (P<0.01) and non-DRM fractions (P<0.001), and a higherGM1 content in DRMs (P < 0.05). Conclusions:Lipid raft-like microdomains were isolated in a discrete region of the gradient. Their high content of saturated fatty acids confers a highly ordered environment. Their composition is modified during in vitro capacitation and acrosome reaction. General significance:These results represent the first characterization of ram sperm DRM, and may contribute to a better understanding of the sperm fertilizing potential acquisition mechanism.

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Section: Discussionsupporting

confidence: 94%

Remodelling of Lipid Rafts during In vitro Capacitation and Acrosome Reaction of Ram Spermatozoa

Colás¹

2013

Biochem Anal Biochem

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“…Previously, we demonstrated that the adsorption of seminal plasma proteins on ram sperm plasma membranes could partially prevent and repair cold-shock sperm membrane damage (Pérez-Pé et al, 2001a,b), and that this adsorption modified the functional characteristics of damaged spermatozoa reproducing those of live cells (Barrios et al, 2000;Pérez-Pé et al, 2001a). More recently (Barrios et al, 2005), we have proven that 2 proteins of approximately 14 (P14) and 20 (P20) kDa are responsible for this protective and restoring effect.…”

Section: Discussionmentioning

confidence: 95%

“…Polyclonal antibodies were raised against the whole seminal plasma fraction 6 (F6) isolated by exclusion chromatography in Sephacryl-100 (Barrios et al, 2000), and the purified P14 and P20 bands were recovered by electroelution from a nondenaturing gel (Barrios et al, 2000) by rabbit immunization with 500 mg (F6) and 300 mg (P14 and P20) of protein in Freunds complete adjuvant. After 15 days they were reimmunized with the same amount dissolved in Freunds incomplete adjuvant.…”

Section: Polyclonal Antibodiesmentioning

confidence: 99%

Immunohistochemical Localization of Sperm‐Preserving Proteins in the Ram Reproductive Tract

Fernández‐Juan

Gallego

Barrios

et al. 2006

Journal of Andrology

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Previously, we reported that the addition of seminal plasma proteins before cold-shock treatment prevents sperm membrane injury, and that 2 proteins of approximately 14 (P14) and 20 (P20) kDa, the main components of fraction 6 isolated by exclusion chromatography, are responsible for this protective effect. The objective of the present study was to localize P14 and P20 in tissues of the ram reproductive tract to determine their origin. Antiserum generated against purified P14 and P20 reacted with proteins in seminal vesicles and vas deferens by Western blot analyses of protein tissue extracts. However, these antisera failed to detect P14 and P20 in testis, prostate, efferent ductules, bulbourethral glands, and epididymis (caput, corpus, and cauda). Immunohistochemical analyses by both indirect immunofluorescence and the avidin-biotin complex technique confirmed that only seminal vesicles showed reactivity, restricted to the secretory cells, with both antibodies. Obtained results indicate that P14 and P20 are secreted specifically in the seminal vesicles. To further confirm that P14 and P20 are specifically expressed in seminal vesicles, we used Northern blot analyses to investigate the expression of both proteins in seminal vesicles and vas deferens. These assays corroborated again that P14 and P20 were specifically expressed in seminal vesicles. Consequently, we suggest referring to these 2 proteins as RSVP14 and RSVP20, respectively, according to their origin and molecular weight.

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“…Asimismo se ha demostrado que el PS completo en exceso tiene un efecto nocivo para la viabilidad espermática (22). Por su parte, Barrios et al (1), describió, en ovinos, que la adición del conjunto de proteínas de PS, aunque no disminuye la viabilidad después de someter los spz a choque térmico por frío, tiene un efecto de recuperación de la viabilidad menor que el ejercido por la adición de la fracción 6 (con proteínas de 14 y 20 kDa), y que la adición de fracciones de PS, que contienen proteínas con pesos moleculares superiores a 60 kDa, no tiene mayor efecto en la recuperación de la viabilidad del semen ovino.…”

Section: Discussionunclassified

“…Estas proteínas, son secretadas por las vesículas seminales (11)(12)(13), y se han empleado para proteger al spz contra el estrés térmico y oxidativo (14,15). Igualmente, se ha demostrado que la incubación de spz in vitro con proteínas de PS, puede contrarrestar los daños ocasionados a las células espermáticas sometidas a choques térmicos (1,13).…”

Section: Introduccionunclassified

Las proteínas del plasma seminal incrementan la viabilidad espermática post-descongelación del semen de toros Sanmartinero

et al. 2013

Rev MVZ Córdoba

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RESUMENObjetivo. El objetivo de este trabajo fue evaluar el efecto de la adición de proteínas del plasma seminal sobre el porcentaje de espermatozoides bovinos viables post-descongelación. Materiales y métodos. Los espermatozoides se congelaron usando dos medios (citrato-fructosa-yema y Bioxcell ® ) y la obtención de proteínas de plasma seminal de bajo peso molecular se realizó por medio de cromatografía líquida de baja presión. Las proteínas de interés eluyeron en las fracciones 21-25 y se sometieron a electroforésis en una y dos dimensiones. Los espermatozoides se incubaron a 37°C durante una hora, con 0.5, 1.0, 1.5 y 2.0 mg de la fracción 21-25. Se incluyeron dos tratamientos adicionales: uno con proteínas totales del plasma seminal y otro sin proteína. Resultados. La electroforésis bidimensional de las fracciones confirmó la presencia de siete puntos de proteína de bajo peso molecular (14-16 kDa y punto Isoeléctrico de 5.0 -5.5). La adición de estas proteínas aumentó 20% (p<0.05), el porcentaje de espermatozoides viables post-descongelación en muestras congeladas en medio citrato-fructosayema (con dosis de 1 ó 1.5 mg de proteína/10 6 espermatozoides), y 25% (p<0.05) en muestras congeladas en medio Bioxcell ® (con dosis de 0.5 mg de proteína/10 6 espermatozoides). Conclusiones. Los resultados de esta investigación sugieren el posible uso de proteínas de bajo peso molecular del plasma seminal, para disminuir el efecto deletéreo de la criopreservación en los espermatozoides.Palabras clave: Capacitación espermática, criopreservación, espermatozoide, plasma seminal, proteínas de choque térmico (Fuente: DeCS).

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Seminal Plasma Proteins Revert the Cold-Shock Damage on Ram Sperm Membrane1

Cited by 195 publications

References 49 publications

Remodelling of Lipid Rafts during In vitro Capacitation and Acrosome Reaction of Ram Spermatozoa

Remodelling of Lipid Rafts during In vitro Capacitation and Acrosome Reaction of Ram Spermatozoa

Immunohistochemical Localization of Sperm‐Preserving Proteins in the Ram Reproductive Tract

Las proteínas del plasma seminal incrementan la viabilidad espermática post-descongelación del semen de toros Sanmartinero

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