Phospholipase A<sub>2</sub> activity in fresh and cryopreserved spermatozoa from boars

Dyck, Michael K.; Buhr, M.M.

doi:10.4141/cjas94-009

Cited by 5 publications

(2 citation statements)

References 17 publications

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“…The cAMP can inhibit phospholipase A 2 activity by bind- ing phospholipase A 2 or replacing the free stored calcium essential for phospholipase A 2 activity (Van Den Bosh, 1980). The semen freezing and thawing process also decreases phospholipase A 2 activity approximately 50% in swine samples when compared with the activity in fresh semen (Dyck and Buhr, 1993).…”

Section: Discussionmentioning

confidence: 99%

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Variations of protein profiles and calcium and phospholipase A2 concentrations in thawed bovine semen and their relation to acrosome reaction

Marques

Goulart

Silva³

2000

Genet. Mol. Biol.

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Just as calcium plays an integral role in acrosome capacitation and reaction, several spermatozoon proteins have been reported as binding to the ovum at fertilization. We examined the relationship between thawed bovine semen protein profiles, seminal plasma calcium ion concentration, spermatozoon phospholipase A2 (PLA2) activity and acrosome reaction. Electrophoretic profile analysis of spermatozoa and bovine seminal plasma proteins (total and membrane) revealed qualitative and quantitative differences among bulls. Variations in PLA2 and seminal plasma calcium concentration indicated genetic diversity among individuals. A 15.7-kDa membrane protein was significantly correlated (r = 0.71) with acrosome reaction, which in turn has been associated with in vivo fertility.
Várias proteínas que constituem o espermatozóide têm sido relatadas como sendo proteínas que se ligam ao óvulo no momento da fertilização, bem como íons cálcio têm um papel importante na capacitação e reação acrossômica. Baseado nisto, este estudo teve como objetivo analisar e correlacionar proteínas do sêmen congelado bovino de diferentes raças, concentração de íons cálcio no plasma seminal e atividade da fosfolipase A2 do espermatozóide com a reação acrossômica, visando encontrar fatores que influenciem no processo de fertilização bovina. Análises do perfil eletroforético das proteínas (totais e de membrana) do espermatozóide e do plasma seminal bovino revelaram variabilidade protéica entre indivíduos na qual diferenças qualitativas e quantitativas foram identificadas. A quantificação da fosfolipase A2, bem como da concentração de cálcio no plasma seminal revelaram diversidade genética entre touros. Uma proteína de 15,7 kDa apresentou correlação significativa (0.71) com a reação acrossômica, que pode estar diretamente relacionada com a fertilização in vivo e deste modo outros experimentos podem ser realizados a fim de investigar a utilidade deste marcador protéico na verdadeira fertilidade

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Section: Discussionmentioning

confidence: 99%

“…The method used for extraction of total spermatozoon proteins was acid extraction as described by Dyck and Buhr (1993). Each 0.5-ml diluted semen sample was thawed at 37°C for 30 s. Seminal plasma and spermatozoon separation was done by centrifugation at 30,000 g for 30 min.…”

Section: Extraction and Detection Of Semen Proteinsmentioning

confidence: 99%

Variations of protein profiles and calcium and phospholipase A2 concentrations in thawed bovine semen and their relation to acrosome reaction

Marques

Goulart

Silva³

2000

Genet. Mol. Biol.

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Premature Capacitation of Bovine Spermatozoa Is Initiated by Cryopreservation

CORMIER,

SIRARD,

BAILEY

1997

Journal of Andrology

160

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Poor motility and abnormal acrosomal morphology only partially explain the reduced fertility of cryopreserved bovine spermatozoa. To test the hypothesis that cryopreservation procedures (dilution, cooling, freeze‐thaw) induce capacitation in bovine spermatozoa, two experiments were conducted using semen diluted in egg yolk—Tris—glycerol extender (EYTG) (Tris, tris(hydroxymethyl)aminomethane). Capacitation was determined prior to and following incubation with various concentrations of heparin using the chlortetracycline (CTC) fluorescence assay or after preexposure to EYTG using in vitro fertilization (IVF) of bovine cumulus—oocyte complexes (COC) in the absence of heparin. Fresh ejaculates were divided into four treatments and the first was diluted with noncapacitating medium, NCM (+ 0.3% polyvinyl alcohol (PVA); control), then maintained at 23°C for 4 hours. The remaining semen was diluted with EYTG; the second treatment was held at 4°C (EYTG‐4), and the third treatment was held at 23°C (EYTG‐23) for 4 hours. The fourth treatment was cooled to 4°C over 4 hours, as per the normal industry protocol, cryopreserved, and thawed (frozen‐thawed). After the 4‐hour maintenance periods or thawing, all treatments were resuspended either in capacitating medium (CM; + 0.6% BSA) for the CTC experiment (n = 3) or in NCM for the IVF experiment (n = 9–11). Prior to incubation in conditions that support capacitation, the percentage of cells exhibiting pattern B (capacitated according to the CTC assay) was similar for all treatments with fresh‐extended spermatozoa. Immediately following the addition of heparin (0, 2, or 10 μg/ml), three times more frozen‐thawed than fresh‐extended spermatozoa exhibited pattern B (P < 0.05). After 3 or 6 hours of incubation, however, the percentages of cells displaying pattern B did not differ among treatments. In the absence of heparin, spermatozoa preexposed to EYTG‐4 fertilized 2.6x more COC than did control cells (P < 0.001) and 9.2 x more than spermatozoa preexposed to EYTG but held at 23°C (EYTG‐23; P < 0.0001). No differences were observed among fertilization rates for fresh‐extended (EYTG‐4) and frozen‐thawed spermatozoa. This study provides evidence that premature capacitation occurs in partially (extended and cooled) and fully cryopreserved bovine spermatozoa.

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Extender Components and Surfactants Affect Boar Sperm Function and Membrane Behavior During Cryopreservation

PETTITT,

BUHR

1998

Journal of Andrology

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To determine how the individual components of extenders affected boar sperm function and membrane structure and to test a new surfactant's cryoprotective ability, boar sperm were cryopreserved in straws in BF5 extender plus or minus egg yolk plus or minus glycerol plus or minus a surfactant (Orvus ES Paste [OEP] or various concentrations of Pluronic F‐127). After thawing, sperm function and fluidity of the isolated head plasma membrane (HPM) were determined. Total motility and adenosine triphosphate content (a measure of viability) were superior postthaw in sperm extended in egg yolk plus glycerol (P < 0.05); neither surfactant improved function. Egg yolk plus any other ingredients improved normal acrosome morphology, whereas a combined measure of motility and normal acrosome morphology was better in the presence of 0.33% OEP or 0.1% Pluronic F‐127 (P < 0.05 vs. controls). Head plasma membrane was isolated from freshly collected spermatozoa and spermatozoa cryopreserved in the various extenders. Membrane fluidity was monitored with the probes cis‐parinaric acid (cPNA), transparinaric acid (tPNA), and 1,6‐diphenyl‐1,3,5‐hexatriene (DPH). The cPNA and the DPH monitor the fluidity of gel and liquid‐crystalline areas of the membrane, whereas the tPNA preferentially monitors the gel‐phase domains of the membrane. Additionally, DPH monitors the hydrophobic core of the bilayer. In the HPM from fresh sperm, the fluidity of each domain changed over time in a manner unique to that domain, and the behavior of the DPH domain varied among boars. The fluidity dynamics of each domain responded uniquely to cryopreservation. The cPNA domain was unaffected, the tPNA domain was altered by four of the eight extenders, and all extenders affected the fluidity of the DPH domain. Membrane structure was significantly correlated with cell function for sperm cryopreserved in extenders that preserved viability and motility. Sperm cryopreserved in egg yolk plus glycerol plus either OEP or 0.1% Pluronic F‐127 functioned best when the bulk domains were less fluid initially and the gel domain solidified more slowly. Therefore, the behavior of domains in the HPM of boar spermatozoa is affected by cryopreservation and is related to the postthaw function of boar sperm cryopreserved in different extenders.

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Phospholipase A₂ activity in fresh and cryopreserved spermatozoa from boars

Cited by 5 publications

References 17 publications

Variations of protein profiles and calcium and phospholipase A2 concentrations in thawed bovine semen and their relation to acrosome reaction

Variations of protein profiles and calcium and phospholipase A2 concentrations in thawed bovine semen and their relation to acrosome reaction

Premature Capacitation of Bovine Spermatozoa Is Initiated by Cryopreservation

Extender Components and Surfactants Affect Boar Sperm Function and Membrane Behavior During Cryopreservation

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Phospholipase A2 activity in fresh and cryopreserved spermatozoa from boars

Cited by 5 publications

References 17 publications

Variations of protein profiles and calcium and phospholipase A2 concentrations in thawed bovine semen and their relation to acrosome reaction

Variations of protein profiles and calcium and phospholipase A2 concentrations in thawed bovine semen and their relation to acrosome reaction

Premature Capacitation of Bovine Spermatozoa Is Initiated by Cryopreservation

Extender Components and Surfactants Affect Boar Sperm Function and Membrane Behavior During Cryopreservation

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Phospholipase A₂ activity in fresh and cryopreserved spermatozoa from boars