Interacting seminal plasma proteins revert capacitation in ram sperm

CESARI, A; LEDESMA, A; HOZBOR, F; FERNANDEZ-ALEGRE, E; MARTINEZ-PASTOR, F

Lisboa

Conferencia; 20th Annual Conference of the European Society for Domestic Animal Reproduction (ESDAR) and the 13th Conference of the Spanish Association for Animal Reproduction (AERA); 2016

European Society for Domestic Animal Reproduction

Cryopreservation induces premature capacita􀦞on in sperm decreasingits fer􀦞lizing capacity. Capacita􀦞on has been associated with proteintyrosine phosphorylation (pY) and it has been demonstrated thatseminal plasma (SP) par􀦞ally reverts this modification in cold- shockedsperm. We developed a methodology to obtain the fraction of SPproteins that bind to the sperm membrane. This fraction, interactingSP proteins (ISPP), may vary according to the season and collection.Our aim was to evaluate whether ISPP obtained by artificial vagina(AV) or electroejaculation (EE) during the breeding and non- breedingseason can simulate the effect of complete SP on thawed ram sperm.Ejaculates from 8 Assaf rams obtained during the breeding or nonbreedingseason were pooled by collection method (AV or EE) andprocessed to obtain SP and ISPP. Cryopreserved semen from 10Assaf rams was incubated either with 20% (v/v) SP or an equivalentamount of ISPP plus 5 mg/ml fructose. The presence of pY wasdetected by Western blot using an􀦞 phosphotyrosine followed byan􀦞- tubuline (loading control). Two protein bands of 45 and 40 kDawere detected. We found that the pY signal induced by cryopreservation was reverted after incuba􀦞on with SP and ISPP with differencesamong treatments. The relative intensity for the 45 kDa bandwas: 0.17 ± 0.03 AV SP, 0.13 ± 0.03 EE SP, 0.21 ± 0.04 ISPP AV,0.36 ± 0.15 ISPP EE, p = 0.04; and for the 40 kDa band: 0.37 ± 0.14AV SP, 0.38 ± 0.14 EE SP, 0.38 ± 0.14 ISPP AV, 0.53 ± 0.14 ISPPEE, p = 0.010. Season showed no effects. We concluded that ISPPobtained by AV and EE reverted premature capacita􀦞on of cryopreservedsperm as complete SP does. This can be a step to developextenders to improve the quality of cryopreserved ram sperm.(Supported by BEC.AR)