Effect of nitric oxide enzyme (NOS) inhibition and effect of nitric oxide (NO) scavenger on motility patterns and hyperactivated population of cryopreserved equine sperm

• DOI: 10.1016/j.jevs.2012.06.023
• Publication Date: 2012-08-10T14:48:33Z
• Authors: R.P. Arruda; D.F. Silva; A.F.C. Andrade; M.C. Caldas-Bussiere; M.A. Alonso; E.C.C. Celeghini; K.M. Lemes; H.F. Carvalho; S.A. Florez-Rodri...
• Keywords: Estrogen receptor beta; Immunofluorescence

collected from10 stallions, aged from10 to 23 years. Seminal plasma was removed by centrifugation and spermatozoa were washed by two successive centrifugations in Tyrode extender. Analysis was performed by immunocytochemistry, western-blot, immunofluorescence and flow cytometry with MC-20 (ESR1), H-150 (ESR2) antibodies. GPER identification in equine species was confirmed by RT-PCR and sequencing. Both estrogen receptors ESR1 and ESR2 are present in stallion spermatozoa. For ESR1, western-blot analysis shows a single 66kDa band corresponding to the wild-type isoform; immunofluorescence analysis showed a flagellar staining. For ESR2, western-blot analysis shows a single 61 kDa band, corresponding to themolecularweight described for the wild type form of this receptor and confocal analysis demonstrated also a flagellar localization. The rate of spermatozoa positive for the detection of both of these receptors was analyzed by flow cytometry. 98% of spermatozoa were positive for ESR1 and 94% positive for ESR2 in samples from 3 stallions collected in April. The estrogen transmembrane receptor, GPERwas also identified. Genome from Equus Caballus contains a GPER-like gene but identification and expression of this gene in horses has not been reported so far. We proceeded to extract RNA from a colt testis and spermatozoa from 3 stallions and with primers designed from the DNA sequence, RT-PCR amplification was performed. The products were sequenced and allowed to describe the expression of GPER in the equine species for the first time. In conclusion, the presence of estrogen receptors in stallion spermatozoa was demonstrated suggesting that sperm are a putative estrogen target. Moreover, GPER was described for the first time in horses. This is opening new possibilities to study estradiol action in male as well as in female reproduction.