Stallion spermatozoa: putative targets of estrogens. Studies of estrogens receptors.
Résumé
Introduction. Among the mammals, stallion appears as the male producing the largest amount of testicular estrogens (Raeside, Can J Biochem., 47:811-5, 1969). This synthesis seems to be submitting to a seasonal regulation as Lemazurier et al. (Gen Comp Endocrinol. 125:272-82, 2002) demonstrated the presence of higher amounts of estrogens in semen in April-May and June compared to non-breeding season. The main sites of estrogen production are the Leydig cells, which show a strong immunoreactivity for aromatase (Almadhidi et al., J. Histochem Cytochem. 43:571-577, 1995). To exert their effects estrogens use specific nuclear receptor named ESR1 and ESR2, which could exert both genomic and non-genomic effects. Estrogen receptors have been localized in testis (Pearl et al., Anim Reprod Sci. 125 :103-11. 2011) as in epididymis (Parlevliet Theriogenology. 2006 ;66 :755-65). The presence of aromatase and estrogen receptors both in testis and in reproductive tract suggests that estrogen may play a role in spermatogenesis as well as in post-testicular maturation. To better define this role and the targets of estrogen we studied the putative presence of estrogen receptors on stallion spermatozoa.
Materials and Methods. Semen was obtained from 10 stallions, aged from 10 to 23 years, housed at Jumenterie du Pin (IFCE). Seminal plasma was removed by centrifugation and spermatozoa were washed by two successive centrifugations in Tyrode. Analysis were performed by immunocytochemistry, Western-blot, immunofluorescence analysis and flow cytometry with MC-20 (ESR1), H-150 (ESR2) antibodies. GPER identification in equine specie was confirmed by RT-PCR and sequencing.
Results. We showed the presence of both estrogen receptors ESR1, ESR2 on 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 showed a single 61 kDa band, corresponding to the molecular weight 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 these both receptors was analyzed by flow cytometry. We showed that 98 % of spermatozoa were positive for ESR1 and 94% positive for ESR2 in samples from 3 stallions obtained in April. The estrogen transmembrane receptor, GPER was also searched. Genome from Equus Caballus contains a GPER-like gene but there was no study about identification and expression of this gene in horse species. We proceeded to RNA extraction from a colt testis and spermatozoa from three stallions and with primers designed from the DNA sequence, we performed RT-PCR amplification. The products obtained were sequenced and allowed to describe the expression of GPER in equine species for the first time.
Conclusion. We described in this study, the presence of estrogen receptors on stallion spermatozoa, allowing to describe it as a putative estrogen target. Moreover, we report the first description of GPER in equine species, opening new possibilities of studies of estradiol action in male as in female reproduction. Future studies will investigate putative use of these receptors to regulate spermatozoa functions like motility.