Stallion spermatozoa : putative targets for estrogens.

Among the mammals, stallion appears as the male producing the largest amount of testicular estrogens (Raeside, 1969). This synthesis occurs mainly in Leydig cells (Almadhidi et al., 1995) and seems to be submitting to a seasonal regulation. Indeed, Lemazurier et al. (2002) demonstrated the presence of higher amounts of estrogens in semen in April-May and June compared to December. To exert their effects estrogens use specific nuclear receptors (ESR1, ESR2), which could exert both genomic and non-genomic actions. Recently, we identified estrogen receptors in ejaculated stallion spermatozoa and in order to determine a putative seasonal response to estrogens a quantification of ESR1 was performed, on semen samples. Semen were obtained monthly between May 2012 and February 2013 from 5 stallions, aged from 10 to 23 years, housed at Jumenterie du Pin (IFCE). Seminal plasma was removed by centrifugation and spermatozoa were washed twice in Tyrode. ESR1 protein expression was studied by Western-blot, confocal analysis and flow cytometry with MC-20 antibody. Results were analyzed by ANOVA followed by Tukey-Kramer multiple comparisons test. Western-blot analysis showed a single 66kDa band corresponding to the wild-type isoform, and then immunofluorescence analysis showed a flagellar staining. We described a high rate of spermatozoa from a sample positive for the detection of ESR1 between May and October then there is a progressive and regular decrease of signal from October to reach a nadir on January, followed by an increase of signal in February. So, we describe for the first time, a seasonal regulation of ESR1 associated to spermatozoa. Further studies will elucidate a putative relation between sperm ability to respond to estrogen and sperm quality.