Estrogens are steroid hormones that affect a wide range of physiological functions. The effect of estrogens on male reproductive tissues and sperm cells through specific receptors is essential for sperm development, maturation, and function. Although estrogen receptors (ERs) have been studied in several mammalian species, including humans, they have not yet been described in bull spermatozoa and reproductive tissues. In this study, we analyzed the presence of all types of ERs (ESR1, ESR2, and GPER1) in bull testicular and epididymal tissues and epididymal and ejaculated spermatozoa, and we characterize them here for the first time. We observed different localizations of each type of ER in the sperm head by immunofluorescent microscopy. Additionally, using a selected polyclonal antibody, we found that each type of ER in bull sperm extracts had two isoforms with different molecular masses. The detailed detection of ERs is a prerequisite not only for understanding the effect of estrogen on all reproductive events but also for further studying the negative effect of environmental estrogens (endocrine disruptors) on processes that lead to fertilization.

Mammalian spermatozoa, produced by the testes, must undergo maturation in the epididymis to gain the ability to fertilize an ovum. This process includes structural modifications, such as changes in the lipid and protein composition of the plasma membrane. The CD81 and CD9 proteins, which are members of tetraspanin superfamily, have been found to be involved in the fertilization process. Our study examined the presence of CD81 in the bull testis and epididymis, and then compared CD81’s distribution profile with that of the molecule CD9. We detected CD81 in the basement membrane along the whole epididymis. In comparison, an abundant expression of CD9 molecule was found in the testis as well as in all three segments of the epididymis. Both molecules are part of the tetraspanin web, and are considered key elements in the sperm-egg interaction.