Role of exosomes in sperm maturation during the transit along the male reproductive tract.

Even tough differentiated spermatozoa are unable of transcriptional or translational activity; the sperm surface undergoes major modifications in macromolecules composition during the transit along the male reproductive tract. This is the result of sequential, well orchestrated interactions between the male reproductive tract secretions and the transiting male gamete. This is particularly true when spermatozoa transit along the epididymis. The epididymis is a long convoluted tubules in which the spermatozoa leaving the testis have to transit. The unraveled epididymal tubule can be as long as 80 m in stallion, and the transit time of spermatozoa is of 3-12 days depending on the species. The epididymis is usually divided in three segments: the caput (proximal part), the corpus, and cauda. While the cauda epididymides acts as a sperm reservoir, the caput and corpus are responsible for sperm maturation. This means that, under androgen control, the epididymal epithelium secretes proteins that will interact sequentially with sperm surface. Some of the sperm proteins acquired during maturation along the excurrent duct behave as integral membrane proteins. In fact, some epididymal originating proteins are glycosylphosphatidylinositol (GPI)-anchored to the sperm plasma membrane. Our laboratory has shown that some of these proteins are secreted in an apocrine manner by the epididymal epithelium and are associated to exosomes, called epididymosomes. Epididymosomes are rich in sphingomyelin and are characterized by a high cholesterol/phospholipids ratio. Many proteins are associated to epididymosomes, some of which are selectively transferred to spermatozoa during the epididymal transit. We have identified some of these exosomes associated proteins transferred to the maturing spermatozoa. These include two enzymes involved in the polyol pathway: an aldose reductase and a sorbitol dehydrogenase. A cytokine named MIF (macrophage migration inhibitory factor) is another protein associated to exosomes who is transferred to spermatozoa during the epididymal transit. We hypothesized that both the polyol pathway and MIF secreted in an apocrine fashion by the epididymal epithelium modulate sperm motility during the transit along the male reproductive tract. Finally, P25b, belonging to a family of sperm surface proteins (P26h/P34H) necessary for the binding to the surface of the egg, is also acquired through the interaction between epididymosomes and the male gamete. In vitro studies have defined the conditions of protein transfer when epididymal spermatozoa are co-incubated with epididymosomes. The transfer of selected proteins to specific membrane domains of spermatozoa is saturable, temperature and pH-dependent, being optimal at pH 6.5. The presence of zinc in the incubation medium, but not of calcium neither magnesium, significantly increases the efficiency of protein transfer. These results show that exosomes play a role in sperm epididymal maturation which is an essential event to produce male gametes with optimal fertilizing ability.