Several studies have shown that dopamine and other catecholamines are present in oviduct luminal fluid. We recently reported that dopamine type 2 receptors (DRD2) are present in a wide range of mammalian sperm, suggesting a role for dopaminergic signaling in events such as fertilization, capacitation, and sperm motility. In the present study, we used Western blot analysis to show that boar sperm express DRD2 and that their activation with dopamine (100 nM) has a positive effect on cell viability that can be correlated with AKT/PKB phosphorylation. Bromocriptine (100 nM) and dopamine (100 nM and 10 μM) increased tyrosine phosphorylation during the capacitation period. Immunofluorescence analysis indicated that DRD2 localization is dynamic and depends on the capacitation stage, colocalizing with tyrosine phosphorylated proteins in the acrosome and midpiece region of capacitated boar sperm. This association was confirmed by coimmunoprecipitation analysis. We also showed that bromocriptine (100 nM) and low-concentration dopamine (100 nM and 10 μM) increased total and progressive motility of sperm. However, high concentrations of dopamine (1 mM) decreased tyrosine phosphorylation and motility in in vitro sperm capacitation assays. This can be explained by the presence of the dopamine transporters (DAT, official symbol SLC6A3) in sperm, as demonstrated by Western blot analysis and immunocytochemistry. Taken together, our results support the idea that dopamine may have a fundamental role during sperm capacitation and motility in situ in the female upper reproductive tract.

This work analysed intracellular calcium stores of boar spermatozoa subjected to ‘in vitro’ capacitation (IVC) and subsequent progesterone‐induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico‐chemical properties, Fluo‐3 and Rhod‐5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo‐3 was located at both the midpiece and the whole head. Rhod‐5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca²⁺ signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O₂ consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca²⁺ labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca²⁺ signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo‐3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca²⁺. The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca²⁺‐induced peak, O₂ consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

Incubation of boar spermatozoa in a capacitation medium with oligomycin A, a specific inhibitor of the F0 component of the mitochondrial ATP synthase, induced an immediate and almost complete immobilisation of cells. Oligomycin A also inhibited the ability of spermatozoa to achieve feasible in vitro capacitation (IVC), as measured through IVC-compatible changes in motility patterns, tyrosine phosphorylation levels of the acrosomal p32 protein, membrane fluidity and the ability of spermatozoa to achieve subsequent, progesterone-induced in vitro acrosome exocytosis (IVAE). Both inhibitory effects were caused without changes in the rhythm of O2 consumption, intracellular ATP levels or mitochondrial membrane potential (MMP). IVAE was accompanied by a fast and intense peak in O2 consumption and ATP levels in control spermatozoa. Oligomycin A also inhibited progesterone-induced IVAE as well as the concomitant peaks of O2 consumption and ATP levels. The effect of oligomycin on IVAE was also accompanied by concomitant alterations in the IVAE-induced changes on intracellular Ca2+ levels and MMP. Our results suggest that the oligomycin A-sensitive mitochondrial ATP-synthase activity is instrumental in the achievement of an adequate boar sperm motion pattern, IVC and IVAE. However, this effect seems not to be linked to changes in the overall maintenance of adequate energy levels in stages other than IVAE.

Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT‐PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre‐ and post‐meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non‐capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology. J. Cell. Biochem. 100: 141–150, 2007.