Alfredo Ramírez

The Presence and Function of Dopamine Type 2 Receptors in Boar Sperm: A Possible Role for Dopamine in Viability, Capacitation, and Modulation of Sperm Motility

Abstract 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.

Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels

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 Ca(2+) 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.

Novel identification of peripheral dopaminergic D2 receptor in male germ cells

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.

Intracellular calcium movements of boar spermatozoa during ‘in vitro’ capacitation and subsequent acrosome exocytosis follow a multiple‐storage place, extracellular calcium‐dependent model

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 sperms 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.

Sperm from Hyh Mice Carrying a Point Mutation in αSNAP Have a Defect in Acrosome Reaction

Hydrocephalus with hop gait (hyh) is a recessive inheritable disease that arose spontaneously in a mouse strain. A missense mutation in the Napa gene that results in the substitution of a methionine for isoleucine at position 105 (M105I) of αSNAP has been detected in these animals. αSNAP is a ubiquitous protein that plays a key role in membrane fusion and exocytosis. In this study, we found that male hyh mice with a mild phenotype produced morphologically normal and motile sperm, but had a strongly reduced fertility. When stimulated with progesterone or A23187 (a calcium ionophore), sperm from these animals had a defective acrosome reaction. It has been reported that the M105I mutation affects the expression but not the function of the protein. Consistent with an hypomorphic phenotype, the testes and epididymides of hyh mice had low amounts of the mutated protein. In contrast, sperm had αSNAP levels indistinguishable from those found in wild type cells, suggesting that the mutated protein is not fully functional for acrosomal exocytosis. Corroborating this possibility, addition of recombinant wild type αSNAP rescued exocytosis in streptolysin O-permeabilized sperm, while the mutant protein was ineffective. Moreover, addition of recombinant αSNAP. M105I inhibited acrosomal exocytosis in permeabilized human and wild type mouse sperm. We conclude that the M105I mutation affects the expression and also the function of αSNAP, and that a fully functional αSNAP is necessary for acrosomal exocytosis, a key event in fertilization.

Marlin-1 is expressed in testis and associates to the cytoskeleton and GABAB receptors.

Marlin‐1 is a GABAB receptor and Jak tyrosine kinase‐binding protein that also associates with RNA and microtubules. In humans and rodents, expression of Marlin‐1 is predominantly restricted to the brain, but expression in lymphoid cells has also been reported. Here, we have studied the distribution of Marlin‐1 in testis and spermatozoa. Our results indicate that Marlin‐1 is highly expressed in testis. The protein is abundant in spermatogonia, spermatocytes, spermatozoa, and Sertoli cells. We also have studied the subcellular distribution in spermatozoa. Marlin‐1 is present in the tail and to a lesser degree in the head of the sperm cell. Finally, we have explored two protein interactions. Our findings demonstrate that Marlin‐1 associates with a microtubule fraction and with GABAB receptors in testis suggesting that the set of protein interactions of Marlin‐1 are conserved in different tissues. J. Cell. Biochem. 103: 886–895, 2008.

Regulation of Cellular Calcium, for Control of Motility and Reduction of Mortality of Bovine Sperm

Modulating the flow of extracellular calcium affects motility and reduces sperm mortality. It has been shown that bovine sperm survival exposure to freezing and thawing (cryopreservation), can be used for complex models of laboratory management and in vitro fertilization (IVF). This modulation can be part of more complex mechanism for the regulation of artificial insemination in bovine. High genetic bovine semen acquired in INSECABIO LTDA was used. Samples were thawed at 30°C during 30 s and suspended between TALP. Sperms were selected by swim-up. The measurement of calcium was performed using the Fluo-3 probe, and the VCL and VSL kinetic parameters were evaluated with the system CASA. Acrosome reaction (AR) was measured in bovine semen using a 2% stain of cromassie blue. Viability in a medium with calcium was reduced after 4 hours, whereas after 6 hours reduction was greater than 50%. The chelation of extracellular calcium with a solution of BAPTA 10 μM managed to inhibit motility, as well as calcium-influx modulation by cadmium. Nevertheless, mortality of the sample was decreased only by BAPTA calcium-modulation. On the other hand, pathways involved in AR depend, in part, on extracellular calcium; interestingly, calcium chelation did not block the AR. In addition, when calcium was restored in the media, control sperm-like motility, progressive motility remains similar to that observed in the control. This evidence suggests that diminishing motility by calcium manipulation generates a reduction in mortality in this model and to do it an in vitro manipulation of the sample.