Role of cholesterol, DOTAP, and DPPC in prostasome/spermatozoa interaction and fusion.

Prostasomes are membranous vesicles present in ejaculated human semen. They are very rich in cholesterol and can interact with spermatozoa. Their physiological roles are still under study. Prostasomes were mixed with liposomes prepared from various lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), DOTAP/1,2-dipalmytoyl-sn-glycero-3-phosphorylcholine (DPPC, 4:1 molar ratio) and DOTAP/cholesterol (4:1, molar ratio) at different pH values (5-8). The mixing of the lipid phases (fusion) was determined by the relief of octadecyl rhodamine B chloride (R(18)) self-quenching and the radii of the vesicles, by light scattering measurements. The mixing of lipids and the radii of prostasomes were both influenced by the addition of liposome, although in a different manner. The ability of prostasomes (modified by previous treatment with liposomes) to transfer lipid to spermatozoa was also measured. Pretreatment with DOTAP decreased the phenomenon and addition of DPPC abolished it. On the other hand, pretreatment of prostasomes with DOTAP/cholesterol liposomes did not affect the transfer of lipid between prostasome and spermatozoa. Therefore, the ability of vesicles to fuse (or, at least, to exchange the lipid component) was affected by the enrichment in either natural or artificial lipid. This may open new possibilities for the modulation of spermatozoa capacitation and acrosome reaction.

Cytosolic calcium levels in spermatozoa are modulated differently in healthy subjects and patients with varicocele.

OBJECTIVE: To study parameters connected to fertility in the semen of patients with varicocele. DESIGN: We examine the ability of spermatozoa obtained from patients with varicocele to respond with an increase of cytosolic Ca2+ ([Ca2+]i) to some stimuli that are connected with spermatozoa activation. SETTING: An academic research environment. PATIENT(S): Ten healthy volunteer donors and 10 patients affected by II or III grade left varicocele. INTERVENTION(S): Spermatozoa and prostasomes (vesicles of prostatic origin obtained from semen) were prepared according to standard procedures. Spermatozoa were stimulated with 1 microM P. The [Ca2+]i was evaluated with the FURA II method. MAIN OUTCOME MEASURE(S): The level of [Ca2+]i. RESULT(S): In resting cells, the level of [Ca2+]i was 120 +/-15 nmol/L (10 determinations). This value increases by > or =100 nmol/L upon stimulation with P. No difference was observed between spermatozoa obtained from healthy donors or from patients with varicocele. S-nitrosocysteine, a nitric oxide donor, and the fusion between spermatozoa and prostasomes increased the effect of P on [Ca2+]i in control spermatozoa but not in spermatozoa obtained from patients with varicocele. CONCLUSION(S): Different responsiveness of varicocele patients' spermatozoa to S-nitrosocysteine and/or to fusion with prostasomes may be among the possible causes of reduced fertility.

Antagonism between olive oil phenolics and nitric oxide on lymphomonocyte cytosolic calcium.

Some biological actions of olive oil phenolics (inhibition of platelet aggregation, decrease of LDL-oxidation, inhibition of bacterial growth and hypertensive action) have been attributed to NOS stimulation in endothelial cells through an increase of cytosolic calcium, notwithstanding the scavenging activity of phenolics on NO and superoxide. In this paper, we determine the concentration of cytosolic calcium in human lymphomonocytes incubated with high concentrations of NO-donors (CysNO) and we evaluate the effects of olive oil phenolics on this parameter. CysNO induces a marked decrease of cytosolic calcium; both olive oil phenolics oppose this action of CysNO. The effects of phenolics and CysNO are independent and additive.

Activity of olive oil phenols on lymphomonocyte cytosolic calcium.

Phenols, present in the Mediterranean diet, have antioxidant properties and are free radical scavengers; however, the molecular mechanisms of their beneficial effects are not yet fully understood. The level of cytosolic calcium ([Ca2+]i) is an important signal also in nonexcitable cells, including immune cells, and regulates fundamental processes. In this paper, we determine [Ca2+]i in human lymphomonocytes incubated with two olive oil phenols: 3,4-(dihydroxyphenyl)ethanol and p-(hydroxyphenyl)ethanol. Both tested phenols increase [Ca2+]i in a dose-dependent way. This effect is antagonized by nifedipine and is noticeable both in the presence and in the absence of calcium in the extracellular medium.

Fusion of prostasomes to human spermatozoa stimulates the acrosome reaction.

OBJECTIVE: To determine the effect of the fusion of prostasomes to spermatozoa on the acrosome reaction. DESIGN: In vitro study of human spermatozoa. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Healthy volunteer men, 25 to 35 years old. INTERVENTION(S): Human semen was fractionated into spermatozoa and prostasomes. Fusion of prostasome to spermatozoa was performed at pH 5.5. Progesterone (1 microM) was added when required. MAIN OUTCOME MEASURE(S): Evaluation of the acrosome reaction by fluorescence microscopy. RESULTS(S): The percentage of spontaneously acrosome-reacted cells was very low unless the Ca(2+)-ionophore A 23187 was added. The treatment of spermatozoa with 1 microM of progesterone scarcely affected the acrosome reaction; a pretreatment in conditions permitting fusion increased it. The addition of progesterone to prostasome-fused spermatozoa further increased the extent of the acrosome reaction. CONCLUSION(S): The H(+)-dependent fusion with prostasomes makes spermatozoa more sensitive to the effect of progesterone on acrosome-reaction induction.

Nitric oxide and fusion with prostasomes increase cytosolic calcium in progesterone-stimulated sperm.

Spermatozoa must undergo a number of reactions before they are able to fertilize the oocyte. Among these is the acrosome reaction, which is related to an increase in cytosolic Ca2+ concentration ([Ca2+]i). It has been reported in the literature that progesterone may achieve this effect through the intervention of extragenomic receptors. Nitric oxide (NO) has been reported to affect spermatozoa; the nature of the effect depends on the concentration of the radical. In a previous paper, we reported that the fusion of spermatozoa with prostasomes may also produce a transient increase in spermatozoa [Ca2+]i; in addition, this phenomenon causes a long-lasting effect that influences the action of progesterone. In this paper, we test the effects of a NO donor (CysNO) and of fusion of the prostasome to spermatozoa on progesterone-induced [Ca2+]i increase. No effect at all was noticed in the absence of progesterone stimulation. In the presence of the hormone, both CysNO and fusion increased the progesterone effect. This phenomenon was much more evident if the two treatments were used together. We conclude that both NO and fusion with prostasomes act on the progesterone-dependent pathway additively. Probably the effects are independent.