Giuseppe Arienti

Fusion of human sperm to prostasomes at acidic pH.

Abstract. Prostasomes are membranous vesicles (150–200 nm diameter) present in human semen. They are secreted by the prostate and contain large amounts of cholesterol, sphingomyelin and Ca2+. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. The possibility that they may fuse to sperm has never been proved. In this work, we studied the fusion of sperm to prostasomes by using various methods (relief of octadecyl Rhodamine B fluorescence self-quenching, fluorescence microscopy and flow cytometry) and we found that it occurs at acidic pH (4–5), but not at pH 7.5 pH-dependent fusion relies on the integrity of one or more proteins and is different from the Ca2+-stimulated fusion between rat liver liposomes and spermatozoa that does not require any protein and occurs at neutral pH. We think that the H+-dependent fusion of prostasomes to sperm may have physiological importance by modifying the lipid and protein pattern of sperm membranes.

BASE-EXCHANGE ENZYMIC SYSTEM FOR THE SYNTHESIS OF PHOSPHOLIPIDS IN NEURONAL AND GLIAL CELLS AND THEIR SUBFRACTIONS: A POSSIBLE MARKER FOR NEURONAL MEMBRANES

Abstract— The calcium‐dependent incorporation of L‐[3‐14C]serine and [1,2−14C]ethanolamine into the phospholipid of isolated neuronal and glial cells from rabbit brain was studied, and the distribution of the enzymic system among the correspondent subfractions was examined. The neuronal cell‐enriched fraction was found to possess a much higher rate of exchange of both bases than the glial cell‐enriched fraction. Among the sub‐fractions isolated from the neuronal and glial cells, those corresponding to neuronal plasma membranes and microsomes showed a noticeably higher exchange of serine and ethanolamine compared to the corresponding subfractions from glia. Neuronal/glial ratios of about 6–8 were found for the exchange activity in both plasma membrane‐enriched fraction and in microsomes. Synaptosomes and synaptosomal subfractions contained low activities. It is concluded that the calcium‐dependent enzymic system for the exchange of serine, ethanolamine and other nitrogenous bases with endogenous phospholipid is concentrated mostly in the neuronal perikaryal membranes, and could be used as a neuronal marker.

The motility of human spermatozoa as influenced by prostasomes at various pH levels.

Human semen contains several components among which spermatozoa, membranous vesicles called ‘prostasomes’, secreted by the prostate gland and unorganized material. Prostasomes possess an unusual lipid composition, contain a number of proteins and small molecules and have been claimed to take a part in the immune response, in seminal fluid liquefaction and in sperm motility. Since sperm may come in contact with an acidic environment in the vagina, it may be of some interest to know whether prostasomes may affect spermatozoon motility or may protect spermatozoa upon the exposure to an acidic milieu. Human semen was supplied by donors. From whole semen we collected spermatozoa by centrifugation and used the supernatant to prepare prostasomes (centrifugation at 105000 g for 120 min, followed by purification step on Sephadex G 200); spermatozoa were then collected by a swim‐up procedure and exposed to an acidic pH medium (from 5 to 7) in the presence or absence of prostasomes. Spermatozoa motility was subsequently assessed with a superimposed image analysis system (SIAS). Results indicate that the motility of spermatozoa was affected by the pH value of the medium. Acidic media reduced the percentage of motile cells and decreased the straight line velocity of spermatozoa (VLS). Prostasomes had a protective effect and increased the percentage of motile cells. However, they did not change the characteristics of motility (curvilinear and straight). Prostasomes may be considered as a system for counteracting the negative effects of acidic pH values that may be present in the vagina after coitus.

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.

Prostasome to sperm transfer of CD13/aminopeptidase N (EC 3.4.11.2)

Prostasomes are membranous vesicles (150-200 nm in diameter) that are present in human semen. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin and Ca2+. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. In previous papers, we demonstrated that lipid can be transferred from prostasomes to sperm by a fusion process occurring at slightly acidic pH. CD (cluster antigens) are ubiquitous proteins; in this paper, we demonstrate that CD13/aminopeptidase N is present is semen, where it is bound to prostasomes. Upon mixing prostasomes and sperm at slightly acidic pH (7 or less), aminopeptidase is transferred from prostasomes to sperm. This evidence comes from enzymatic activity determinations and from the use of the monoclonal antibody, anti-human CD13. The transfer was about 8% of total prostasomal activity at pH 5 and with a prostasome to sperm ratio of 2 (on a protein basis). The transfer did not occur at pH 8.0, but was measurable at pH 7. Therefore, this mechanism may represent a means of modifying the composition and the biological properties of ejaculated sperm.

Transfer of CD26/dipeptidyl peptidase IV (E.C. 3.5.4.4) from prostasomes to sperm.

Prostasomes are vesicles present in human semen. They are secreted by the prostate and contain large amounts of cholesterol and sphingomyelin. Some of their proteins are enzymes. Prostasomes are involved in a number of biological functions. In previous papers we demonstrated that lipid can be transferred from prostasomes to sperm by a fusion process occurring at neutral or slightly acidic pH. In this paper we demonstrate that CD26/dipeptidyl peptidase IV, an enzymatic activity absent in sperm, is transferred to sperm from prostasomes. This may be of particular interest since, by this procedure, sperm may acquire new membrane‐bound enzymes and modify the catalytic activity of their surface.

NO Synthesis in Human Saliva

Human saliva contains nitrate that is converted into nitrite by the activity of facultative, anaerobic bacteria of the oral cavity. Nitrite can be reduced to NO in the acidic gastric milieu; some NO may also form in the mouth at acidic pH values. In this paper, we show that bacteria ( S. salivarius, S. mitis and S. bovis ) isolated from saliva, may contribute to NO production in human saliva. NO formation by bacteria occurs at neutral pH values and may contribute to the antibacterial activity of saliva.

Transfer of aminopeptidase activity from prostasomes to sperm

Prostasomes are membranous vesicles (150-200 nm diameter) present in human semen. They are secreted by the prostate and contain large amounts of cholesterol, sphingomyelin and Ca2+. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. It has been demonstrated that lipid can be transferred from prostasomes to sperm by a fusion process occurring at slightly acidic pH. In this paper, we show that an aminopeptidase activity is transferred from prostasome to sperm. This may be of particular interest since it indicates the involvement of protein in the process of fusion and because sperm may acquire new membrane-bound proteins by this procedure.

Metabolism of Phosphoglycerides

Glycerophospholipids are present in all living organisms and are necessary for membrane assembly and function. Their presence in nervous tissue has been recognized for many years,1 and, with the development of suitable analytical techniques, their metabolism has been amply studied and is being investigated in different aspects in several laboratories.

Acetyl-l-carnitine influences the fluidity of brain microsomes and of liposomes made of rat brain microsomal lipid extracts

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) was measured in different preparations (bovine spinal cord phosphatidylserine liposomes, rat brain microsomes, liposomes made with rat brain microsomal lipid having different phospholipid:cholesterol ratios) at temperatures ranging from 10° to 55°C. Phosphatidylserine liposomes exhibited an exponential relationship of rversus temperature, whereas the relationship shown by microsomes and liposomes prepared with microsomal lipid extracts was a linear one. The removal of protein and high phospholipid:cholesterol ratios decreased the slope of the lines (fluidity increased), although the intercept was unaffected. This means that differences were better appreciated at high temperatures and were well evident at 37°C. Acetyl-l-carnitine decreased r in rat brain microsomes and in liposomes made with microsomal lipids with different phospholipid:cholesterol ratios. The fluidifying effect of acetyl-l-carnitine was mild but statistically significant and could explain, at least in part, the data reported in the literature of acetyl-l-carnitine acting on some parameters affected by ageing. Besides, acetyl-l-carnitine seemed to oppose the changes of viscosity due to lipid peroxidation, which has been reported to increase in ageing and dementia.l-carnitine shares the properties of its acetyl ester, but only in part.