Enrico Carlini

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.

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.

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.

Distribution of lipid and protein in human semen fractions.

Human semen is formed by the secretions of different glands. We fractionated semen by centrifugation and obtained four main fractions: (a) spermatozoa, (b) material precipitating at 10¿ omitted¿000xg, (c) prostasomes (precipitate at 105¿ omitted¿000xg), and (d) a soluble fraction. When required, fractions were purified further. We find that most semen protein (about 85%) is in the soluble fraction, 7% in spermatozoa and the remainder is scattered in the other fractions. We compared the electrophoretic pattern of soluble protein with the protein of prostasomes and found marked differences. On the other hand, prostasomes, that comprises only about 3% of total semen protein, contain about 45% of cholesterol and almost 15% of lipid phosphorus with a cholesterol to phospholipid molar ratio greater than 2. On the contrary, phospholipid is largely bound to the fraction containing spermatozoa (about 46% of total lipid phosphorus). This fraction is poor in cholesterol and has a cholesterol to phospholipid molar ratio of about 0.2. The distribution of lipid phosphorus among lipid classes shows some similarity in the soluble fraction and in prostasomes; in both fractions, sphingomyelin is the most abundant phospholipid (about 50%). On the other hand, phosphatidylcholine is the main phospholipid in spermatozoa-enriched fractions (about 35% of total lipid phosphorus). We conclude that the various fractions of seminal plasma obtained by centrifugation differ markedly from each other as to lipid and protein content.

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.

INTERACTIONS BETWEEN PROSTASOMES AND LEUKOCYTES

Prostasomes are membranous vesicles (150-200 nm diameter) 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 sperm and are involved in a number of biological functions. In a previous work, we found that prostasome can fuse to spermatozoa at slightly acidic pH values, as demonstrated by the transfer of the lipophilic octadecylrhodamine probe. In this paper, we study the interactions of two leukocyte populations (polymorphonuclear and mononuclear) with prostasomes and find a pH-dependent adhesion (revealed by microscopic observation), but no fusion. These phenomena may be relevant for the functions of leukocytes in human reproduction.

Transport of phosphatidylserine from microsomes to the inner mitochondrial membrane in brain tissue.

Abstract: Phosphatidylserine was labeled by incubating rat brain homogenates with [3‐14C]serine in the presence of Ca2+ (base‐exchange conditions). Some labeled phosphati‐dylethanolamine also forms, in spite of the inhibition of Ca2+ on phosphatidylserine decarboxylase. Phosphatidylserine labeling and decarboxylation also occur on incubating a mixture of purified mitochondria and microsomes, suggesting that no soluble factors are necessary for the synthesis and the decarboxylation of phosphatidylserine. Ca2+ favors the transfer of phosphatidylserine from microsomes (where it forms) to mitochondria (where it is decarboxylated). The specific radioactivity of the phosphatidylserine transferred to mitochondria is higher than that of microsomal phosphatidylserine. This finding supports the hypothesis that the lipid is compartmentalized in microsomes and that radioactive, newly synthesized phosphatidylserine is much better exported than the bulk of microsomal phospholipid.