Sperm Cytoplasmic Droplets Research Articles

Smoking is associated with the retention of cytoplasm by human spermatozoa

Objectives. To determine whether cigarette smoking is associated with the abnormal retention of residual sperm cytoplasm in infertile men. Methods. Semen samples were obtained from 87 consecutive non-azoospermic men with idiopathic infertility (18 smokers and 69 nonsmokers) and from 20 men presenting for vasectomy (fertile controls). Standard semen parameters and the percentage of spermatozoa with residual cytoplasm (on Papanicolaou smears) were recorded. Results. Subject age, semen volume, and sperm density, motility, and morphology were not significantly different between the two groups of infertile men. However, a significant difference was found in the mean ± SEM percentages of sperm with cytoplasm droplets between smokers and nonsmokers (12.9% ± 1.7% and 8.1% ± 0.9%, respectively; P < 0.001). Conclusions. Our data suggest that cigarette smoking is associated with retention of sperm cytoplasmic droplets in infertile men, a morphologic characteristic associated with impaired sperm function.

Age‐related changes in the hamster epididymis

Reproductive ability is decreased in aged animals and in men. Little is known about the changes taking place in the epididymis, and the possible influence on the loss of sperm quality. We studied the age-related alterations in the epididymis and in epididymal spermatozoa of hamsters. Adult (6-month-old), middle-aged (18-month-old), and aged (24-month-old) hamsters were used. Serum samples were obtained to determine testosterone levels. Testes and epididymides were removed and studied by light and electron microscopy. Epididymal sperm was also obtained and the motility, position of cytoplasmic droplet, and concentration were evaluated. Measurements of the height of the epithelium, length of stereocilia, external tubular diameter, and thickness of the muscular wall were performed. The proliferative activity was also studied. An ANOVA analysis was used to compare quantitative differences between epididymal zones and age groups. Aged hamsters presented involutive changes in the epididymis. A decrease in tubular diameter was found in cauda; principal cell ultrastructure showed changes including the appearance of damaged mitochondria, bundles of filaments, and the accumulation of lipofuscin. Some clear cells showed an unusual morphology by the presence of large electrondense vacuoles. A reduction in sperm quality was also observed, including a decrease in sperm motility and concentration, and alterations in the migration of sperm cytoplasmic droplet. Testosterone levels and cellular proliferative activity did not change. Aging causes a morphological alteration of hamster epididymis (mainly in the cauda), and a decrease in sperm quality. Anat Rec 256:335–346, 1999. © 1999 Wiley-Liss, Inc.

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.

Biochemical studies of metalloendoprotease activity in the spermatozoa of three mammalian species.

Ejaculated porcine and human spermatozoa, hamster spermatozoa from the cauda epididymidis, isolated hamster sperm heads and hamster cytoplasmic droplets contained activity that hydrolyzed the metalloendoprotease substrate ABZ-Ala-Gly-Leu-Ala-NBA (AAGLAN). Hamster sperm heads were isolated by treating spermatozoa with proteinase K and removing sperm tails with Dowex-50W beads. Hamster sperm activity was characterized using spermatozoa from which cytoplasmic droplets were removed by sonication and centrifugation. Porcine sperm preparations were essentially free of cytoplasmic droplets, while human sperm preparations retained somewhat more droplet material. Activity from all of these sources was inhibited by the metalloendoprotease inhibitors phosphoramidon, 1,10-phenanthroline, CBZ-D-Phe and CBZ-L-Phe but was not competitively inhibited by the metalloendoprotease substrate CBZ-Ser-Leu-amide. The AAGLAN hydrolyzing activity found in intact spermatozoa of all three species had a pH optimum of 6.2, while the optimum of the hamster sperm cytoplasmic droplet activity was 7.0. In addition, hamster sperm preparations were inhibited by ZnCl2 and dithiothreitol, but were not affected by toluene, benzamidine or chymostatin. The AAGLAN hydrolyzing activity of hamster sperm preparations was reduced, but not eliminated, by dialysis. It is concluded that spermatozoa from all three species, hamster sperm heads and hamster cytoplasmic droplets contain metalloendoprotease activity. Furthermore, metalloendoprotease activity found in hamster cytoplasmic droplets is different from that found in spermatozoa.

Phagocytosis of sperm cytoplasmic droplets by a specialized region in the epididymis of the brushtailed possum, Trichosurus vulpecula.

During sperm maturation in the brushtailed possum, Trichosurus vulpecula, cytoplasmic droplets are shed from maturing spermatozoa in the distal regions of the head of the epididymis. Examination of luminal contents from various regions of the epididymis showed that the proportion of detached droplets in the luminal contents was reduced from about 45% in the proximal corpus epididymidis to less than 10% in the distal corpus and cauda epididymides. In contrast, the proportion of droplet-free spermatozoa increased from about 45% to more than 90% in the luminal contents. Disappearance of detached cytoplasmic droplets from the lumen was found to be associated with a region of specialized principal cells lining Regions 6 and 7 of the epididymis which selectively sequester and phagocytose free droplets from the luminal milieu. The luminal surfaces of these cells are characterized by a complex system of interdigitating processes which appear as waves of microfolds . These processes contrast with the stereocilia which cover the luminal surfaces of principal cells in adjacent, nonphagocytic regions of the duct. Cytoplasmic droplets are phagocytosed with their limiting membrane intact and gradually become condensed as they are transported deeper into the cell. Membrane lamellae are gradually compacted, transformed into concentrically arranged membrane stacks and then condensed into small electron-dense vesicles, which are probably degraded by the epithelial cells. The presence of a specific recognition factor on cytoplasmic droplets is suggested by the observation that phagocytic principal cells are able to selectively remove detached cytoplasmic droplets from the lumen in the presence of sperm-associated droplets and spermatozoa.

Boar sperm cytoplasmic droplets: Their ultrastructure, their numbers in the epididymis and at ejaculation and their removal during isolation of sperm plasma membranes

Cytoplasmic droplets of the boar are progressively lost from the flagellum of boar spermatozoa during epididymal transit, at ejaculation and during the nitrogen cavitation technique for isolation of plasma membranes. Apparently very fragile, these structures are broken up in the fluids of the reproductive tract and in the buffer used during the nitrogen cavitation procedure. The maximal potential contamination of cytoplasmic droplet internal vesicular membranes in plasma membrane fractions was determined to be 2.2% of the entire membrane surface area collected. The highly sensitive silver-stained, two-dimensional (2-D) polyacrylamide (PAGE) gels of boar sperm plasma membranes did not reveal cytoplasmic droplet, internal membrane, marker polypeptides, further demonstrating the high purity of plasma membrane preparations. In addition, freeze-fracture demonstrates that the internal membranes of the cytoplasmic droplet show few intramembranous particles and these may contribute little protein to plasma membrane preparations. The presence of two forms of vesicular elements in boar sperm Cytoplasmic droplets (typical vesicles and collapsed vesicles) is described.

Novel affinity chromatographic methods for purification of β- N-acetylhexosaminidase from rabbit sperm cytoplasmic droplets

1. 1. Two affinity chromatography columns for β- N-acetylhexosaminidase were prepared by coupling desialylated Cowper's gland mucin of the boar and desialylated fetuin to Sepharose 4B. These affinity adsorbents completely retained the enzyme. 2. 2. The enzyme was readily eluted either by changing the ionic strength of the buffer or by washing the columns with yN-acetylglucosamine. 3. 3. This method is simple and gives 5–10-fold purification of N-acetylhexosaminidase with 80–85% recovery of the enzyme activity. 4. 4. The β- N-acetylhexosaminidase from rabbit sperm cytoplasmic droplets showed maximum activity at pH 4.O. 5. 5. The K mn and V max values for the hydrolysis of p-nitrophenyl N-acetyl β- d-glucosaminide were 1.42 mM and 2.0 μmol/min per mg protein and for the corresponding galactosaminide 0.75 mM and 0.48 μmol/min per mg protein respectively. 6. 6. The enzyme is strongly inhibited by Hg 2+ and by p-chloromercuribenzoate.

Pyruvate metabolism in boar spermatozoa.

Summary. Boar spermatozoa utilize pyruvate efficiently both anaerobically and aerobically. Pyruvate metabolism, in marked contrast to fructolysis, continues in spermatozoa immobilized by cold shock. Sperm cytoplasmic droplets alone do not metabolize pyruvate. Carbon dioxide, lactate, acetate, succinate and acetoacetate were identified as anaerobic products of pyruvate metabolism; the ratio between pyruvate utilized and lactate and acetate formed did not fit into the stoichiometry of a simple dismutation. Carbon dioxide and lactate were formed from pyruvate aerobically. The rate of pyruvate disappearance, as well as the percentage of pyruvate converted to lactate, were higher aerobically than anaerobically. Very little acetoacetate was utilized by boar spermatozoa aerobically. Lactate was oxidized but at a much lower rate than pyruvate. Acetate (0·18 μmol/109 cells) was extracted from exhaustively washed spermatozoa; this acetate is assumed to be derived from acetyl compounds of the sperm cells.