Inhibition of acrosin by oviductal secretory immunoglobulin A

A major inhibitor of acrosin in rhesus monkey and rabbit oviduct fluid, isolated by isoelectrofocusing in sucrose gradients, displayed a broad peak in the acidic region of the column and was demonstrated to contain secretory IgA specific for acrosin. Its identity was established by immunodiffusion, by the removal of acrosin inhibition with antisera to IgA (α-chain), and by its correct molecular weight during ultracentrifugation. Purified human serum IgA also inhibited rabbit, rhesus monkey, and human acrosins, but neither purified human IgG nor IgM had any inhibitory effect on these acrosins. Neither oviduct fluid secretory IgA nor purified human serum IgA inhibited the activity of bovine pancreatic trypsin. The high specificity of secretory IgA for acrosin and its presence in every rabbit and rhesus monkey oviduct fluid specimen examined suggests a possible regulatory role for this antibody in reproduction.     

The effects of taurine and hypotaurine on in vitro fertilization in the golden hamster

Taurine and hypotaurine were examined for their efficacy in replacing sperm motility factor (SMF), prepared from bovine adrenal cortex, for in vitro fertilization in the golden hamster. Combinations of these amino acids at concentrations of 0.001, 0.01, 0.1, and 1 mM together with 16 μM isoproterenol (a catecholamine β-agonist) were added to the sperm incubations. After three hours of sperm preincubation, oviductal eggs were added to the sperm suspensions and examined for penetration and stage of fertilization after three or five hours of culture. At 0.001 mM, neither taurine or hypotaurine was capable of maintaining motility of hamster sperm for four to 4½ hours or of inducing fertilization. With all other concentrations, both amino acids were found to maintain motility of sperm as well as SMF. Hypotaurine stimulated motility to a greater extent than taurine and both required isoproterenol for the greatest motility. A low proportion of cumulus-free ova were fertilized when sperm were preincubated with either amino acid alone over the range of 0.01 to 1 mM; however, over 80% fertilization was consistently obtained when isoproterenol was also present during sperm incubation. Proportions of ova fertilized with taurine or hypotaurine present during sperm preincubation were comparable to those achieved with SMF. The possibility that taurine or hypotaurine is the sperm motility factor is discussed. After three hours of sperm/egg incubation, a lag in the early events of fertilization was observed in experimental groups treated with one of the amino acids (0.01 mM) alone compared with groups treated with isoproterenol present. However, if sperm/egg incubation was extended from three to five hours, no increase in number of eggs penetrated was found. Therefore, the delay observed at three hours was considered a function of fewer numbers of capacitated sperm present in the absence of isoproterenol rather than of the need for an extended capacitation time.     

Influence of sialic acid on cell surface properties in I-cell disease fibroblasts

Summary Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity of the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing. This project was supported in part by National Institutes of Health (NIH) BRSG Grant RR-05493, NIH Grant 1-R01-HD-11453-01-A1, National Science Foundation Grant PCM 77-05733, and Maternal and Child Health Service Project 417. Georgirene D. Vladutiu is the recipient of Research Career Development Award 1K04 HD 00312-01A1 from the National Institutes of Health.     

Selective binding of specific rat epididymal secretory proteins to spermatozoa and erythrocytes

Tissue pieces from the caput epididymidis of the rat were incubated in vitro with (³⁵S) methionine to produce radioactive secretory proteins. The radioactive secretory proteins so formed were tested for their ability to bind to washed rat spermatozoa collected from the rete testis and cauda epididymidis, and to rat erythrocytes. The sperm and erythrocytes bound approximately 5% of the total radioactive protein. Binding was protein-specific in that only selected proteins became associated with the cells. Binding was not cell-specific, however, since testicular spermatozoa, caudal spermatozoa, and erythrocytes all bound the same proteins to a similar degree.     

Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

F-actin in guinea pig spermatozoa: its role in calmodulin translocation during acrosome reaction.

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS-PAGE and Western blots immunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility.     

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

羊精子体外获能

本文发展了一种羊精子体外获能培养基——mTs或RSCM。羊精子在该培养基中(39℃.pH7.8和5％ CO_2,95％空气)预先培养7小时,可使大部分精子获能。获能精子呈现超激活运动,并可穿透去透明带仓鼠卵,穿透率分别为78.5±14.3％和96.7±2.3％,这种作用可被同种精浆逆转。获能精子与同种卵的受精率为83.3％。16个受精卵等量移入2只假孕兔输卵管壶腹部中。72小时后回收到12个胚胎。其中6个胚胎已发育为4—8细胞阶段,将这些细胞等量移入2只受体母羊输卵管壶腹部。其中1只妊娠,并维持到2个月之久。     

A simple method forin situ freezing of anchorage-dependent cells including rat liver parenchymal cells

We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish. The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at –80°C. After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.     

Primary culture and cryopreservation of mouse astrocytes under serumfree conditions

The methods of primary culture and cryopreservation of mouse astrocytes under serum-free conditions were examined. Cerebra from newborn C3H/He mice were employed as the source of astrocytes. The cultured cells were able to grow in a serum-free, chemically defined medium containing transferrin, hydrocortisone, biotin, sodium selenite, insulin, fibroblast growth factor and epidermal growth factor. After the culture was maintained in the medium for 3 weeks, purity was assessed using immunofluorescence staining. The great majority of the cells (>98%) contained glial fibrillary acidic protein and S-100 protein which are cell markers of astrocytes. To cryopreserve the enriched astrocytes under serum-free conditions, various cryoprotectants were examined. The combination of 10% dimethylsulfoxide and 0.1% methylcellulose gave the highest survival rate. These methods of primary culture and cryopreservation will be useful in physiological and biochemical studies which require mouse astrocytes.