Category Archives: Sperm

Developmental Profile of a Caltrin-Like: MATERIALS AND METHODS(4)


The slides were immersed in a blocking solution (5% nonfat skim milk in PBS) in a moisture chamber at 25°C for 1 h and washed with PBST (0.05% Tween in PBS) four times, each for 15 min. The testis sections on slides were preincubated with 3.0 ^M P12 in PBS at 25°C for 1 h, and the slides were washed with PBST as above. The slides prepared from each tissue were incubated for 2 h in the presence of the P12-induced antiserum diluted 1: 250 in blocking solution. After the slides had been gently agitated in four changes of PBST for 15 min each, the antibody to P12 were immunodetected with alkaline phosphatase-conjugated anti-rabbit IgG (Sigma, St. Louis, MO) diluted 1:1000 in the blocking solution. The sections were covered with 50% glycerol in PBS and photographed with a microscope (AH3-RFCA; Olympus, Tokyo, Japan) after they had been washed with three changes of PBS for 15 min each. birth control yasmin

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Developmental Profile of a Caltrin-Like: MATERIALS AND METHODS(3)

The cell suspension was gently filtered through two layers of nylon gauze, layered on top of a linear gradient of 10-90% Per-coll (Pharmacia), and centrifuged at 150 X g for 10 min. Two distinct cell layers were formed. The lower layer, which contained more than 95% of viable cells with progressive motility, was diluted with three volumes of modified Tyrode’s solution and centrifuged at 150 X g to remove Percoll solution. The cells were resuspended in modified Tyrode’s solution (1.0 X 107 cells/ml) and used for the study. Throughout this study we used only the preparation in which more than 70% of cells were motile. buy flovent inhaler

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Developmental Profile of a Caltrin-Like: MATERIALS AND METHODS(2)


The protein extract of tissue homogenate was resolved on a 15% polyacrylamide gel slab (6.5 cm X 10.5 cm X 0.075 cm) by the method of Laemmli. The proteins on the gel were stained with Coomassie brilliant blue dye or transferred to a nitrocellulose membrane. After transfer, protein blots were immunodetected by Western blot procedures, using the P12-induced antisera or the partially purified antibody as the primary antibody and goat anti-rabbit IgG conjugated with horseradish peroxidase as the secondary antibody.

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Developmental Profile of a Caltrin-Like: MATERIALS AND METHODS(1)

Preparation of P12 and Its Derivatives

The seminal vesicles of mature male mice (ICR, 8-12 wk; Charles River Laboratories, Wilmington, MA) killed by cervical dislocation were carefully dissected free of the adjacent coagulating glands. The secretions collected from 100 mice were expressed directly into 100 ml of ice-cold 5% acetic acid. P12 was purified from SVS according to our previous procedure. A fusion protein of GST-P12 was prepared from the expression of a recombinant DNA in E. coli. A recombinant P12 with the full activity of the naturally occurring P12, with regard to its inhibitory effect on trypsin activity, was recovered from the chimerical polypeptide. canadian

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Developmental Profile of a Caltrin-Like Protease Inhibitor, P12, in Mouse Seminal Vesicle and Characterization of Its Binding Sites on Sperm Surface

Sperm Surface

Existence of protease inhibitors (PIs) in the sexual glands of mammals is well known. They have physiological functions in addition to their inhibitory effect on protease activity. It is believed that they are important for the protection of genital tract epithelium against proteolytic damage and/or have a regulatory role in the fertilization process. The trypsin-like activity seems to involve the binding of mouse spermatozoa to the zona pel-lucida. In rat seminal vesicle, one calcium transport inhibitor (caltrin), which is identical to pancreatic secretory trypsin inhibitor, is able to suppress Ca2+ uptake by spermatozoa to prevent a premature acrosome reaction far from the oviduct. Hence, the study of PIs in the genital tract becomes an important subject of reproductive biology.

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