[1] 22w matophore collapses, hardens, and becomes yellowish brown. It may remain attached to the female genital opening for a few hours or days.

[2] 96w The various stages of the formation of the spermatophore were closely followed under a stereoscopic microscope. I ascertained their temporal sequence by interrupting copulation at various stages. Although the whole process of extruding the ecto-spermatophore, filling it with sperm, and extruding the endo-spermatophore takes only 20 to 30 seconds, by close observation and quick manipulation it was possible to interrupt spermatophore formation when the ecto-spermatophore was either empty or filled only with sperm. In either case, once the male had started to copulate, it continued the chain of instinctive actions, even if they were in vain.

[3] 39w If, at the right moment, the stillempty ecto-spermatophore is removed from the male genital aperture, the male will eject sperm, large proteinic granules, and adlerocysts, and will extrude the endo-spermatophore on the surface of its own body (Fig. 2).

[4] 109w If the ecto-spermatophore is removed when it is already full of sperm, the male will eject adlerocysts and extrude the endo-spermatophore, the adlerocysts adhering closely to the exterior surface of the latter. When the endo-spermatophore is extruded from the male genital aperture to the outside (after the ecto-spermatophore has been removed), its shape is also that of a bilobate bottle, but the two lobes are considerably larger than they are inside the ecto-spermatophore (Fig. 2), although they are not as large as when they are filled with matophore collapses, hardens, and becomes yellowish brown. It may remain attached to the female genital opening for a few hours or days.

[5] 96w The various stages of the formation of the spermatophore were closely followed under a stereoscopic microscope. I ascertained their temporal sequence by interrupting copulation at various stages. Although the whole process of extruding the ecto-spermatophore, filling it with sperm, and extruding the endo-spermatophore takes only 20 to 30 seconds, by close observation and quick manipulation it was possible to interrupt spermatophore formation when the ecto-spermatophore was either empty or filled only with sperm. In either case, once the male had started to copulate, it continued the chain of instinctive actions, even if they were in vain.

[6] 39w If, at the right moment, the stillempty ecto-spermatophore is removed from the male genital aperture, the male will eject sperm, large proteinic granules, and adlerocysts, and will extrude the endo-spermatophore on the surface of its own body (Fig. 2).

[7] 119w If the ecto-spermatophore is removed when it is already full of sperm, the male will eject adlerocysts and extrude the endo-spermatophore, the adlerocysts adhering closely to the exterior surface of the latter. When the endo-spermatophore is extruded from the male genital aperture to the outside (after the ecto-spermatophore has been removed), its shape is also that of a bilobate bottle, but the two lobes are considerably larger than they are inside the ecto-spermatophore (Fig. 2), although they are not as large as when they are filled with 2 JUNE 1967 sperm in the uterus. If the endospermatophore is dropped into saline on a slide, one can see the huge number of adlerocysts which cover the lobes of the endo-spermatophore.

[8] 260w The processes that normally occur in the female genital tract after the deposition of the spermatophore on the female genital aperture can be studied in vitro. If a completely formed spermatophore is removed from the male before it is deposited on the female and if it is put into saline or left on a dry slide, within 1 to 2 minutes the endo-spermatophore evaginates from the ecto-spermatophore. The evagination occurs whether the spermatophore was taken off the male genital opening or off the male chelicerae. The evagination starts without any intervention. At first, a stem pops out (Fig. Id), and immediately afterwards the two lobes of the endospermatophore are everted and evaginated (Fig. le). At this stage arise most of the minor differences between 0. savignyi and 0. tholozani, to which this description refers, and some other species of Argasid ticks. The everted neck of the endo-spermatophore extends and elongates. During evagination, the lobes turn inside out, and adlerocysts and sperm which were in the ecto-spermatophore, but outside the endo-spermatophore, stream quickly into the two everted lobes of the endo-spermatophore (Fig. If) until no sperm remains in the ectospermatophore. Only after the lobes have entirely been filled up with sperm do they separate slowly to form two distinct capsules, but both are still connected to the ecto-spermatophore by the neck (Fig. Ig). The sperm and adlerocysts, which were entirely separated in the ecto-spermatophore, are mixed in these capsules. Thus, during the whole process of transfer of sperm from the male to the female, the semen remains in a closed system.

[9] 87w In the normal course of copulation (but not in vitro), the two capsules are soon disconnected from the ectospermatophore. The separation occurs at the neck, near the capsules. The longer part of the neck, which originally belonged to the endo-spermatophore, remains attached to the ectospermatophore and can be seen when it falls off the female (Fig. lh). The sperm in the uterus. If the endospermatophore is dropped into saline on a slide, one can see the huge number of adlerocysts which cover the lobes of the endo-spermatophore.

[10] 260w The processes that normally occur in the female genital tract after the deposition of the spermatophore on the female genital aperture can be studied in vitro. If a completely formed spermatophore is removed from the male before it is deposited on the female and if it is put into saline or left on a dry slide, within 1 to 2 minutes the endo-spermatophore evaginates from the ecto-spermatophore. The evagination occurs whether the spermatophore was taken off the male genital opening or off the male chelicerae. The evagination starts without any intervention. At first, a stem pops out (Fig. Id), and immediately afterwards the two lobes of the endospermatophore are everted and evaginated (Fig. le). At this stage arise most of the minor differences between 0. savignyi and 0. tholozani, to which this description refers, and some other species of Argasid ticks. The everted neck of the endo-spermatophore extends and elongates. During evagination, the lobes turn inside out, and adlerocysts and sperm which were in the ecto-spermatophore, but outside the endo-spermatophore, stream quickly into the two everted lobes of the endo-spermatophore (Fig. If) until no sperm remains in the ectospermatophore. Only after the lobes have entirely been filled up with sperm do they separate slowly to form two distinct capsules, but both are still connected to the ecto-spermatophore by the neck (Fig. Ig). The sperm and adlerocysts, which were entirely separated in the ecto-spermatophore, are mixed in these capsules. Thus, during the whole process of transfer of sperm from the male to the female, the semen remains in a closed system.

[11] 117w In the normal course of copulation (but not in vitro), the two capsules are soon disconnected from the ectospermatophore. The separation occurs at the neck, near the capsules. The longer part of the neck, which originally belonged to the endo-spermatophore, remains attached to the ectospermatophore and can be seen when it falls off the female ( The submaxillary gland of the male mouse contains a number of proteins that have specific growth-promoting properties. One of these, the nervegrowth factor, stimulates the growth and differentiation of embryonic sensory nerve cells and embryonic and mature sympathetic nerve cells (1). Subsequently, another protein, the epidermal-growth factor which promotes the growth of epidermal cells, was purified from the submaxillary gland (2).

[1] 98w The material responsible for the latter effects was also considered to be a pro- tein on the basis of properties noted during its isolation and partial purification. To characterize this material further and to determine its possible mode of action on embryonic muscle tissue, we have purified the factor and examined its enzymatic properties. The highly purified material is a protein containing both esterase and peptidase activities. Several properties of this enzyme have been measured and compared with another protein that was purified from the salivary gland of the mouse during the isolation of the biologically active material.

[2] 136w In previous fractionation of salivary gland extracts, the biologically active factor was eluted from a DEAE-Sephadex column with 0.1M NaCl. Accordingly, partially purified material which had been first fractionated with ammonium sulfate and passed over a Sephadex G-100 column (3) was dialyzed against 0.01M tris-Cl, pH 7.4, and pumped onto a DEAE-cellulose column (Fig. 1). Those fractions with high biological activity were pooled, dialyzed, and pumped again onto a DEAE-cellulose column. Fraction A (Fig. 1, II) had high biological activity and gave essentially a single protein band when examined by acrylamide-gel electrophoresis (Fig. 2). A similar electrophoretic analysis of other eluates from these column separations showed that tube 35 of column I contained primarily a single protein band (B). This protein was subsequently found to have active esterase activity and some effect on the embryonic tissue.

[3] 135w The highly purified proteins, A and teolysis, but in the absence of proteases they can reassociate to form native material unaffected by proteolytic enzymes (4). Despite the apparent proteolytic activity of B, it has only one-tenth the peptidase activity of A. Even more striking is the effect of the protease inhibitor, phenylmethanesulfonyl fluoride (5). Under conditions in which trypsin is inhibited 90 percent by this chemical, A is inhibited to 80 percent, whereas B is unaffected. Thus, these two proteins must have distinctive catalytic centers despite their relative similarity in substrate specificities. Protein A has been examined in the analytical Spinco ultracentrifuge (Model E) and found to have a sedimentation coefficient (so0,,) value of 3.3S. If we assume this to be a globular molecule, the molecular weight is estimated to be between 30,000 and 40,000.

[4] 174w The mechanism of action of protein A on embryonic muscle tissue has not been ascertained. After 24 hours in the presence of protein A, fragments of embryonic skeletal muscle tissue are radically modified in their structure when compared with a control culture. The dissociation of the muscle fibers into individual myoblasts with a loss of myosin is a striking feature of the treated tissue, readily observable in histological sections (Fig. 3). Tissue exposed to chemically inhibited protein A, however, shows no substantial difference from the control culture (Fig. 3c). Thus, inhibition of enzymatic activity of protein A also leads to a loss of biological activity. Possibly, protein A cleaves specific peptide bonds essential to maintenance of the differentiative state of the tissue and thus leads to a series of events that result in the loss of cell specificity and disappearance of myosin. Unlike trypsin which causes eventual tissue disaggregation, the presence of protein A results in the formation of a very compact mass of undifferentiated cells, even when it is added in large amounts.

[5] 63w The addition of protein B to embryonic muscle brings about a modification which is clearly distinct from that of A in the first 24 hours of incubation (Fig. 3d) but is somewhat similar to the effect with very low doses of trypsin. Protein B and trypsin cause a disaggregation of muscle fibers with sloughing of cells from the periphery of the tissue fragment.

[6] 26w In previous studies with the partially purified fraction of the mouse gland, a variety of tissues of mesodermal origin were found to be affected to various

[7] 47w SCIENCE, VOL. 156 tidase activities inhibited by phenylmethanesulfonyl fluoride, a chemical reported to inhibit proteolytic activity strongly. The observation that the inhibited protein also loses its biological activity supports the conclusion that the action of this protein on muscle tissue is a result of its enzymatic activity.

[8] 108w Table 1. Comparison of enzymatic activities of purified salivary gland proteins and trypsin. Reaction 1: hydrolysis of benzoylarginine ethyl ester. Reaction 2: hydrolysis of benzoyltyrosine ethyl ester. Reaction 3: hydrolysis of benzoylarginine naphthyl amide. Reaction 4: inhibition of reaction 1 by phenylmethanesulfonyl fluoride (mole ratio inhibitor to protein, 10:1). Reaction 5: inhibition of reassociation of alkaline phosphatase subunits of Escherichia coli. Reaction 6: proteolysis of casein. Results of reactions (R) Test substance R* R R3t R4 R5? R6" (unit/mg) (,ug/min) ( % ) (%) (% ) Fraction A 164 0 24 80 14 2 Fraction B 344 0 2.5 0 46 Trypsin 15 0 11.2 90 99 100

[1] 53w There the two capsules remain attached to each other for various lengths of time depending on external conditions, such as subsequent copulation and feeding by the female. The capsules then separate, and every two capsules found in the uterus represent one copulation. The sperm remains viable in the capsules for many months (6).

[2] 13w Abstract. A protein from the salivary gland of mice has been highly purified.