Summary. The total protein content and the activities of lysosomal hydrolases (arylsulphatase, alkaline and acid phosphatases, β-glucuronidase, β-N-acetylhexosaminidase, α-L-fucosidase and β-galactosidase) in the uteri of ovariectomized rabbits treated with different concentrations of progesterone, oestradiol-17β and a combination of progesterone and oestradiol were determined. The enzyme activities were also measured in the reproductive organs of rabbits induced to superovulate by PMSG and hCG. In superovulated and steroid-treated rabbits, the changes in lysosomal hydrolases were more obvious in the endometrium than the myometrium. Except for the myometrial alkaline phosphatase and β-galactosidase and the endometrial alkaline phosphatase, there were no significant changes in the solubilities of hydrolases after treatment with steroids. β-Galactosidase levels were significantly higher in the ovariectomized rabbits treated with progesterone. An antagonistic effect of oestradiol and progesterone was observed with respect to uterine weight, protein content and enzyme activities in the ovariectomized rabbits treated simultaneously with oestradiol and progesterone.
H. Rahi and P. N. Srivastava
C. H. Yang and P. N. Srivastava
High concentrations of α-chlorohydrin were found to inhibit hyaluronidase, β-glucuronidase, and aryl sulphatases in bull and rabbit spermatozoa, but not acrosin and neuraminidase. Preincubation of the enzyme and α-chlorohydrin was essential to achieve the maximum inhibition which was irreversible.
E. F. HARTREE and P. N. SRIVASTAVA
Material extracted from ram spermatozoa with 0·0125 n-NaOH was separated into lipid and glycoprotein fractions. By use of an anionic detergent (Hyamine 2389) acrosomes were separated from ram spermatozoa and also fractionated into lipid and glycoprotein. The chemical compositions of the two glycoprotein fractions, as well as of the two lipid fractions, show marked similarities. Taking into consideration the chemical changes that may occur during the isolation of these fractions it is deduced that they provide a reasonable approximation to the composition of the acrosomes. Of the amino acids glutamic acid predominates. The following sugars are present: mannose, galactose, fucose, glucosamine, galactosamine and sialic acid. The residue left after extracting ram spermatozoa with n-NaOH contains polysaccharide of which the constituent sugars are glucose, galactose and mannose. All the sialic acid of ram spermatozoa appears to be contained in the acrosome. The sialic acid content of the spermatozoa of other species has been measured and the possible role of sialic acid in sperm physiology is discussed.
CHUL HAK YANG and P. N. SRIVASTAVA
Ram sperm acrosomes were disrupted by Hyamine and Triton treatment and extracts were initially fractionated with a Sephadex G-100 column at pH 3·5. Acrosin, a proteolytic enzyme, was completely separated from hyaluronidase by this step. Highly active hyaluronidase, specific activity of 1320 units/mg protein (38,280 National Formulary units/mg protein), was obtained by DEAE chromatography but two minor contaminants were present. The partially purified enzyme had an optimum at pH 4·3. The enzyme showed no activity at pH 3·0 and only 10% of its activity at pH 8·0. Heparin and chondroitin sulphate B inhibited 50% of its activity. The molecular weight was estimated to be 62,000 by sodium dodecyl sulphate gel electrophoresis.
R. P. Scott, V. Ninjoor and P. N. Srivastava
Summary. Cathepsin B (EC 18.104.22.168) has been purified from rabbit testes to apparent homogeneity by chromatography on DE-52, affinity chromatography on organomercurial agarose and subsequent gel filtrations on Sephadex G-75. The enzyme is composed of a single polypeptide of M r 23 000. Thiol blocking agents and leupeptin abolished the activity of the enzyme completely. The enzyme showed maximum activity at pH 6·0 and 43°C, required 2 mm-cysteine for the optimal activity and had a K m1·45 × 10−3 m using Z-Arg-β-naphthylamide as the substrate. However, Z-Arg-Arg-β-naphthylamide was 12 times more sensitive as a substrate than was Z-Arg-β-naphthylamide. Rabbit testicular cathepsin B hydrolysed intact proteins. An endogenous inhibitor isolated from the rabbit testes inhibited purified Cathepsin B.
P. N. SRIVASTAVA, C. E. ADAMS and E. F. HARTREE
Cell-free enzyme preparations consisting of lipoglycoprotein were obtained from acrosomes of ram, bull and rabbit spermatozoa. The ram and bull preparations showed proteolytic and hyaluronidase activities. Preparations from the three species brought about dispersal of the cumulus oophorus and corona radiata of newly ovulated rabbit eggs. In some cases the zona pellucida was also removed. Iodoacetate and a polyanionic hyaluronidase inhibitor each reduced the denuding activity of the ram preparation and to a lesser extent of the bull preparation. Heating to 100° C also reduced the activity of the ram and bull preparations. It is concluded that proteolytic enzymes in spermatozoa contribute to the denudation of rabbit eggs and probably facilitate penetration of the zona pellucida.
L. J. D. ZANEVELD, P. N. SRIVASTAVA and W. L. WILLIAMS
The existence of a trypsin-like enzyme (TLE) in acrosomes of epididymal spermatozoa was confirmed and was further demonstrated to be present in acrosomes of ejaculated and capacitated spermatozoa. TLE rapidly removes the zona pellucida of the ovum. Extracts of acrosomes of ejaculated spermatozoa contain an inhibitor that is separated from the TLE by purification of the TLE.
The inhibitor of TLE is also present in seminal plasma. This enzyme— enzyme inhibitor relationship appears analogous to the corona-removing enzyme-decapacitation factor relationship and part of capacitation very likely involves removal of the inhibitor from TLE and decapacitation factor from the corona-removing enzyme. TLE is inhibited by soybean trypsin inhibitor and less effectively by mercaptoethanol.
P. N. SRIVASTAVA, J. F. MUNNELL, C. H. YANG and C. W. FOLEY
A method involving the treatment of ram spermatozoa with MgCl2 followed by Hyamine 2389 and Triton X-100 is described, which gave selective removal of the acrosomal membranes and acrosomal enzymes. The effect on the individual membranes of ram spermatozoa was evaluated by electron microscopy. Treatment with MgCl2 removed or altered the integrity of the plasma and outer acrosomal membranes, allowing the release of material from the acrosome. The inner acrosomal membrane and the electron-dense material of the equatorial segment were frequently unaffected by this treatment. Subsequent treatment of these spermatozoa with Hyamine and Triton removed the inner acrosomal membrane and the electron-dense material from the equatorial segment. The MgCl2 extract contained acrosomal proteinases and hyaluronidase. The detergent extract contained sperm neuraminidase.
The specific activities and yields of the enzymes obtained in the MgCl2 step were much higher than those of the enzymes obtained by the detergent treatment. Slight alterations in the conditions of the treatment had different effects on the acrosomal membranes in that one or more of the membranes were removed or altered in varying proportions resulting in the release of one or more enzymes. Only the plasma membrane and the acrosome appeared to be affected by these treatments as selected mitochondrial enzymes were not detectable in the extracts.
N Srivastava, R Santhanam, P Sheela, S Mukund, SS Thakral, BS Malik and SK Gupta
Dog zona pellucida glycoprotein 2 (dZP2), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned and expressed as a polyhistidine fusion protein in Escherichia coli to evaluate the immunocontraceptive efficacy of ZP glycoproteins. The recombinant dZP2 (rec-dZP2) revealed a 70 kDa band corresponding to the full length transcript, as well as several low molecular mass fragments in western blot analysis. In addition to rec-dZP2, E. coli expressed recombinant dog ZP glycoprotein 3 (rec-dZP3), which has also been evaluated for its efficacy to block fertility in a homologous system. Three groups of female dogs (n = 4 per group) were immunized with rec-dZP2 conjugated to diphtheria toxoid (rec-dZP2-DT), rec-dZP3 conjugated to DT (rec-dZP3-DT) and DT alone. Immunization of female dogs with rec-dZP2-DT and rec-dZP3-DT led to generation of antibodies against the respective ZP proteins as well as to DT. Subsequent to mating, the four female dogs immunized with rec-dZP2-DT all conceived, which is indicative of failure of the anti-rec-dZP2 antibodies to block fertility. In the group of dogs immunized with rec-dZP3-DT, three of four animals did not conceive when mated with males of proven fertility. The block in fertility was associated with anti-dZP3 antibody titres. Ovarian histopathology revealed that the block in fertility in the group immunized with rec-dZP3-DT is probably manifested by inhibition in the development of follicles and is due to atretic changes in the zona pellucida. These results, although preliminary, indicate that immunization with dZP3 may be a feasible proposition to control dog populations provided that adequate antibody titres are achieved.