After ejaculation, mammalian spermatozoa must undergo capacitation to fertilize. Capacitation of bovine spermatozoa occurs in vitro in medium supplemented with heparin. Semen cryopreservation is an important tool for assisted reproduction, although the fertility of frozen-thawed spermatozoa is reduced, possibly due to precocious capacitation-like changes that are known to occur. Our purpose was to clarify the mechanisms involved in bull sperm cryocapacitation induced by cryopreservation. Our general hypothesis is that the signaling pathways that lead to capacitation are triggered by the cryopreservation procedure. Ejaculated bovine semen was divided into two aliquots and diluted in extender; one was then kept fresh, whereas the second was cryopreserved. Western blots of extracted sperm proteins with anti-phosphotyrosine antibody showed that capacitation, induced by either heparin in fresh sperm or cryopreservation (cryocapacitation), is associated with a differential profile of phosphotyrosine-containing proteins. Immunolocalization of phosphotyrosine-containing proteins in the fresh and cryopreserved spermatozoa showed that, after thawing, cryocapacitated sperm displayed labeling over the acrosomal region, whereas for fresh sperm, this labeling appeared after 5-h incubation with heparin. The chlortetracycline assay and the ability of the sperm to undergo the lysophosphatidylcholine-induced acrosome reaction were used to confirm that a subpopulation of cryopreserved sperm is capacitated at thawing, irrespective of heparin inclusion. Since glucose is known to inhibit heparin-induced capacitation, the semen extender was modified to include glucose as a means of inhibiting cryocapacitation; however, cryocapacitation was not prevented according to the chlortetracycline assay and profile of phosphotyrosine-containing sperm proteins.

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Bailey, J., Morrier, A. and Cormier. N. 2003. Semen cryopreservation: Successes and persistent problems in farm species. Can. J. Anim. Sci. 83: 393-401. Artificial insemination has arguably been the most important practice contributing to the advancement of animal production. The numerous advantages of artificial insemination are augmented when the semen is cryopreserved and conserved for long periods. Unfortunately, the utility of cryopreserved semen is limited because for most mammals, even cattle, a considerable proportion of sperm loose their fertility during freezing-thawing. In many species, this loss of fertility is substantial, rendering cryopreserved semen impractical for routine use. Our goals are to characterize the nature of the sublethal sperm damage caused by cryopreservation, and to develop a method to improve the functional fertility of thawed semen. This review describes our theory that sperm are prematurely activated by cryopreservation ("cryo-capacitation") and discusses strategies for preventing this damage.Key words: Sperm, artificial insemination, capacitation, fertility Bailey, J., Morrier, A. et Cormier, N. 2003. La cryoconservation de la semence : les succès et les problèmes persistants chez les espèces de la ferme. Can. J. Anim. Sci. 83: 393-401. L'insémination artificielle a grandement contribué à l'avancement des productions animales. Les nombreux avantages qu'offre cette technique sont augmentés lorsque la semence est cryoconservée et entreposée pour de longues périodes. Malheureusement, l'utilisation de la semence cryoconservée est limitée puisque chez la majorité des mammifères, incluant la vache, une large proportion des spermatozoïdes perdra leur motilité pendant le processus de congéla-tion-décongélation. Chez plusieurs espèces, cette perte de fertilité est suffisamment substantielle pour rendre impraticable l'utilisation routinière de la semence cryoconservée. Les objectifs de notre laboratoire portent sur la caractérisation des dommages sous létaux subis par les spermatozoïdes lors de la cryoconservation et le développement de méthodes permettant d'améliorer la fertilité des spermatozoïdes décongelés. Cette revue décrit notre théorie selon laquelle les spermatozoïdes seraient prématurément activés par la cryoconservation ("cryo-capacitation"). Différentes stratégies permettant de prévenir ce dommage y sont également abordées.

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These studies demonstrated that the inbred BALB/c mouse strain can be optimized for the assessment of vaccinia virus virulence, growth, and spread from the site of inoculation and immune protection from a lethal vaccinia virus challenge. The studies established that manipulation of the vaccinia virus genome generated mutants exhibiting a wide range of attenuated phenotypes. The nine NYCBH vaccinia virus mutants had intracranial 50% lethal doses that ranged from 2 to >7 log1o units. The decreased neurovirulence was due to decreased replication in brain tissue. Three mutants had a decreased ability to disseminate to the lungs, brains, livers, and spleens of mice after intranasal infection. One mutant had a decreased transmission from mice infected by tail scarification to naive cage mates. Although the mutants, with one exception, grew to wild-type titers in cell culture, they showed a growth potential on the scarified skin of mice that was dramatically different from that of the wild-type virus. Consequently, all of the mutants had significantly compromised immunogenicities at low virus immunization doses compared with that of the wild-type virus. Conversely, at high immunization doses most mutants could induce an immune response similar to that of the wild-type virus. Three Wyeth vaccine strain mutants were also studied. Whereas the thymidine kinase, ribonucleotide reductase, and hemagglutinin mutants had a reduced virulence (50%o lethal dose), only the thymidine kinase mutant retained its immunogenicity.

The cystatin-related epididymal spermatogenic (CRES) protein is related to the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, CRES lacks sequences important for cysteine protease inhibitory activity and is specifically expressed in reproductive and neuroendocrine tissues. Thus, CRES is distinct from cystatins and may perform unique tissue-specific functions. The purpose of the present study was to determine whether CRES functions as a protease inhibitor in in vitro assays. In contrast to mouse recombinant cystatin C, recombinant CRES did not inhibit the cysteine proteases papain and cathepsin B, suggesting that it probably does not function as a typical cystatin. CRES, however, inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system, whereas cystatin C showed no inhibition. CRES did not inhibit subtilisin, trypsin, or the convertase family members, PC1 and furin, indicating that it selectively inhibits PC2. Kinetic analysis showed that CRES is a competitive inhibitor of PC2 with a K(i) of 25 nM. The removal of N-terminal sequences from CRES decreased its affinity for PC2, suggesting that the N terminus may be important for CRES to function as an inhibitor. These studies suggest that CRES is a cross-class inhibitor that may regulate proprotein processing within the reproductive and neuroendocrine systems.

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Sperm-zona adhesion is an essential event in mammalian fertilization, failure of which causes sterility. However, the molecular mechanisms involved in this process are still poorly understood. It has been suggested by few laboratories studying gamete interaction that acrosomal molecules are implicated in sperm -zona pellucida adhesion prior to the acrosome reaction (AR). Zonadhesin, a spermspecific protein located in the acrosome is critically involved in zona binding. Here we describe the cellular and molecular interaction of zonadhesin during fertilization and also discuss its role in species-specific gamete interaction-an intriguing question in biology. We propose a model in which sperm could transiently expose acrosomal molecules that adhere to the zona independently of the AR in a 'kiss and run' mechanism. This could be a valuable framework for further investigations and a detailed understanding of the molecular events during gamete adhesion is likely to provide new approaches for the design of more effective male contraceptives and better diagnostic methods for sperm dysfunction.