This increasing gelatin digestion observed in fresh samples agrees well with the results of Kawakami et al. Figure 3: Photomicrograph magnification: x of canine spermatozoa after incubation on gelatin substrate slides A, spermatozoa showing good acrosin activity, large halo diameter; B, spermatozoa showing regular acrosin activity, medium and small halo diameter ; C, spermatozoa showing no halo formation. Apparently, activation of proacrosin to acrosin does not occur within the intact acrosome, but rather is initiated after physical disruption of the acrosome.
The higher rate of unlabeled spermatozoa in frozen-thawed sperm in comparison to fresh spermatozoa during all culture periods is more likely due to an acrosome alteration induced by freezing-thawing procedures, as has been described previously in non-capacitated dog spermatozoa Florman et al. Acrosin is considered a diffusible molecule Harrison et al.
Calcium exerts a stimulatory effect on acrosin activity in boar Kennedy and Polakoski, and bovine Nagdas, ; Rosatti et al.
Capacitation is described as a temperature-dependent process, in some species like human, sperm incubation at room temperature does not allow capacitation. In contrast, storage of ram semen at room temperature has a marked capacitating effect on a subpopulation of spermatozoa Perez et al. At both temperatures, fresh and frozen- thawed sperm showed the same trend in the percentage of immunolabeling over time. However, temperature significantly influenced the rate of acrosin release in cryopreserved sperm.
Sirivaidyapong et al. Energy metabolism is influenced by temperature increasing glucose consumption and glucose has been reported to favor acrosome reaction in dog spermatozoa Fraser, However, acrosomal proteolytic activity determined by means of the gelatinolytic technique was not influenced by preincubation temperature in either fresh or frozen dog sperm. Freezing-thawing acts mainly by disordering the lipid structure of the membranes and loss of cholesterol from the sperm plasma membrane, which has a destabilizing effect on sperm and may be part of the molecular mechanism of capacitation in vivo Iborra et al.
Removal of cholesterol from bovine sperm increases sensitivity to a lysophosphatidylcholine challenge and induction of the acrosome exocytosis Foote and Parks, Since sperm capacitation, and thus acrosome reaction, is required for fertilization, premature release and activity of acrosin may impair fertilization, because they cannot retain their fertilizing ability over time Barros et al. In summary, our results provide support that there is a difference between fresh and frozen dog sperm in terms of the release of acrosin and the level of acrosin activity of acrosin over the course of in vitro capacitation.
Premature activation and early and high release of acrosin could be other functional parameters affected by cryopreservation. Biol Res Mol Reprod Dev J Androl Anim Reprod Sci Theriogenology Reprod Domest Anim 44 Suppl 2 : Fertil Steril Reprod Fertil Develop 5: Hum Reprod 10 Suppl 1 : J Reprod Fertil Synthesis of a novel fertilization inhibitor.
Dev Biol Biol Reprod J Vet Med Sci Biochemical Gamete Res 1: Andrologia Reprod Domest Anim Biocell Hoppe Seyler's Z Physiological Chemical , J Reprod Develop M Wassarman pp Chicago, CRC Press. Five acrosin inhibitors soybean trypsin inhibitor SBTI , leupeptin, benzamidine, N-p-tosyllysin-chloromethyl ketone TLCK and phenylmethylsulphonyl fluoride PMSF and one non-acrosin inhibitor N-p-tosylphenylalanine chloromethyl ketone TPCK were tested at non-toxic levels below motility-disturbing concentrations.
These inhibitors were added at three different times: 1 during the acrosome reaction of spermatozoa, 2 during sperm-oocyte contact and fusion, and 3 soon after sperm-oocyte fusion was completed.
TLCK prevented sperm-oocyte fusion by inhibiting the acrosome reaction. PMSF inhibited gamete fusion, without inhibiting the acrosome reaction. SBTI, leupeptin and benzamidine also inhibited gamete fusion, but they had no effect if spermatozoa were allowed to acrosome-react in inhibitor-free medium. During the acrosome reaction the contents of the acrosome are released outwardly. The cell membrane of the spermatozoon fuses with the outer membrane of the acrosome. The contents of the acrosome flow out through the resulting pores.
Quiz That the zona-binding represents a decisive step in the fertilization cascade can perhaps be seen in the fact that the zona-binding is species specific ; the subsequent binding of the oocyte membrane onto the oolemma, on the other hand, is not.
When the acrosome reaction has been completed, the spermatozoon is now covered at its upper end only by the former inner membrane of the acrosome. For the further progress of the fertilization this is decisive because structures are thereby uncovered which are necessary for contact with the oocyte.
One consequence of this is that changes appear, especially in the post-acrosomal membrane area of the spermatozoon.
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