( RIA ) ELISA [3] SDS-PAGE

HTML
DOWNLOAD

Μέγεθος: px

Εμφάνιση ξεκινά από τη σελίδα:

Download "( RIA ) ELISA [3] SDS-PAGE"

Ἰορδάνης Μήτζου
8 χρόνια πριν
Προβολές:

1 24 Vol MARINE SCIENCE BULLETIN Dec No. ( ) ( Scylla serrata ) 3.5 µiu/ µIU / 10 ( ) P735 A (2005) [1] [2] ( RIA ) ELISA [3] SDS-PAGE [4] [5,] [5] [7-10] [11, 12] [13,14] Kennedy [5] Nα--DL--p- ( Nα-benzol-DL-arginine-p-nitroanilide, BAPNA ) ( Benzamindine ) 400 ( Ficoll 400 ) ( Sigma ) N-2- -N-2- ( Hepens ) ( DMSO ) ( 2002AA03013 )

2 a ) Ficoll 11 Ficoll 0.12 mol/l NaCl0.025 M Hepes ph g NaCl0.20 g Hepes 3.7 g Ficoll 30 ml HCl NaOH ph ml ( Sodium azide ) 33.3 mg Ficoll b ) 0.01 Triton X mol/l Hepes0.055 mol/l NaCl ph g Hepes0.32 g NaCl1 ml 0.1 Triton X ml 1 mol/l NaOH ph ml 100 mg c ) ( Benzamidine Hydrochloride ) 500 m M 87.3 mg 1 ml 2 d ) DMSO 23 mmol/l BAPNA 10 mg BAPNA 1 ml DMSO 510 min e ) 4.5 ml ( B ) 0.5 ml BAPNA/DMSO 5 ml Ficoll 1.4 a ) ( Ca 2+ -FSW ) ( Leung-Trujillo & Lawrence, 1987 ) b ) ( 210 ) µl c ) 0.5 ml Ficoll 5 ml Ficoll d ) g 30 min e ) Ficoll 100 µl Ficoll f ) 100 µl g ) 1 ml ( E ) 1 ml 11 h ) h i ) 3 h 100 µl ( C ) j ) g 30 min k ) 410 nm 3 d l ) 23 1 µmol BAPNA/min µiu/10 µiu /10 = OD-OD10 {1485 } 1 ( OD )

3 89 1 Tab.1 The Acrosin Activity of Spermatozoa in Spermathecae of Scylla serrata - -1 / µiu 10 / ml = = [5] ( Radioimmunoassay, RIA ) [12] Triton X-100 ( ) mu/10 [7] ( 140 ) µiu/10 ( ) µiu/10 ( 735 )µiu/ µiu/10 [5] ( ) µiu/10 [15] ( Polyodon spathula ) ( ) µiu/10 ( µIU/10 n =8 ) [8] ( chymotrypsin-like activity ) Triton X-100 [8] [13] [14] [10] ph [8] [11] ( 1995 ) 10 Polyodon spathula [8] [12] [1] [17]

4 90 24 [18] [19] [1] Baba T, Kashiwabara S, Watanabe K et al. Activation and maturation mechanisms of boar acrosin zymogen based on the deduced primary structure [J]. Jouranl of Biochemical, 1989, 24: [2] Fritz H, Tschesche H, Fink E. Proteinase inhibitors from boar seminal plasma [J]. Meth Enzymol, 197, 45: [3] Nagdas SKBovine proacrosin from cauda epididymal sperm: Purification, characterzation and partial sequencing at N- terminus,international [J]. Jouranl of Biochemical, 1992, 24: [4] Henkel R, Muller C, Miska W et al. Acrosin activity of human spermatozoa by means of a simple gelatinolytic technique: a method useful for IVF [J]. Jouranl of Andrology, 1995, 1: [5] Kennedy WP, Kaminski JM, Van der Ven HH et al. A simple, clinical assay to evaluate the acrosine activity of human spermatozoa [J]. Jouranl of Andrology, 1989, 10: [] Cross NLA modified and improved assay for sperm amidase activity [J]. Journal of Andrology. 1990, 11: [7] Ciereszko A, Ottobre J S, Glogowski J. Effects of season and breed on sperm acrosin activity and semen quality of boars, Animal Reproduction Science [J]. 2000, 4 (1-2): [8] Ciereszko A, Dabrowski K, Mims SD et al. Characteristics of sperm acrosin-like activity of paddlefish ( Polyodon spathula Walbaum ) [J]. Comparative Biochemistry and Physiology Part B, 2000b, 125 (2): [9] Ball B A, Fagnan M S. Dobrinski I. Determination of acrosin amidase activity in equine spermatozoa [J]. Theriogenology, 1997, 48 (7): [10] Glogowski J, Demianowicz W, Piros B et al. Determination of acrosin activity of boar spermatozoa by the clinical method: Optimization of the assay and changes during short-term storage of semen [J].Theriogenology, 1998, 50 (): [11],,. [J]., 1995, 21(5): [12],,. [J]., 199, 22(): [13] Jeyalectumie C, Subramoniam T. Biochemistry of seminal secretions of the crab Scylla serrata with reference to sperm metabolism and storage in the female [J]. Molecular Reproduction and Development, 1991, 30: [14] Sainte-Marie G, Sainte-Marie B. Reproductive products in the adult snow crab (Chionoecetes opilio). II. Multiple types of sperm cells and of spermatophores in the spermathecae of mated females[j]. Canadian Journal of Zoology, 1999, 77 (3): [15] Ball BA, Fagnan MS, Dobrinski I. Determination of acrosin amidase activity in equine spermatozoa [J]. Theriogenology, 1997, 48 (7): [1] Benhalima K, Moriyasu M. Prevalence of bacteria in the spermathecae of female snow crab, Chionoecetes opilio (Brachyura : Majidae) [J]. Hydrobiologia, 2001, 449 (1-3): [17] Gwo JC. Cryopreservation of aquatic invertebrate semen: a review [J]. Aquaculture research, 2000, 31(3): [18] Bray WA,Lawrence AL.Male viability determinations in Penaeus vannameri: evaluation of short-term storeage of

5 91 spermatophores up to 3 h and comparison of Ca 2+ -free saline and seawater as sperm homogenate media [J]. Aquaculture, 1998, 10: 3-7. [19],. [J]., 2002, 1(2): ( 1972 ) 5 Acrosin Activity of Spermatozoa in Spermathecae of Scylla serrata GUAN Weibing, WANG Guizhong, LI Shaojing, CHEN Jingmin ( Department of Oceangraphy, Institute of Subtropical Oceanography, Key Laboratory for Marine Environmental Science of the Ministry of Education, Xiamen University, Xiamen 31005, Fujian, China ) AbstractAcrosin, a sperm-specific arosomal proteinase, has an essential role in the fertilization process and is thought to be important in the acrosome reaction, sperm-zona binding and zona penetration. The spermatozoa acrosin activity of Scylla serrata is measured with a simplified clinical assay of total sperm acrosin activity. This method is based on the measurement of amidase activity of sperm suspensions. The acrosin activity assay has been used in many animals. Results of these studies indicated that use of this assay method could provide valuable information concerning semen quality. The spermatozoa acrosin activity of Scylla serrata is about 3.5µIU/10 and 4.248µIU/10. The acrosin activity is lower than some mammals such as boars, human beings and horses. It shows that the significant difference between acrosin activity of different kinds of species and also reflect the spermatozoa of Scylla serrata may survive with the restraining state in spermathecae. This result indicates that the clinical assay of acrosin activity can be used for crab spermatozoa to evaluate the quality of crab semen. KeywordsScylla serrta; acrosin; spermatozoa; spermathecae

Σχετικά έγγραφα

ΙΩΑΝΝΗ ΑΘ. ΠΑΠΑΪΩΑΝΝΟΥ

ΑΡΙΣΤΟΤΕΛΕΙΟ ΠΑΝΕΠΙΣΤΗΜΙΟ ΘΕΣΣΑΛΟΝΙΚΗΣ ΣΧΟΛΗ ΓΕΩΠΟΝΙΑΣ, ΔΑΣΟΛΟΓΙΑΣ ΚΑΙ ΦΥΣΙΚΟΥ ΠΕΡΙΒΑΛΛΟΝΤΟΣ ΤΜΗΜΑ ΓΕΩΠΟΝΙΑΣ ΤΟΜΕΑΣ ΑΓΡΟΤΙΚΗΣ ΟΙΚΟΝΟΜΙΑΣ ΙΩΑΝΝΗ ΑΘ. ΠΑΠΑΪΩΑΝΝΟΥ Πτυχιούχου Γεωπόνου Κατόχου Μεταπτυχιακού