- Email: [email protected]
Acrosin and hyaluronidase assay in single mammalian sperm for detection of reprotoxic agents
ACROSINAED RYALWROBIDASEASSAY IN SINGLE MAMMALIAN SPERM FOR DETECTIONOF REPRDTOXIC AGEWTS.
FIGSOR G, WAIBRL R, GIBSBERGL. D~~~E~ UNIVERSITY.RALWAZOO, HI 49003.
OF BI~I~L
SCIENCES,WRSTERN MI~HIG~
With machine coating technologywe prepared gelatin or hyaluronlcacid substrate-films which are uniformlythin to be digaated through by acrosin or hyaluronidasepresent In singlemaamsliansperm. The order of acrosin activityas determined by the diameterof digest halos was h~an?dog~squi~el monkey>mouserrat for acroafn and ~u8e~h~an for hyaluronldase. Ninety and 99% of mouse sperm have acrosin and hyaluronidaseactivity, respectively. When groups of 6-10 mitomycin C (MC) or ethylnitrosourea(ENU) treatedmice were killed 5 weeks after treatmentthe percentageof sperm without acrosin activity increased significantly. (Prior to killing, these mica were mated with two untreatedfemales viable testing profor genotoxicevaluationof their progeny. See Ficsor et al. i'Dominant tocol for detectionof genotoxicagents in mice" in these Abstracts.) Hyaluronidasewas Inasmuch as acrosin and tested only with ENIJwhich produceda similar loss of activity. hyaluronidaseare required for fertilization,their induced loss in a significantproportion of spermatozoafrom treatedanimalsmay be indicativeof a reprotoxiceffect. Potentially these enzyme assays may also be used to detect damage to human germ cell%. This work vas supportedby NIEES grant 1 ROl ES02607-02.
DOMINANTVIABLE TESTING PROTOCOLFOR RRTECTIONOF GENOTOXICAGENTS IN NICE:
DEPARTMEBTOF BIOMEDICALSCIENCES,WESTERN FICSOR,G., PABDA, B. AND GINSBERG,L. HXCBIGANUNIVERSITY.EALA%UO, !+I149003.
In the specificlocus test (morphological or biochemical)and in the dominantcataract test,mutant F are usuallyviable. Such Fl carry the Induced lesion and may serve as the source of the $esion for subsequentgenetic analysis. In these tests, however,only a few genes are evaluatedwhich necessitatesthe raising of thousandsof F progeny to detect a mutagen. In the dominant lethal test a larger proportionof the gen&Ie is evaluatedand hence fewer animals are required,but the mutant progeny die. In our experimentaldesign controlor treatedmales are mated to untreated females of the same strain. (For results of sperm evaluationof the treatedand control F generationsee Ficsor et al. elswhere in these abstracts). The Fl progeny are then teat& for negative geotaxis at 9 and 13 days, for water escape at 14 and 21 days, for open field behavior at 43 and 45 days of age. Adult F, mslea are then mated to untreatedfemales and subsesuentlvare killed for snerm enxyme &td motilitydeterminations. In one experimentsix I& UCRj males were injected with saline,3 with 2.5 m&g/kgmitomxin C (MC), 10 with 100 mnlkn ethylnitroaourea&NU). Each male was mated to tvo untreatedfamalee. The MC Fl progeny-exhibitedsignificant deficit in water escape and the END Fl showed a significantincrease in motor activity in the open field teat. The eperm of approximately20 F males from control,WC- or El4U treatedfatherswere evaluatedfor sperm motility, acrosfn, succinic dehydrogenaaeand alpha glycsrolphosphate dehydrogenase(AGPD) activity. With the exceptionof the AGPD END group, all end-pointsshowed an increasedgenetic effect from the treatments. We conclude that the significantresponse to the mutagens iu our small progeny sample is due to domiaant mutationsin any one of the many genes neadad for sperm functions and normal behavior. This work vm supported by NIMS grant 1 ROl ES02607-02.
FIGSOR G, WAIBRL R, GIBSBERGL. D~~~E~ UNIVERSITY.RALWAZOO, HI 49003.
OF BI~I~L
SCIENCES,WRSTERN MI~HIG~
With machine coating technologywe prepared gelatin or hyaluronlcacid substrate-films which are uniformlythin to be digaated through by acrosin or hyaluronidasepresent In singlemaamsliansperm. The order of acrosin activityas determined by the diameterof digest halos was h~an?dog~squi~el monkey>mouserrat for acroafn and ~u8e~h~an for hyaluronldase. Ninety and 99% of mouse sperm have acrosin and hyaluronidaseactivity, respectively. When groups of 6-10 mitomycin C (MC) or ethylnitrosourea(ENU) treatedmice were killed 5 weeks after treatmentthe percentageof sperm without acrosin activity increased significantly. (Prior to killing, these mica were mated with two untreatedfemales viable testing profor genotoxicevaluationof their progeny. See Ficsor et al. i'Dominant tocol for detectionof genotoxicagents in mice" in these Abstracts.) Hyaluronidasewas Inasmuch as acrosin and tested only with ENIJwhich produceda similar loss of activity. hyaluronidaseare required for fertilization,their induced loss in a significantproportion of spermatozoafrom treatedanimalsmay be indicativeof a reprotoxiceffect. Potentially these enzyme assays may also be used to detect damage to human germ cell%. This work vas supportedby NIEES grant 1 ROl ES02607-02.
DOMINANTVIABLE TESTING PROTOCOLFOR RRTECTIONOF GENOTOXICAGENTS IN NICE:
DEPARTMEBTOF BIOMEDICALSCIENCES,WESTERN FICSOR,G., PABDA, B. AND GINSBERG,L. HXCBIGANUNIVERSITY.EALA%UO, !+I149003.
In the specificlocus test (morphological or biochemical)and in the dominantcataract test,mutant F are usuallyviable. Such Fl carry the Induced lesion and may serve as the source of the $esion for subsequentgenetic analysis. In these tests, however,only a few genes are evaluatedwhich necessitatesthe raising of thousandsof F progeny to detect a mutagen. In the dominant lethal test a larger proportionof the gen&Ie is evaluatedand hence fewer animals are required,but the mutant progeny die. In our experimentaldesign controlor treatedmales are mated to untreated females of the same strain. (For results of sperm evaluationof the treatedand control F generationsee Ficsor et al. elswhere in these abstracts). The Fl progeny are then teat& for negative geotaxis at 9 and 13 days, for water escape at 14 and 21 days, for open field behavior at 43 and 45 days of age. Adult F, mslea are then mated to untreatedfemales and subsesuentlvare killed for snerm enxyme &td motilitydeterminations. In one experimentsix I& UCRj males were injected with saline,3 with 2.5 m&g/kgmitomxin C (MC), 10 with 100 mnlkn ethylnitroaourea&NU). Each male was mated to tvo untreatedfamalee. The MC Fl progeny-exhibitedsignificant deficit in water escape and the END Fl showed a significantincrease in motor activity in the open field teat. The eperm of approximately20 F males from control,WC- or El4U treatedfatherswere evaluatedfor sperm motility, acrosfn, succinic dehydrogenaaeand alpha glycsrolphosphate dehydrogenase(AGPD) activity. With the exceptionof the AGPD END group, all end-pointsshowed an increasedgenetic effect from the treatments. We conclude that the significantresponse to the mutagens iu our small progeny sample is due to domiaant mutationsin any one of the many genes neadad for sperm functions and normal behavior. This work vm supported by NIMS grant 1 ROl ES02607-02.