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Molecular cloning and expression of mammalian acrosin cDNA
497
496 Isolation and culture of rat gonoeytes: An in vitro model for the spermatogonial proliferation/differentiation? F.M.F- van Dissel-Emilianil, D.G. de Rooijl & M.L. Meistrich2 1 Dept. Cell Biology, Utrecht, the Netherlands, 2 Dept. Exptl. Radiotherapy, M.D. Anderson Hospital, Houston, USA A method was developed to isolate gonocytes from 18 days post-coitum (p.c.) rat embryos. Cells were separated on the basis of size by the Staput technique of velocity sedimentation at unit gravity. Populations of 600.000 gonocytes (70-75% purity), sedimentinq at about i2 mm/h, could be obtained from 30-35 fetal rats within 8 h after killing. The isolated gonocytes were cultured alone or on a monolayer of Sertoli cells from 21 days old rats. In the absence of Sertoli cells, the gonocytes degenerated after 3 days. However, in the presence of Sertoli cells, and with FSH (I IU Metrodin/ml) and testosterone (10-6M]added to the culture medium, the gonocytes remained viable for at least 11 days, as determined by the Trypan blue dye exclusion test and by their adherence to the monolayer. The system seen~to be a useful model for the in vitro study of factors involved in the late embryonal/early post-natal spermatogonial differentiation and may lead to a better understanding of the nature of the
Effect of Estradiol and HY-Antigen in Roller Cultures of Chick Embryo Gonads U. DREWS, M. LAMMERDING, F. LOHMANN A n a t o m i s c h e s I n s t i t u t , 7400 T i i b i n g e n In o r d e r to t e s t t h e p r e s u m e d effect of a n t i g e n , we p r e p a r e d roller c u l t u r e s g o n a d s of 8 d a y chick e m b r y o s . A f t e r d a y s small a g g r e g a t e s develop which organotypic differentiation.
HYfrom two show
D u r i n g r e a g g r e g a t i o n of t h e cell s u s p e n s i o n s of t e s t i s and left o v a r y t h e c u l t u r e s were t r e a t e d with e s t r a d i o l a n d a p u r i f i e d f r a c t i o n of H Y - a n t i g e n p r o v i d e d b y Dr. Wiberg, I n s t i t u t e of H u m a n g e n e t i k F r e i b u r g ] B r e i s g a u . Morphometric a n a l y s i s of t h e c u l t u r e s r e vealed t h a t in t h e r e a g g r e g a t e s of o v a r y e s t r a d i o l i n d u c e d p r o l i f e r a t i o n of g e r m cells. In t h e r e a g g r e g a t e s of t e s t i s , e s t r a d i o l p r o d u c e d e n l a r g e m e n t of g e r m cells indicating a b o r t i v e initiation of female d e v e l o p m e n t . In t h e e x p e r i m e n t s with a DEAE f r a c t i o n of HYa n t i g e n from rat t e s t i s , in t h e female a g g r e g a t e s t h e g e r m cells were significantly larger than in t h e c o n t r o l s . T h e o r g a n o t y p i c male or female p a t t e r n was not a l t e r e d b y t h e HYantigen treatment.
stem spermatogonia of the adult testis.
498
499
Molecular cloning and expression of mammalian acrosin cDNA W. ENGEL, I.M. ADHAM, U. KLEMM, H. KREMLING, S. KEIME, M. HUMMEL,Institut fir Humangenetik Gosslerstrasse 12D, D-3400 G~ttingen, FRG Mammalian fertilization is the result of a poorly understoOd complex series of biochemical events that occur when an ovum and a spermatozoa fuse. Acrosin, a serine [x~Celnase located in the acrosome of the sperm, has been implicated in the recognition and binding of the sperm to the zona pellucida of the ovum and the sperm penetration through the zona pellucida. Immunoflourescent studies indicate that the biosynthesis of acrosin/ proacrosin starts in early haploid spermatids. Despite the great importance of the enzyme in the fertilization process, only very few data as to its molecular structure are available. We succeeded in the isolation and sequencing of cDNAs for preproacrosin of boar, human, mouse and rat. From these results a 361 amino acids containing molecule is deducible, conraining a hydrophobic leader sequence and a p r o l i n e - r i c h domain at the COOH-terminal end which can be resbonsible for the binding of the sperm to the zona pellucida. Northernblot experin~nts and in situ hybridization experiments on testis sections i~dicate to a haploid expression of the preproacrosin gene in mammalian spermatids. First results of genetic defects of the gene presented which might be responsible for male infertility.
SEASONAL CHANGES IN FORMATION AND MATURATION OF SPERMATOZOA IN A DESERT RODENT, THE SAND
RAT (P~Ctmraoraq~ obe.~uz ). GERNIGON Th., MALAPRADE D., MESBAH A. In adult male sand rats inhabiting the Beni Abbes area (Algeria) in early summer (junejuly) the seminiferous tubular diameter decreased (135 ~m) and spermiogenesis decreased or stopped. In Leydig cells, lipid cytoplasmic droplets are very numerous. In principal cells epididymis, dense granules are surrounded by a concentric rough endoplasmic reticulum. In breeding season, the seminiferous tubular diameter is equal or superior to 200 pm, the spermiogenesis very abundant. In Leydig cells, the dominant organelles are the smooth endoplasmic reticulum very dilated adn mitochondria with tubular cristae. In the principal cells epididymis, rough endoplasmic reticulum showed dilated cisternae filled with dense secretion and Golgi apparatus is very extensive. The decreasing value of testosterone in the spring have cellular repercussion only in june and july and do not act on the structural morphology of Sertoli cells and clear cells.
S152
496 Isolation and culture of rat gonoeytes: An in vitro model for the spermatogonial proliferation/differentiation? F.M.F- van Dissel-Emilianil, D.G. de Rooijl & M.L. Meistrich2 1 Dept. Cell Biology, Utrecht, the Netherlands, 2 Dept. Exptl. Radiotherapy, M.D. Anderson Hospital, Houston, USA A method was developed to isolate gonocytes from 18 days post-coitum (p.c.) rat embryos. Cells were separated on the basis of size by the Staput technique of velocity sedimentation at unit gravity. Populations of 600.000 gonocytes (70-75% purity), sedimentinq at about i2 mm/h, could be obtained from 30-35 fetal rats within 8 h after killing. The isolated gonocytes were cultured alone or on a monolayer of Sertoli cells from 21 days old rats. In the absence of Sertoli cells, the gonocytes degenerated after 3 days. However, in the presence of Sertoli cells, and with FSH (I IU Metrodin/ml) and testosterone (10-6M]added to the culture medium, the gonocytes remained viable for at least 11 days, as determined by the Trypan blue dye exclusion test and by their adherence to the monolayer. The system seen~to be a useful model for the in vitro study of factors involved in the late embryonal/early post-natal spermatogonial differentiation and may lead to a better understanding of the nature of the
Effect of Estradiol and HY-Antigen in Roller Cultures of Chick Embryo Gonads U. DREWS, M. LAMMERDING, F. LOHMANN A n a t o m i s c h e s I n s t i t u t , 7400 T i i b i n g e n In o r d e r to t e s t t h e p r e s u m e d effect of a n t i g e n , we p r e p a r e d roller c u l t u r e s g o n a d s of 8 d a y chick e m b r y o s . A f t e r d a y s small a g g r e g a t e s develop which organotypic differentiation.
HYfrom two show
D u r i n g r e a g g r e g a t i o n of t h e cell s u s p e n s i o n s of t e s t i s and left o v a r y t h e c u l t u r e s were t r e a t e d with e s t r a d i o l a n d a p u r i f i e d f r a c t i o n of H Y - a n t i g e n p r o v i d e d b y Dr. Wiberg, I n s t i t u t e of H u m a n g e n e t i k F r e i b u r g ] B r e i s g a u . Morphometric a n a l y s i s of t h e c u l t u r e s r e vealed t h a t in t h e r e a g g r e g a t e s of o v a r y e s t r a d i o l i n d u c e d p r o l i f e r a t i o n of g e r m cells. In t h e r e a g g r e g a t e s of t e s t i s , e s t r a d i o l p r o d u c e d e n l a r g e m e n t of g e r m cells indicating a b o r t i v e initiation of female d e v e l o p m e n t . In t h e e x p e r i m e n t s with a DEAE f r a c t i o n of HYa n t i g e n from rat t e s t i s , in t h e female a g g r e g a t e s t h e g e r m cells were significantly larger than in t h e c o n t r o l s . T h e o r g a n o t y p i c male or female p a t t e r n was not a l t e r e d b y t h e HYantigen treatment.
stem spermatogonia of the adult testis.
498
499
Molecular cloning and expression of mammalian acrosin cDNA W. ENGEL, I.M. ADHAM, U. KLEMM, H. KREMLING, S. KEIME, M. HUMMEL,Institut fir Humangenetik Gosslerstrasse 12D, D-3400 G~ttingen, FRG Mammalian fertilization is the result of a poorly understoOd complex series of biochemical events that occur when an ovum and a spermatozoa fuse. Acrosin, a serine [x~Celnase located in the acrosome of the sperm, has been implicated in the recognition and binding of the sperm to the zona pellucida of the ovum and the sperm penetration through the zona pellucida. Immunoflourescent studies indicate that the biosynthesis of acrosin/ proacrosin starts in early haploid spermatids. Despite the great importance of the enzyme in the fertilization process, only very few data as to its molecular structure are available. We succeeded in the isolation and sequencing of cDNAs for preproacrosin of boar, human, mouse and rat. From these results a 361 amino acids containing molecule is deducible, conraining a hydrophobic leader sequence and a p r o l i n e - r i c h domain at the COOH-terminal end which can be resbonsible for the binding of the sperm to the zona pellucida. Northernblot experin~nts and in situ hybridization experiments on testis sections i~dicate to a haploid expression of the preproacrosin gene in mammalian spermatids. First results of genetic defects of the gene presented which might be responsible for male infertility.
SEASONAL CHANGES IN FORMATION AND MATURATION OF SPERMATOZOA IN A DESERT RODENT, THE SAND
RAT (P~Ctmraoraq~ obe.~uz ). GERNIGON Th., MALAPRADE D., MESBAH A. In adult male sand rats inhabiting the Beni Abbes area (Algeria) in early summer (junejuly) the seminiferous tubular diameter decreased (135 ~m) and spermiogenesis decreased or stopped. In Leydig cells, lipid cytoplasmic droplets are very numerous. In principal cells epididymis, dense granules are surrounded by a concentric rough endoplasmic reticulum. In breeding season, the seminiferous tubular diameter is equal or superior to 200 pm, the spermiogenesis very abundant. In Leydig cells, the dominant organelles are the smooth endoplasmic reticulum very dilated adn mitochondria with tubular cristae. In the principal cells epididymis, rough endoplasmic reticulum showed dilated cisternae filled with dense secretion and Golgi apparatus is very extensive. The decreasing value of testosterone in the spring have cellular repercussion only in june and july and do not act on the structural morphology of Sertoli cells and clear cells.
S152