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An intracellular free Ca2+ wave following fertilization in frog (xenopus laevis) eggs
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AN INTRACELLULAR FREE Ca 2+ WAVE FOLLOWING FERTILIZATION IN FROG (XENOPUS LAEVIS) EGGS. W.B. Busa and R. Nuccitelli, Dept. of Zoology, University of California, Davis, CA 95616 We have applied Ca2+-selective microelectrodes to detect a transient increase in free intracellular Ca 2+ concentration ([Ca2+]i) in fertilizing frog eggs. As observed with an electrode in the animal hemisphere, [Ca2+]i in the resting egg was 0.4 uM (pCa 6.4 ± 0.02, mean ± S.E.; n=7). Beginning ca. i min following onset of the fertilization potential, [Ca2+]i increased over the course of two min to 1.2 uM (pCA 5.93 i 0.06), and recovered to its original level over the next i0 min. No further changes occurred through the first cleavage division. In eggs impaled with 2 Ca 2+ electrodes, the Ca 2+ pulse was observed to travel as a wave from the animal to the vegetal hemisphere at 9.7 ± 1.5 um/sec at 22°C (n=4) and the apparent onset of the wave at the sperm entry point occurred 63 ± 5.5 sec following onset of the fertilization potential. The time for [Ca2+] i to increase from resting to peak level was significantly shorter in the animal (2.2 i 0.3 min; n=8) than in the vegetal hemisphere (3.0 i 0.3 min).
CYTOSKELETAL AND NUCLEAR LAMIN ORGANIZATION IN MOUSE OOCYTES AND SEA URCHIN FF/3S DURING FERTILIZATION. O. Schatten, G. Maul*, H. Schatten and C. Simerly. Dept of Biology, Flor. State Univ., Tallahassee, FL 32306. *Wistar Inst., Philadelphia, PA 19104. The organization of the cytoskeleton and nuclear skeleton in m o u s e o o c y t e s and sea urchin eggs during fertilization was investigated with immunofluorescence and electron microscopy, and motility was studied with time lapse video microscopy. The configurations of microtubules differs remarkedly. In contrast to the sea urchins, the mammalian pronucleate egg has numerous cytoplasmic asters nucleated from maternal sources. At mitosis an unusual plant-like anastral spindle appears to be nucleated independent of the sperm centrioles: this seems to violate the dogma regarding the paternal inheritance of MTOCs (microtubule organizing centers) during fertilization. Microfilaments, active during sperm incorporation in sea urchins, do not appear to participate in mammalian sperm incorporation, but may be involved in pronuclear centration. The male and female pronuclei acquire nuclear lamins as they develop whereas all chromosomes and nuclei have a richromin-likeantigen. Supported by H, NSF, UICC.
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PARTIAL CHARACTERIZATION OF A PROTEIN KINASE ACTIVITY WHICH INCREASES AT GERMINAL VESICLE BREAKDOWN OF STARFISH AND SURF CLAM OOCYTES. K.Sano~ H.Kanatani2and S.S. Koide~ M.B.L.,Woods Hole, 1Kitasato Univ. Sch.of Med.,Sagamihara 228,Japan, 2Natl. Inst. Basic Biol.,Okazaki 444,Japan, 3population Council,New York 10021,U.S.A. To confirm whether a protein kinase is activated at germinal vesicle breakdown (GVBD), we compared protein kinase activity (PKA) in GVBD-induced oocytes with that of immature oocytes from starfish and surf clam. In starfish, Asterina pectinifera, the supernatant (Sup) of GVBDinduced oocyte showed a 7-fold higher PKA than that of immature oocyte Sup when Sup from immature oocyte was used as substrate. This high PKA had a high substrate specificity for HI histone as an exogenous substrate. We observed similar increase of HI histone-specific PKA in GVBD-induced oocytes of surf clam. The specific PKA in GVBD-induced starfish oocytes was Ca2+and cyclic AMP-independent, and very labile. These show that a Ca 2+ and cyclic AMP-independent, labile protein kinase appears at GVBD of starfish oocytes. Supported by US-Japan Cooperative Science Program, NSF INT-821150, Rockefeller Foundation grant GA PS8418.
TRANSLATIONAL REGULATION DURING OOGENESIS AND VERY EARLY EMBRYOGENESIS IN THE TOBACCO HORNWORM, MANpUCA $E×TA. BRUCE JARNQT AND SPENCER ~. BERRY, DEPARTMENT OF BIOLOGY, WESLEYAN U N I V E R S I T Y , MIDDLETOWN, CONNECTICUT 0 6 4 5 7 THE OOGENIC AND EARLY EMBRYOGENIC PATTERN OF EVENTS DISPLAYED BY M. SEXTA ARE S I M I L A R TO THOSE DESCRIBED FOR SEA URCHINS AND A M P H I B I A N S . MESSENGER RNA (MRNA) TRANSCRIBED DURING OOGENESIS I S NOT TRANSLATED U N T I L AFTER F E R T I L I Z A T I O N . TRANSCRIPTION OF THE EMBRYO GENOME I S NOT I N I T I A T E D U N T I L CELLULAR BLASTODERM STAGE. THUS, PROTEIN SYNTHESIS I N THE EARLY EMBRYO R E L I E S ON THE A C T I V A T I O N OF STORED MRNA AND TRANSLATIONAL M A C H I NERY WHICH HAD BEEN M A I N T A I N E D I N A QUIESCENT STATE I N THE OOCYTE. WE HAVE ADRESSED SEVERAL POSSIBLE MECHANISMS INVOLVED I N TRANSLATIONAL I N I T I A T I O N AND REGULATION, I T S ' SUPRESSION I N THE OOCYTE, AND A C T I V A T I O N SHORTLY AFTER FERTILIZATION. THESE INCLUDE CHANGES I N THE PROTEIN COMPLEMENT OF THE STORED MRNA, PH FLUCTUATIONS AT F E R T I L I Z A T I O N , ALTERATION OF THE 5 ' MRNA CAP, AND A V A I L A B I L I T Y OF FREE RIBOSOMAL SUBUNITS.
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AN INTRACELLULAR FREE Ca 2+ WAVE FOLLOWING FERTILIZATION IN FROG (XENOPUS LAEVIS) EGGS. W.B. Busa and R. Nuccitelli, Dept. of Zoology, University of California, Davis, CA 95616 We have applied Ca2+-selective microelectrodes to detect a transient increase in free intracellular Ca 2+ concentration ([Ca2+]i) in fertilizing frog eggs. As observed with an electrode in the animal hemisphere, [Ca2+]i in the resting egg was 0.4 uM (pCa 6.4 ± 0.02, mean ± S.E.; n=7). Beginning ca. i min following onset of the fertilization potential, [Ca2+]i increased over the course of two min to 1.2 uM (pCA 5.93 i 0.06), and recovered to its original level over the next i0 min. No further changes occurred through the first cleavage division. In eggs impaled with 2 Ca 2+ electrodes, the Ca 2+ pulse was observed to travel as a wave from the animal to the vegetal hemisphere at 9.7 ± 1.5 um/sec at 22°C (n=4) and the apparent onset of the wave at the sperm entry point occurred 63 ± 5.5 sec following onset of the fertilization potential. The time for [Ca2+] i to increase from resting to peak level was significantly shorter in the animal (2.2 i 0.3 min; n=8) than in the vegetal hemisphere (3.0 i 0.3 min).
CYTOSKELETAL AND NUCLEAR LAMIN ORGANIZATION IN MOUSE OOCYTES AND SEA URCHIN FF/3S DURING FERTILIZATION. O. Schatten, G. Maul*, H. Schatten and C. Simerly. Dept of Biology, Flor. State Univ., Tallahassee, FL 32306. *Wistar Inst., Philadelphia, PA 19104. The organization of the cytoskeleton and nuclear skeleton in m o u s e o o c y t e s and sea urchin eggs during fertilization was investigated with immunofluorescence and electron microscopy, and motility was studied with time lapse video microscopy. The configurations of microtubules differs remarkedly. In contrast to the sea urchins, the mammalian pronucleate egg has numerous cytoplasmic asters nucleated from maternal sources. At mitosis an unusual plant-like anastral spindle appears to be nucleated independent of the sperm centrioles: this seems to violate the dogma regarding the paternal inheritance of MTOCs (microtubule organizing centers) during fertilization. Microfilaments, active during sperm incorporation in sea urchins, do not appear to participate in mammalian sperm incorporation, but may be involved in pronuclear centration. The male and female pronuclei acquire nuclear lamins as they develop whereas all chromosomes and nuclei have a richromin-likeantigen. Supported by H, NSF, UICC.
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PARTIAL CHARACTERIZATION OF A PROTEIN KINASE ACTIVITY WHICH INCREASES AT GERMINAL VESICLE BREAKDOWN OF STARFISH AND SURF CLAM OOCYTES. K.Sano~ H.Kanatani2and S.S. Koide~ M.B.L.,Woods Hole, 1Kitasato Univ. Sch.of Med.,Sagamihara 228,Japan, 2Natl. Inst. Basic Biol.,Okazaki 444,Japan, 3population Council,New York 10021,U.S.A. To confirm whether a protein kinase is activated at germinal vesicle breakdown (GVBD), we compared protein kinase activity (PKA) in GVBD-induced oocytes with that of immature oocytes from starfish and surf clam. In starfish, Asterina pectinifera, the supernatant (Sup) of GVBDinduced oocyte showed a 7-fold higher PKA than that of immature oocyte Sup when Sup from immature oocyte was used as substrate. This high PKA had a high substrate specificity for HI histone as an exogenous substrate. We observed similar increase of HI histone-specific PKA in GVBD-induced oocytes of surf clam. The specific PKA in GVBD-induced starfish oocytes was Ca2+and cyclic AMP-independent, and very labile. These show that a Ca 2+ and cyclic AMP-independent, labile protein kinase appears at GVBD of starfish oocytes. Supported by US-Japan Cooperative Science Program, NSF INT-821150, Rockefeller Foundation grant GA PS8418.
TRANSLATIONAL REGULATION DURING OOGENESIS AND VERY EARLY EMBRYOGENESIS IN THE TOBACCO HORNWORM, MANpUCA $E×TA. BRUCE JARNQT AND SPENCER ~. BERRY, DEPARTMENT OF BIOLOGY, WESLEYAN U N I V E R S I T Y , MIDDLETOWN, CONNECTICUT 0 6 4 5 7 THE OOGENIC AND EARLY EMBRYOGENIC PATTERN OF EVENTS DISPLAYED BY M. SEXTA ARE S I M I L A R TO THOSE DESCRIBED FOR SEA URCHINS AND A M P H I B I A N S . MESSENGER RNA (MRNA) TRANSCRIBED DURING OOGENESIS I S NOT TRANSLATED U N T I L AFTER F E R T I L I Z A T I O N . TRANSCRIPTION OF THE EMBRYO GENOME I S NOT I N I T I A T E D U N T I L CELLULAR BLASTODERM STAGE. THUS, PROTEIN SYNTHESIS I N THE EARLY EMBRYO R E L I E S ON THE A C T I V A T I O N OF STORED MRNA AND TRANSLATIONAL M A C H I NERY WHICH HAD BEEN M A I N T A I N E D I N A QUIESCENT STATE I N THE OOCYTE. WE HAVE ADRESSED SEVERAL POSSIBLE MECHANISMS INVOLVED I N TRANSLATIONAL I N I T I A T I O N AND REGULATION, I T S ' SUPRESSION I N THE OOCYTE, AND A C T I V A T I O N SHORTLY AFTER FERTILIZATION. THESE INCLUDE CHANGES I N THE PROTEIN COMPLEMENT OF THE STORED MRNA, PH FLUCTUATIONS AT F E R T I L I Z A T I O N , ALTERATION OF THE 5 ' MRNA CAP, AND A V A I L A B I L I T Y OF FREE RIBOSOMAL SUBUNITS.
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