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Ovulation Triggers Activation of Drosophila Oocytes

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Ovulation Triggers Activation of Drosophila Oocytes

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    1. Ovulation Triggers Activation of Drosophila Oocytes Yael Heifetz, Jing Yu, and Mariana F. Wolfner Speaker: Julie Atkinson

    2. Introduction and Background Information Fertilization

    3. Fertilization: the activation of development Cell surface interactions between egg and sperm changes in egg membrane potential Wave of calcium

    4. Interactions between egg and sperm Sperm is specialized to facilitate penetration of the egg. (ie. Acrosomal reaction) Penetration activates blocking mechanism in egg to prevent further sperm from entering egg.

    5. In mammalian eggs: 1st barrier- cumulus cells embedded in a sticky mass of hyaluronic acid. 2nd layer- zona pellucida composed of a layer of glycoproteins.

    6. Acrosomal Reaction Acrosomal Reaction: phenomenon whereby hydrolytic enzymes released from within the head of the sperm dissolve the vitelline coat of the egg. Polymerization of actin facilitates contact with the egg membrane.

    7. Polyspermy The entry of more than one sperm into the egg. Blocked by changes in egg membrane. Release of cortical granules from egg, which contain enzymes that block further sperm from binding to the zona pellucida once first sperm has fused with the egg’s plasma membrane.

    8. In sea urchins Depolarization of egg plasma membrane from –70mV to +20mV. Return to normal potential while fertilization membrane is formed. If depolarization does not occur, polyspermy is free to occur (in mouse fertilization, no change in membrane potential).

    9. Signal Transduction

    10. Sperm penetration Cortical granule exocytosis Formation of fertilization envelope by lifting of the vitelline envelope away from the egg plasma membrane. Sperm nucleus free to enter egg.

    11. Calcium Wave Fertilization and egg activation in mammals and sea urchins associated with release of Ca 2+ ions. Wave triggered by sperm entry and crosses egg at speed of 5-10 µm/sec. Preventing Ca2+ increase blocks activation.

    13. Summary Hyaluronidase activity on surface of sperm penetrate cumulus layer. Acrosomal reaction involving sperm head and zona pellucida. Sperm membrane fuses with egg membrane. Wave of Calcium. Sperm nucleus delivered into cytoplasm. Egg undergoes final stages of meiosis and development occurs

    14. Drosophila Background Oocytes are arrested in the ovary. Oocytes are still activating when they are fertilized but activation does not require fertilization. Egg activation is the process whereby the egg becomes metabolically active, initiates protein and DNA synthesis, and undergoes structural changes to its cortex and cytoplasm.

    15. Purpose of Experiment Hypothesis To determine the in vivo triggers and events of egg activation in Drosophila. -occurs rapidly and within the female fly Egg activation initiates during ovulation while the egg is largely in the ovary and has gone through the early stages of activation by the time they reach the uterus.

    16. Materials and Methods Mated virgin Drosophila melanogaster (wild type Oregon R) flies. 3-10h postmating, female reproductive tracts were dissected. Eggs removed from differing regions of reproductive tract and treated with 50% hypochlorited (bleach) to examine their permeability state.

    17. Regions -the lateral oviducts (positions I-III) -junction of the lateral and common oviducts (IV-V) -uterus

    18. Tests Performed Permeability to bleach -activated eggs are more resistant to lysis by bleach Vitelline membrane cross-linking -to determine cross-linking status of major vitelline membrane protein (VMP). Resumption of meiosis -to determine cell cycle stage of stage 14 oocytes and eggs.

    19. Microscopy -laser scanning confocal microscopy Statistics -One-way analyses of variance (ANOVA) used to rank EPI regions of dissected eggs. -Paired-sample Student’s t-test used to determine if significant difference between eggs at specific positions

    20. Results As ovulation proceeded and eggs moved down oviduct, EPI increased significantly. Eggs positioned at the pedicel region of the ovary (position I) lysed in the bleach at the same rate as stage 14 oocytes at the base of the same ovary before release. When egg reached common oviduct (pos. V), totally released from ovary, it was impermeable to bleach.

    21. Results cont’d EPI of eggs at pos. 2 significantly greater than that of mature oocytes in ovary ?egg activation had already initiated when egg reached pos. 2. Egg activation is detectable soon after beginning of ovulation.

    22. Vitelline Cross-linking test Amount of soluble sV23 varies from egg to egg. Trend of increased cross-linking as eggs move down reproductive tract. Ovarian oocytes have the highest levels of soluble sV23.

    23. Vitelline Cross-linking test All uterine eggs tests were fully impermeable by the EPI assay. sV23 cross-linking not complete in all uterine eggs ?suggests impermeability does not require complete cross-linking

    24. Meiosis Resumes Meiosis I spindles of eggs at pos. I-IV were oriented parallel Change in spindle orientation to perpendicular at pos. V (characteristic of meiosis resumption)

    25. Meiosis Resumes cont’d Meiosis initiates prior to egg’s entry into the uterus. Earliest presence of sperm tail occurred in uterus when egg was in Metaphase II

    26. Meiosis Resumes cont’d Data suggest that fertilization occurs after meiosis.

    27. Discussion and Significance Activation is triggered during ovulation -not mutually exclusive Hydration in the oviduct environment might signal activation, or Mechanical trigger such as pressure on the egg’s surface during ovulation. Changes in egg envelope sufficient to cause impermeability to bleach cannot serve as the block for polyspermy

    28. Further study Signals and signal transduction pathway(s) during egg activation not yet known in Drosophila. Identifying the precise signal for egg activation. Comparing that signal and the pathway that that converts it, to those in other systems. Derive a method to determine whether the permeability changes in the eggs are correlated with levels of cross linking of sV23.

    29. The End Have a nice day!!!

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