Early Invertebrate Development (1) Flashcards
What is oogenesis?
- meiosis in females
- primary oocyte (2N) -> first metaphase (1N) -> second metaphase -> meiosis complete
At what stage of oogenesis, does fertilization occur?
- fertilization happens at different stages for different organisms
- for cnidarians and sea urchins, fertilization happens once meiosis is complete
- for amphibians. mammals and fish, fertilization happens at the second metaphase and triggers the completion of meiosis
How does unification of male and female DNA?
- male pronucleus decondenses
- actin-based fertilization cone brings m/f pronuclei together
- DNA synthesis (S-phase) resumes in both male and female pronuclei
- male/female centrosomes form microtubule-based asters
- chromatin condenses into chromosomes and mitosis occurs
What other events take place after sperm entry?
- many events from sperm entry to first cleavage or “egg activation”
What makes sea urchins a good model organism?
- thousands of eggs easily obtained
- in vitro fertilization
- all embryos synchronized in their development
- genome is sequenced (S. purpuratus)
- simple model but same cell types as humans
What is a “fishing expedition”?
- biochemical analysis
- trying to find genes that are important
- analysis of protein expression using 2 dimensional gel electrophoresis analysis of protein expression
How does an SDS work?
- proteins run based on size on a gel in the direction of a charge
- run left to right based on pH (isoelectric focusing point), useful if you have lots of proteins and they are showing up as bands in the same vertical
What do we see in a “fishing expedition” comparing unfertilized and fertilized proteins?
- some dots disappear after fertilization
- this is because proteins can be degraded and proteases can become activated
What do we see in a “fishing expedition” using a western blot to compare unfertilized and fertilized proteins?
- a western blot uses an antibody to see which proteins are unphosphorylated
- we can compare blots to see that some proteins are present but not phosphorylated
What is phosphorylation and how is it regulated?
- phosphorylation acts as a switch to attract proteins or inhibit protein-protein interaction
- kinases and phosphatases work hand in hand to regulate phosphorylation
How do phosphorylated proteins compare in unfertilized and fertilized eggs?
- after two min the amount of phosphorylated proteins doubled
- this affects the activity and metabolism of the cell
What does egg activation lead to?
- cell division
What is equal division cleavage called? What is unequal division cleavage called?
- equal divisions: isolecithal, sea urchins
- asymmetrical: mesolecithal, amphibians
What is typical of holoblastic cleavage?
- initial cleavages rapid
- many organisms have no mRNA transcription
- instead proteins synthesized from “maternal mRNA’s” stored in the egg
Another “fishing expedition” examined early sea urchin protein synthesis, what did they do and find?
- used/added 35 S-methionine and added sperm for IVF
- examined newly synthesized proteins at various times points (using autoradiography)
- see proteins that are being synthesized following fertilization
- line 35 shows a parabolic pattern of darkness (protein increases, decreases, then increases)
