Ovulation, Gamete Transport, and Fertilization Flashcards

Question

WHAT IS THE ARx?

Answer 1

EXOCYTOTIC RELEASE OF ACROSOMAL CONTENTS OCCURS BY MULTIPLE MEMBRANE FUSIONS OF OAM AND PM ZP3 INDUCES ARx 1ST DESCRIBED BY BARROS 1967 ET AL FOR MAMMALS SPERM PM FUSES WITH OAM CREATES MANY SMALL PORES IN SPERM SURFACE AND ALLOWS ACROSOMAL CONTENTS TO LEAK OUT EVENTUALLY SPERM SHEDS THE MEMBRANE VESICLES = Arxed Arx MUST OCCUR FOR SPERM TO PENETRATE THE ZP WHERE DOES Arx OCCUR? ZONA GEL - 3 GLYCOPROTEINS CUT OUT BANDS AND TESTED EACH TO SEE IF THEY STIMULATE ARX ONLY ZP3 STIMULATES Arx RX IS THOUGHT TO OCCUR ON ZP SURFACE ZP3 ALSO HAS SPERM BINDING CAPABILITY CONTRIBUTES TO ZP STRUCTURE BINDS SPERM TO ZP INDUCES Arx ON ZP SURFACE Acrosome is a vesicle. Only the cap vesiculates (Not the equitorial region). Teh acrosome causes the membranes to fuse together and allow the contents of teh acrosome to fuse out. Proteins on the equatorial segment are modified so they can fuse to the plasma membrane of teh oocyte. Acrosome might do this.

Answer 2

Despite the importance of the events leading to formation of a zygote- and despite decades of research- the molecular basis for sperm–egg fusion has remained poorly understood, and the field is littered with the carnage of erstwhile molecular candidates.” ~15 years of intense study, many mistakes were made In mammals, teh membrane on teh equatorial segment is where gamete membrane fusion begins.

Answer 3

GAMETE MEMBRANE FUSION OCCURS TO A RESTRICTED PART OF SPERM’S PM PM OVERLYING EQ REGION OF SEPRM IS FUSOGENIC OOCYTE PM FORMS MV - TIPS OF MV FUSE WITH SPERM PM ADAMs FAMILY= A DISINTEGRIN AND METALLOPROTEASE DOMAIN – EXPLAIN FERTILIN AND CYRITESTIN ARE ADAMs DISTINEGRIN DOMAIN BINDS TO INTEGRINS IN OOLEMMA BUT KO OF FERTILINS AND INTEGRINS DO NOT SUPPORT THE MODEL CD9 = A TETRASPANIN – SPANS MEMBRANE 4 TIMES FOUND IN OOLEMMA CD9 KO – SPERM DO NOT FUSE WITH OOCYTE – SUGGESTS CD9 IS KEY MOELCUE IN FUSION CD9 RESCUE – INJECT KO OOCYTES WITH WILD TYPE CD9 - mRNA RESCUES PHENOTYPE – NOW PSERM CAN FUSE Mrna with mutaion in large extracellular loop – phenotype not restored – so large loop IMPORTANT IN SETTING UP FUSION TETRASPANINS ORGANIZE MUTLIMOLECLUAR COMPLEXES ON CELL SURFACES ALSO MAY BIND A LIGAND ON AN ADHERING CELL DATA SUGGEST THAT TETRASPANINS ARE A KEY MOLECULE IN GAMETE MEMBRANE FUSION Fertilin (alpha and beta) belongs to ADAM family. Fertilin was the first molecule discovered (in this) there are integrins in oocyte membrane (alpha 6 beta 1) KO beta fertilin, integrins, sperm still fused Fertilin and integrin not involved in fusion Red protein CD9 in Oocyte - tetra spannin (goes through membrane 4 times) KO for CD9 infertile

Answer 4

Japanese shrine dedicated to marriage Inoue et al 2005 Nature 434:234 Had a monoclonal AB (antibody) that inhibited GMF Used MAB (similar to western blot) to identify antigen in blots of 2D gels Isolated antigen, sequenced by mass spectrometry IDed sequence in database Named protein IZUMO-implicated in gamete membrane fusion IZUMO = a member of the immunoglobin superfamily Is a type I membrane protein with extracellular immunoglobin domain MW = 56.4 kDa for mouse Is not detectable on sperm surface until after the ARx

Answer 5

Experiment: Izumo KO was fertile with injection of sperm, but they cannot bind to the membrane. We don't know what Izumo binds to (yet) ICSI (intracytoplasmic sperm injection) was used in this experiment so that the nuclei of the sperm and the oocyte could fuse to make the embryo. SUZI is subzonal insertion(into the PVS)

Answer 6

Essentially, oocyte's answer to fetilization AFTER GAMETE MEMBRANE FUSION OOCYTES UNDERGO CRx MANY VESICLES ARE PRESENT BENEATH PM OF UNFERTILIZED OOCYTE AT FERTILIZATION THEY DUMP THEIR CONTENTS INTO PVS SOME CONTENTS DIFFUSE INTO ZP AND MODIFY IT CALL THE ZONA REACTION SPERM CAN NO LONGER BIND TO THE ZP SETS UP A BLOCK TO POLYSPERMY SOME CONTENTS STAY IN THE PVS FORM A NEW COAT - THE CORTICAL GRANULE ENVELOPE MAY ALSO SERVE TO BLOCK POLYSPERMY - REDUNDANT BLOCK MAY ALSO FUNCTION IN PREIMPLNATATION DEVELOPMENT Some stay in PV space, some change the zona pellucida (in the zona reaction) so taht sperm can no longer bind. This blocks polyspermy. ZP2 cleaved--\>no sperm binding

Answer 7

Localization of ovastacin in normal and AstlNull mice. (A) Unfertilized eggs and two-cell embryos from normal, AstlHet, and AstlNull mice were collected and viewed by confocal and differential interference contrast (DIC) microscopy. Eggs and embryos were stained with rabbit anti-ovastacin (Ovst) antibody, LCA-FITC, and Hoechst 33342. Peripherally located cortical granules stained with LCA-FITC (white arrows) were observed in normal and AstlHet but not AstlNull eggs. The presence or absence of ovastacin correlated with the detection of LCA-FITC (black arrows in differential interference contrast/merge). Astl=ovastacin

Answer 8

ZP2 cleavage and sperm binding to AstlNull two-cell embryos. (A) Immunoblot of lysates from eggs (20) and two-cell embryos (20) from normal, AstlHet, and AstlNull mice probed with an mAb specific for the C-terminal region of mouse ZP2 (M2c.2). Intact ZP2 is 120 kD, and the cleaved C-terminal fragment of ZP2 is 90 kD. (B) Eggs and two-cell embryos from AstlNull and two-cell embryos from normal mice were incubated (1 h) with capacitated sperm. After washing with a wide-bore pipette to remove all but two to six sperm on normal two-cell embryos (negative control), eggs and embryos were stained with Hoechst 33342, and bound sperm were presented as z projections of 5-µm confocal optical sections and differential interference contrast (DIC) images.

Answer 9

MALE AND FEMALE PRONUCLEI FORM ## Footnote SYNGAMY-fusion of male and female nuclei Fertilized oocyte=zygote

Answer 10

NEW INDIVIDUAL DIPLOIDY RESTORED SEX DETERMINED OOCYTE ACTIVATED sex dependent on sperm. we can't tell yet. fertilization saves the oocyte from degradation. 18-21 hours later, the zygote divides.

Answer 11

SUMMARY OF DAY 1 EVENTS FERTILIZATION SECOND POLAR BODY IS EXTRUDED - OOCYTE FINISHES MEIOSIS MALE AND FEMALE PRONUCELI FORM - EACH IS HAPLOID PRONUCELI FIND EACH OTHER AND UNITE CREATING A DIPLOID ZYGOTE BY 18-21 HAS DIVIDED MITOTICALLY TO FORM 2C STAGE DURING 1ST 18-21 HOURS mRNA IS NOTSYNTHESIZED THINGS RUN ON MATERNAL mRNA ALREADY STORED IN EGG POSTTRANSLATION MODIFICATIONS OF MATERNAL PROTEINS (GLYCOSYLATION AND PHOSPHORYLATION) REFERED TO AS MATERNAL CYTOPLASMIC INHERITENCE OVULATION GAMETE TRANSPORT FERTILIZATION AND CRx PRONUCLEAR FORMATION SYNGAMY 2C BY 18-21 HRS

Answer 12

1st human-1960's. R.G. Edwards (researcher) and Patrick Steptoe (physician) They were successful in 1969 with Louise Brown Now, there have been about 4 million babies born via IVF 2010-Edwards won the Nobel Prize. Steptoe had already died.

Answer 13

Harvest oocytes (takes a ton of injections.) they are then cultured. Sperm are take from the father and they are mixed (typically by ICSI) Some of these become zygotes. The best are implanted in the mother's uterus. Maybe one implants. At first, there was only a 20% success rate, now it's about 60% It costs somewhere between 10,000 and 100,000 per attempt. to keep zygotes separate by parent, scientists can stick barcode-like device onto zona pellucida (isn't happening yet) holding pipette attaches to ZP, holds Oocyte still while sperm is injected.