APEX OF PREOVULATORY FOLLICLE
RUPTURE SITE MUST FORM IN APEX - INFLICTS WLOUND ON ITSELF WALL IS THICK AND MANY LAYERS MUST BREAK DOWN
Basal lamina=continuous layers of connective tissue
Actual Follicle Wall (rabbit) 10 Hours Before Ovulation
MECHANISM OF OVULATION
PROGRAMMED CELL DEATH OR APOPTOSIS
HYDROLASES DEGRADE APEX
SMOOTH MUSCLE CELL CONTRACTION AT BASE OF FOLLICLE
3 MECHANISMS IMPORTANT IN OVULATION
1. PROGRAMMED CELL DEATH/APOPOTOSIS
3. SMC CONTRACTION
1. CELLS IN SURFACE EPITHELIUM DIE BY APOPTOSIS AND ARE LOST
2. HYDROLASES BREAK DOWN TISSUES/CELLS
HYDROLASES AND OVULATION
Lutenizing hormone stimulates the production of plasminogen activator (granulosa cells) in follicle cells. Plasminogen activator converts plasminogen to plasmin. Plasmin can either degrade the apex directly or activate procollagenase. Procollagenase, once activated, becomes collagenase which breaks down collagen. At the end of the degredation, the rupture site has formed and the oocyte is ovulated.
Actual Follicle 1 Hour Before Ovulation
Actual Follicle Minutes Before Ovulation
SMOOTH MUSCLE CELL CONTRACTION AND OVULATION IN MAMMALS
SMC ARE PRESENT IN THE BASE OF FOLICLE
CONTRACT JUST BEFORE OVULATION
CAUSES ANTRUM TO BECOME SMALLER
FORCES OCC TOWARDS APEX
WEAKENS APEX AND OPENS RUPTURE SITE
Products of Ovulation
zona pellucida, oocyte arrested in meiosis II, culumus cells (used to be follicle cells) are ovulated as the oocyte cumulus complex. Held together by hyaluronan or hyaluronic acid. Brings water in.
Hyaluronan or hyaluronic acid is a polymer of disaccharides, themselves composed of D-glucuronic acid and D-N-acetylglucosamine . It is a glycosaminoglycan between cells in the OCC.
OVIDUCT HAS THREE REGIONS - INFUND, AMPULLA, ISTHMUS
OCC IS OVULATED INTO PERITONEAL CAVITY
IS QUICKLY TRANSPORTED TO OVIDUCT WHERE FERTILZATION OCCURS
CILIA ON OUTER SURFACE OF INFUNDIBULUM PICKUP OCC
MOVES OCC INTO AMPULLA WITHIN MINUTES
PROVIDES OTPIMAL ENVIRONMENT FOR FERTILZIATION, PI DEVELOPMENT
OCC STAYS IN AMUPLLA SEVERAL DAYS - WAITING FOR SPERM
AMPULLA = SITE OF FERTILIZATION - NORMALLY
SPERM ARE DEPOSTIED IN BASE OF CERVIX
100 MILLION/EJACULATE -XENOESTOGEN/MALE FISH W/ OVARIES
COLONIZE CERVIX AND UTEROTUBAL JUNCTION
ONLY 1-1000 SPERM GET TO AMPULLA
AS SPERM MOVE THRU FEMALE TRACT - UNDERGO CAPACITATION AND ARx
AFTER FERTILIZED - MOVES THRU ISTHMUS - SEVERAL DAYS
ISTHMUS HAS LOTS OF SECRETORY CELLS
TAKES 4-5 DAYS TO PASS THRU OVIDUCT - TIMING REGULATED
co-inventor of birth control pills:
Evidence for Capacitation
AUSTIN AND CHANG DISCOVERED INDEPENDENTLY MICE AND RATS
PLACE SPERM DIRECTLY IN AMPULLA W/ FRESH EGGS
WAIT 1-2 HRS AND SEE IF EGGS ARE FERTILIZED - SURPRISINGLY THEY WERE NOT
DO SAME EXPERIMENT BUT WAIT 5-6 HOURS - THEN LOOK AT EGGS
NOW EGGS WERE FERTILIZED
SOMETHING HAPPENS TO SEPRM IN THE FEMALE REPRODUCTIVE TRACT THAT ENBALES THEM TO FERTILIZE = CAPACITATION
Austin-Mice M.C. Chang-Rats
injecetd sperm into female oviduct, waited 1-2 ours, looked at oocytes. Found that oocytes were unfertilized.
Repeated same experiment, but waited 5-6 hours instead, and saw fertilized ocyte and sperm on the oocyte.
In mammals, sperm have to reside in female tract a certain amount of time (humans: 6-8 hrs) to "incubate" and develop the capacity to fertilize. This process is known as capacitation.
allows sperm to undergo the acrosome reaction. Incubation can take place either in a dish or inside the female reproductive tract.
Graphically, this is represented by a %ARx vs. Time plot where the time without acrosome reaction is the capacitation interval.
WHAT IS CAPACITATION?
DEFINITION = CAPACITATION = CHANGES THAT OCCUR IN SPERM THAT ANABLE THEM TO UNDERGO AN ACROSOME REACTION
DURING THE CAPACITATION INTERVAL SEVERAL CHANGES OCCUR IN SPERM
HYPERACTIVATION OF MOTILITY
COATING MOLECULES COME OFF THE PM - DESTABILZE PM - MAKES IT EASIER FOR Arx TO OCCUR
PERMEABILITY TO Ca INCREASES - CA INFLUX IS NEEDED FOR Arx
MAY BE OTHER EVENTS BUT THE CHANEGS ACTUALLY REQUIRED FOR OCCURRENCE OF Arx ARE NOT WELL UNDERSTOOD
Before capacitation, sperm swim in serpintine fashion. Afterwards, they move with wide, wiplash beating of flagella.
seminal plasma proteins tend to keep sperm infertile, capacitation helps remove these proteins
During capacitation, the sperm plasma membrane increases its calcium permeability, allowing it to undergo acrosome reaction and penetrate the cumulus complex layers.
Before capatation, there is an acidic environment inside the sperm, capacitation allows ion channels to open and increase the pH
~1-2K sperm can get to the junction between the uterus and the oviduct. There, they bind to the oviductal epithelium and continue capacitation. If the oocyte moves to the ampulla, sperm are released from the epithelium, so they can fertilize. ONly ~1-1000 typically get to the oocyte
The Oocyte has to be fertilized to live. In humans, capacitance takes about 6-8 hrs. Seminal plasma secreted by glands added to sperm during ejaculation has to be removed.
Increased Intracellular pH Accompanies Capacitation and Hyperactivation
Proton concentration inside uncapacitated sperm is high (1000x more protons inside cell than outside)
Pores in sperm PM are created by HV1 protein- when this pore opens during capacitation, protons can flow out of sperm
This increase pH inside of sperm cell – need for hyperactivation, ARx
HV1 can be opened in different ways – depolarization, alkaline environment, anandamide, removal of Zn
Also marijuana opens channels – may be why users of marijuana have fertility problems – channels may open prematurely and sperm burn out before reaching the oocyte
Knowing channel may allow control of male fertility. Could inhibit opening of channel- prevent fertilization. Could stimulate opening of channel in subfertile sperm and give them a boost so they may become capable of fertilizing.
The stages of sperm capacitation in vivo.
The stages of sperm capacitation in vivo.
(1) At insemination hundreds of millions of spermatozoa are released into the female tract. At this stage in their life history these cells are progressively motile, yet uncapacitated.
(2) As spermatozoa traverse the uterine cavity, the initial stages of capacitation occur characterized by the loss of decapacitation factors, largely acquired from epididymal and seminal plasma, from the sperm surface.
(3) Spermatozoa are subsequently thought to establish a reservoir in the isthmic region of the Fallopian tubes (Baillie et al., 1997). While bound to these epithelial cells the spermatozoa become quiescent and are stored in readiness for ovulation.
(4) An endocrine signal coincident with ovulation induces a sudden change in sperm biochemistry characterized by an increase in reactive oxygen species (ROS) generation, intracellular cyclic adenosine monophosphate levels and tyrosine phosphorylation. In response to these signals, calcium is released from an intracellular store in the redundant nuclear envelope in a pulsatile manner inducing the expression of hyperactivated motility.
(5) In this hyperactivated state, spermatozoa are released from the oviductal epithelium and migrate up the Fallopian tube towards the oocyte where they engage the cumulus mass.
(6) Spermatozoa may acrosome react within the cumulus mass or may migrate towards the zona surface and bind to this structure via surface-orientated zona-binding complexes localized within lipid rafts and featuring a number of potential zona-binding molecules including arylsulfatase A (ARSA) and the zona pellucida binding protein (ZPBP2) (Redgrove et al., 2011, 2012).
PENETRATION OF THE
ZONA PELLUCIDA, AND OOLEMMA
WHEN CAPACITATED SPERM ARRIVES AT OCC IT IS ABLE TO PENETRATE CUMULUS LAYER
SPERM PENETRATION OF CUMULUS
IS BOUND TO SPERM SURFACE – GPI LINKED
MAY AID IN PENETRATION OF PVS
SOME EVIDENCE SUGGESTS IT FUNCTIONS IN SECONDARY BINDING OF SPERM TO ZP
Sperm travel between cells. Smooth muscle in the uterus and oviduct help sperm move towards teh ampulla. After that, sperm are on their own.
Hyaluronic acid (keeps Oocyte Cumulus Complex together) -sperm have an enzyme called hyaluronidase to break it down.
It was originally thought that hyaluronidase on sperm got rid of hyaluronic acid, but it was shown to be false.
knockout gene for Ph20 should have reduced fertilized egg count, but it actually just slowed fertilization down.
Therefore, Ph20 facilitates fertilization.
MODEL OF ZONA PELLUCIDA STRUCTURE
ZONA PELLUCIDA FORMS -AN ECM
A. ISLANDS OF FIBRILLAR MATERIAL ARE DEPOSITED BETWEEN OOCYTE AND F.C
B. MATERIAL ORIGINATES FROM OOCYTE - IN SITU HYBRIDIZATION
C. COALESCES TO FORM CONTINUOUS ACELLULAR COAT
D. ZP. PROTECTS OOCYTE AND CLEAVING PE
E. MADE OF 3 GLYCOPROTEINS ZP1, ZP2, ZP3
F. WORKING MODEL OF ZONA STRUCTURE
humans: 7 microns thick.
perivitelline space (PVS) between oocyte and zonna pellucida.
Paul Wassarman and Jurrien Dean
Paul Wasserman was the first to isolate the zona pellucida (Had to get 4,000 oocytes for enough material). Discovered 3 proteins in teh mouse zona - ZP1, ZP2, and ZP3.
Wasserman model: ZP2 and ZP3 alternate to make filament, cross linked by dimers of ZP1 (may be incorrect)
The Structure of ZP3 at 2.0Ǻ Resolution
Composition of zona pellucida varies by species.
Determined by crystallizing ZP3 and studying its structure with X-ray crystallography
ZP-N and ZP-C domains interact with corresponding domains in adjacent molecule to create a dimer of ZP3 – link these together over and over – leads to formation of a filament
Can not dimerize until the EHP domain is cleaved off the polypeptide
Figure 1. Analysis of expression by PCR, of human ZP genes ZP1, ZP2, ZP3, ZPB, and housekeeping genes β‐actin and HPRT in amplified cDNA preparations from four samples of human oocytes.
Figure 1. Analysis of expression by PCR, of human ZP genes ZP1, ZP2, ZP3, ZPB, and housekeeping genes β‐actin and HPRT in amplified cDNA preparations from four samples of human oocytes. Sizes of expected PCR products are given to the right of the Figure in base pairs (bp). Blank (–): omitting DNA sample to the PCR reaction.
Zona proteins in various mammals
Sperm binding to ZP-Wassarman/Dean
Human zona proteins are incorporated into the zona pellucida of transgenic mouse eggs. Ovulated eggs from human ZP1 (huZP1), huZP2, and huZP3 rescue as well as huZP4 transgenic mice were fixed and stained with mAb to human ZP1 (huZP1), huZP2, huZP3, huZP4, mouse ZP1 (moZP1), moZP2, and moZP3. Antibody binding to human and mouse zona proteins was detected by confocal microscopy and faux colored dark blue (huZP1), red (huZP2), green (huZP3), orange (huZP4), magenta (moZP1), light blue (moZP2), or yellow (moZP3). Fluorescent and DIC images were merged.
Isolate zonas, put in acid to dissolve.
Make solutions of proteins, run on gel, separate, cut out bands. Yields purified proteins
Incubate sperm with proteins, wash off unbound protein, let sperm incubate with egg (full with zona)
1. sperm fertilize the egg-->Protein does not bind to sperm
2. Sperm do not fertilize-->protein binds to sperm, blocks receptors.
Which ZP does sperm bind to?
WHAT IS THE ARx?
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.
GAMETE MEMBRANE FUSION
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.
Which gamete membranes are involved in fusion?
What Molecules are Involved in Fusion?
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
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
CREATED KNOCK OUT MOUSE TO DETERMINE FUNCTION OF IZUMO
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)
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
Ovastacin Plays a role in blocking polyspermy
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).
ZP2 cleavage and sperm binding to AstlNull two-cell embryos.
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.
FINAL STEP IN FERTILIZATION
MALE AND FEMALE PRONUCLEI FORM
SYNGAMY-fusion of male and female nuclei
RESULTS OF FERTILIZATION
sex dependent on sperm. we can't tell yet.
fertilization saves the oocyte from degradation.
18-21 hours later, the zygote divides.
REVIEW OF DAY 1
SUMMARY OF DAY 1 EVENTS
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
FERTILIZATION AND CRx
2C BY 18-21 HRS
Fertilization in vitro
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.
What is In Vitro Fertilzation
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.