Fertilisation and the Initiation of Development Flashcards
What happens to semen after it is deposited in the vagina?
- Semen deposited in the vagina, at the cervical os
- Semen coagulates, becoming gelatinous (enzymes released from the prostate act on fibrinogen-like substances to create a fibrin-like gel)
- Gel retains spem and may buffer them against the acid cervical fluid (pH 5.7) and vagina (pH4.7) – important, as it would immobilize the sperm
- Within one minute, sperm are detected in the cervix → around 1 million
- 99% sperm are lost from the vagina
- Around 102 make it to the fallopian tube
Describe the characteristics of human cervical mucous
• Protects cervix from hostile vaginal environment, eg) pH, leukocytes, pathogens
• Retricts sperm entry to the periovulatory period
− During the periovulatory period, mucous has greatest water content (98%)
− Greatest mucin glycoprotein concentration
− Largest mucin glycoprotein:serum protein ratio
− Maximum ferning and spinnbarket (also known as fibrosity – rheology term referring to the stringy property of the mucous)
− Only in the absence of progesterone can sperm penetrate mucous
• Restricts entry of abnormal sperm morphs
• Provides a potential auxillary energy source
• Removes anti-capacitation factors from sperm
• Sperm swimming through mucus takes on distinctive morphology – figure of 8
• Sperm form groups (phalanges) – beat tails in a cooperative fashion to get through mucous effectively
• Within 1 minute of intercourse, sperm found in cervical crypts → can survive here 24 hours
• Crypts may act as a sperm reservoir, or provide low resistance mucous channels to the uterus
How are sperm lost on the way to the oviduct?
- Many sperm wont enter the cervical mucous
- Of those that do, passage into the uterus possible only for those with vigorous progressive motility, morphological normality and a plasma membrane functioning appropriately in response to the environmental conditions
- In the uterus, some will undergo a premature acrosome reaction, others will succumb to leukocyte-produced ROS
- At most, several hundred sperm will reach the oviducts and attach to the oviductal epithelium
- The cumulous and zona pellucida on the oocyte act as the final sperm filter, so that perhaps 10 or 20 sperm reach the zona surface.
How do sperm move to the fallopian tube
• Sperm move by their own motility
• Possibly aided by uterine cilia-driven currents or uterine muscular movements
− Kunz et al, 1997 → peristaltic contractions initiating in the cervical region of the uterusand propagating to the fundal region with increasing frequency as the follicular phase progresses towards ovulation
• NOT moved by prostaglandin action
• Sperm become immotile at the isthmus of the oviduct – perhaps binding oviductal epithelium (Seems to be the case in animals).
What are the events occuring during capacitation?
Fast
1. Activation of sperm motility at the time of ejaculation
• Although sperm in the epididymis consume oxygen at a high rate, they are immotile. Flagellar movements start when sperm come into contact with high HCO3- and Ca2+ in the seminal fluid.
• Depends on increase PKA activity mediated by Ca2+ and HCO3- stimulation of the atypical adenyl cyclase SACY.
• HCO3- and Ca2+ are transported into the sperm by a Na+/HCO3- cotransporter and a sperm-specific Ca2+ channel CatSper present in the principle piece of the tail.
• Sperm membrane becomes hyperpolarized
Slow (occurring in female reproductive tract)
2. Removal of seminal glycoproteins
• Glycodelin S, Tocopherol, cholesterol stabilize the uncapacitated state
• Seminal plasma has been shown to prevent sperm acquiring acrosomal responsiveness to fusion promoting agonists.
• Removal of the glycoproteins causes increases in membrane fluidity, that is more permeable to Ca2+
− Sterol binding proteins initiate loss of cholesterol
− Suggested that movement through the mucous has a ‘membrane scrubbing effect’
− Tocopherol protects against oxidation induced damage. Cervical mucous contains leukocytes which produce ROS, and sperm also generate ROS.
− Tocopheral removal would make immature sperm vulnerable to ROS, rendering them inviable and removing them from the functional cohort.
− Removal from normal sperm could potentially facilitate beneficial ROS mediated capacitation.
- Induction of hyperactive motility → vigorous, whiplash type, frantic, high amplitude
• Further Ca2+ influx and release of internal Ca2+ stores (requiring external HCO3-) results in increased intracellular cAMP levels, and a switch to hyperactive motility
• Also promotes cAMP dependent tyrosine phosphorylation of sperm proteins
− The tripeptide FPP (fertilization promoting peptide) inceases adenyl cyclase activity in the sperm and is essential for capacitation
• CatSper is constitutively active, but stimulated by progesterone to induce hyperactivation and asymmetric (directed) motility (Lishko et al 2011)
− Sperm responds to progesterone in less than a second
• ROS thought to play a role in aiding some of these steps, eg, increased tyrosine phosphorylation and ability to respond to progesterone. - Sperm detach from the oviductal epithelium in isthmus to reach oocyte
• Aided by the hyperactive motility – sperm can more easily break free - Export of protrons
- Cytoskeletal chances
- Exposure of sperm receptors, notably those for the zona pellucida proteins ZP2 and ZP3
• Capacitation is a priming step for the acrosome reaction → Ca2+ influx, ROS, increased adenyl cyclase activity and tyrosine phosphorylation involved in induction of the acrosome reaction.
Describe fertilisation from penetrating the cumulous and binding to the zona
- Sperm penetrate the cumulus cells
• Hyperactivated motility
• GPI-anchored surface hyaluronidase PH-20 enzyme → digests hyaluronic acid, craating a channel. - Step 1: Initial binding to the zona
• Is b-1-4-galacyotsyl transferase (Gal-T) independent
• Other sperm receptors identified, eg) SED1 → synthesized by devel sperm and acquired in epididymis – can competitively inhibit sperm zona attachment
• Is responsible for firm sperm-zona binding and may induce aggregation of sperm receptor Gal-T which binds ZP3 - Step 2: Gal-T dependent binding to the zona
• Gal-T on the sperm head binds to ZP3 on the egg zona pellucida
• ZP3 receptor binding leads to clustering of the receptors to the side of the sperm head
• Removal of the carbohydrate residues on ZP3 prevents sperm binding
Is this zona binding required for subsequent acrosomal reaction?
• Originally thought Gal-T binding was required
• Recent experiments found sperm that undergo acrosome reaction while penetrating the cumulus (before zona binding) can still fertilise
• So binding to zona facilitates acrosome reaction, but can occur without it.
Describe the acrosome reaction (Step 4) and penetration through the zona (5-6)
- Sperm undergo the acrosome reaction
• Facilitated by Gal-T (controversial) binding to the ZP3 oligosaccharide chain
• Sperm plasma membrane and outer acrosome membrane undergo multiple fusions
• Sperm head at the anterior region becomes covered by what was originally the acrosomal membrane
• Mechanism:
− Requires clustering of the sperm receptors (Gal-T enzyme)
− Other ZP3 receptors and cumulus factors may be involved
− Ca2+ dependent → activation of CatSper leads to Ca2+ influx, that activates G proteins and phospholipase C
➢ PLC activation converts PIP2 to IP3, which releases internal calcium stores and leads to sustained Ca2+ influx
➢ Ca2+ induced exocytosis of acrosomal cap
➢ Causes the conversion of the enzyme proacrosin to acrosin – acrosin is the active protease. - ZP2 receptor binding ZP2 keeps the sperm attached to the zona
- Penetration through the zona
• Facilitated by motility, acrosin protease and glycosidase activity
Describe sperm-oocyte membrane binding (step 7)
• NB: many more acrosomally reacted sperm will bind than will eventually fuse with the oocyte. Often outnumbered 10-1.
• Sperm binds to the oocyte membrane to a micro-villi free region away from the metaphase chromosomes
• Sperm binds by the equatorial segment
• ADAM1 has been suggested to be involved, but its integrin ligands on the oocyte are not
− Several ADAM proteins expressed by sperm
− ADAM integrin ligands a6b1 KO mice are fertile → so they must be binding to something else
• Sperm associated DE
− Secreted by the epithelial cells of the epididymis
− Found tightly bound to sperm surface
− Binding inhibits fusion
• Acrosome released vitronectin may act as a bridge facilitating other binding
Describe sperm oocyte fusion (step 8) and final fertilisation.
- Sperm oocyte fusion – involves fusion proteins
• CD9 – a tetraspanin protein
− On the oocyte membrane in the microvilli
− CD9 KO mice cant fuse with sperm
− Theories suggest it is involved in organizing membrane proteins – may be needed to maintain proper structure of egg microvili
• CD81
− Another tetraspanin, resembles CD9
− Its KO effects are milder than CD9
• GPI anchored oocyte protein
• Sperm protein Izumo
− A novel Ig family protein
− Izumo null sperm can penetrate the zona, but cant fuse - Upon fertilization, the sperm head, and much of the middle piece and tail move into the oocyte cytoplasm
− Sperm brings the centriole, forming the sperm aster for the first mitotic division.
What does activation of the egg involve?
- Sperm activates the egg by triggering a series of oscillations in intracellular free calcium → calcium transients.
- Transients start within a few minutes of sperm- egg interaction
- Rise in free calcium (10x increase) lasts about a minute from first peak
- Frequency varies form 3-15 minutes
• Transients require internal calcium release from ER, but they stop in Ca2+ free medium → suggesting influx of Ca2+ also important
• Transients required for cortical granule extrusion, resumption of meiosis and pronuclear formation (the latter 2 events also need CAM kinase II)
− CAM kinase II activity stimulated at fertilisaiton
− Phosophorylates Emi2, targeting it for destruction and sets in train the activation of the anaphase promoting complex.
- Increased intracellular calcium spreads out from the point of sperm entry in a wave
- Repeated calcium wave essential to activation of the egg –if it is blocked, no further development occurs
- The calcium wave is regenerative, sperm-egg binding leads to cytoplasmic events that increase sensitivity to calcium-induced calcium release.
What are the possible methods of generating calcium transients
- Sperm factor
• Most likely possibility, suggested by Swann and colleages
• Idea that fusion of sperm and egg results in release of a molecule present in the sperm cytosol to the oocyte
• Evidence: - Sperm-oocyte fusion precedes 1st calcium transient
- Injection of sperm extract into oocyte leads to calcium transient
- Injection of sperm extract increases sensitivity to calcium-induced calcium release
- Injection of a single sperm into oocyte cytoplasm for ICSI results in oocyte activation and calcium transients
• This factor appears to by PLC ζ - an isoform expressed exclusively in the testis
− Swann have shown it to trigger Ca2+ oscillations
− Knockdown reduces the activation rate of fertilisation
Why are there oscillations at all?
• Has been known that a ethanol can activate eggs by causing a single increase in Ca2+
• May be that oscillations are simply a consequence of non-linear feedback loop of Ca2+ activated IP3 production
− Must be noted that eggs of sea urchins and frogs have the same signaling mechanisms and yet display a single Ca2+ incease
• Could be to do with efficiency
− A single Ca2+ increase generated by electroporation keeping Ca2+ high for 5-10 mins is not very efficient at activating eggs →
− Giving a single large Ca2+ followed by a series of smaller pulses of Ca2+ was a much better protocol
− This may be linked with later stages in development
➢ Eggs activated by a single Ca2+ pulse do not develop as well after implantation
➢ Pattern of Ca2+ oscillations influences the size and morphology of post-impantation rabbit embryos
➢ Implies there may be some range of patterns of Ca2+ oscillations that is best for embryo development, and not linked to the immediate task of getting the embryo through the first cell cycle.
How is the block to polyspermy put in place?
• Has been known that a ethanol can activate eggs by causing a single increase in Ca2+
• May be that oscillations are simply a consequence of non-linear feedback loop of Ca2+ activated IP3 production
− Must be noted that eggs of sea urchins and frogs have the same signaling mechanisms and yet display a single Ca2+ incease
• Could be to do with efficiency
− A single Ca2+ increase generated by electroporation keeping Ca2+ high for 5-10 mins is not very efficient at activating eggs →
− Giving a single large Ca2+ followed by a series of smaller pulses of Ca2+ was a much better protocol
− This may be linked with later stages in development
➢ Eggs activated by a single Ca2+ pulse do not develop as well after implantation
➢ Pattern of Ca2+ oscillations influences the size and morphology of post-impantation rabbit embryos
➢ Implies there may be some range of patterns of Ca2+ oscillations that is best for embryo development, and not linked to the immediate task of getting the embryo through the first cell cycle.
