FERTILIZATION

                                                                                                

         

                                                      

                                                                        

      

                                       

                                                                        

      

                                       

Sperm transportSemen is ejaculated into vagina, cervical os and may reach cervical canalSemen coagulate by coagulating enzymes from prostate gland which interacts with fibrinogenous substrate from seminal vesiclesCoagulum protects spermatozoa from vaginal acidity and prevent loss of spermatozoaCoagulum liquefies after 15 to 20 minutesLiquefaction time is one criteria for semen qualitySperm may live up to 48 – 72 hours in female reproductive tract

Abnormal sperm

Barriers (1)Spermatozoa have to confront three barriers before reaching ampulla, the site for fertilizationFirst barrier – cervical mucusMucus will filter and choose spermatoza – dead and immotile spermatozoa are discarded, normal spermatozoa are stored in the cervical crypts (first reservoir)Filtered spermatozoa discarded in the vaginal secretion post-coitalConsistency of cervical mucus assist in sperm motility upwardsMucus thick, sperm could not penetrateMucus thin and more stringy, sperm is assisted in motility by swimming in channels form by the mucus

Barriers (2)

Endometrial glands – second barrierWill choose and filter spermatozoaChosen spermatozoa are stored here (second reservoir)Here, capacitation occurs due to prostaglandins from endometrium

Barriers (3)

Third barrier – utero-tubal junctionChosen spermatozoa are finally stored in the isthmus (third reservoir) to wait for the ovum to travel downOvulated ovum will be caught by infundibulum when fimbriae comes close to ovaryOvum will travel down the infundibulum to the ampulla by oviductal contraction and presence of cilia

                                                                        

      

                                     

Capacitation (1)

A time dependent phenomenon which is species-specificTakes more than 24 hours in humanReversible process (if capacitated spermatozoa are placed in epididymal fluid or seminal plasma, will be decapacitated – contains decapacitating factor) only in vitroMust occur to enable acrosome reaction to occurSubstances like cholesterol, glycosaminoglycans and glycoproteins are stripped from plasma membrane of sperm head

                                                                        

      

                                       

Capacitation (2)

Two elements in this process:1. Hyperactivated motility – sperm starts

to show whiplashing movement to enable sperm to move forward faster

2. Change to membrane surface – membrane stability decreases. More permeable to calcium ions. Tyrosine kinase activity increases. Adenyl cyclase activity in spermatozoa heightens and causes protein phosphorylation which are cAMP dependent

Acrosome reaction

Occurs right after capacitationTotally dependent on calcium uptake into cells and increase in intracellular pH (pH 7.1 to pH 7.5)The acrosome swells and the outer acrosomal membrane fuses with the overlying plasma membraneVesiculation occurs and pores are formedAcrosomal contents (hyaluronidase, acrosin etc) are released

Acrosome reaction

Two types:True acrosome reaction – reaction occurs at zona pellucidaFalse acrosome reaction – degeneration of sperm due to death (enzymes from acrosome will self-desctruct sperm)

Initiators of the acrosome reaction (1)

High calcium influxZP3 (zona pellucida glycoprotein 3)Progesterone etcZP3 in ovum will bind to sperm binding protein (receptor) on sperm plasma membraneThis binding site may contain galactosyl transferase activityWhen binding occurs, G protein involvement will stimulate calcium influx and the rise in pH initiates the acrosome reaction

Initiators of the acrosome reaction (2)

Progesterone will also stimulate calcium influx which then stimulates adenyl cyclase and cAMPProgesterone can stimulate acrosomal leakage to release hyaluronidaseHyaluronidase will digest hyaluronic acid which binds cumulus cellsWhen these cells breaks apart, spermatozoa can bind to zona pellucidaProgesterone has been reported to initiate capacitation also

Sperm binding properties to zona

Outer acrosomal membrane has receptor to ZP3Inner acrosomal membranes has receptor to ZP2Equatorial segment and post-acrosomal region is the part of the spermatozoon that enters the ovumTail and midpiece left outside ovum

ZP3 binding with receptor on outer acrosomal membrane of sperm

ZP2 binding to receptor on inner acrosomal membrane of sperm

                                                                        

      

                                      

Gamete fusionSperm penetration of the zona takes between 5-20 minutesSperm lies tangent at the ovum surface between the zona pellucida and oolemma at the perivitelline spaceMicrovilli at the oocyte surface will engulf the sperm headThe equatorial segment and the post-acrosomal region of the sperm fuses with the ovumAfter fusion, zygote forms and male and female pronucleiSyngamy occurs when both male and female chromosomes combines and then form 2-cell conceptus

Formation of male and female pronuclei

Block to PolyspermyImportant to prevent more than one sperm fertilising the ovum. This phenomenom ensures: Prevention of triploidy (2 sperm fertilising an ovum)Prevention of polyploidy (many sperm fertilising an ovum) Usually only one sperm (n) can fertilize an ovum (n) to achieve a diploid (2n) zygote Fusion of ovum to sperm initiates the release of free intracellular calcium from calcium stores and this causes calcium spikes.

Increase in calcium is due to change in the pattern of protein phosphorylation in the ovum and the involvement of enzymes e.g., tyrosine kinase and phospholipase BHigh calcium influences the cortical reaction; cortical granules in the zygote cortex will fuse with the oolema.This causes release of the cortical substances into the cortical spaceThe substances (enzymes) will act on the zona pellucida by ‘hardening’ itThis hardening (zona pellucida reaction (tindakbalas zona) is the ‘masking’ of the ZP3 and ZP2 so that other sperm could not digest the zona with acrosin and also reduce the sperm binding properties to the zona

Mammalian sperm factor – PLC-zeta

Schematic representation of the basic hypothesis for how PLC initiates Ca2+ release in mammalian eggs.

Stimulus of oocyte activation

After fusion of the sperm and egg plasma membranes, the sperm-derived PLC protein diffuses into the egg cytoplasm. This hydrolyses PIP2, from an unknown source, to generate InsP3.

It is possible that the subsequent rise in Ca2+ leads to the regulation of PLC activity.

When sperm activate eggs at fertilization the signal for activation involves increases in the intracellular free Ca2+ concentration. In mammals the Ca2+ changes at fertilization consist of intracellular Ca2+ oscillations that are driven by the generation of inositol 1,4,5-trisphosphate (InsP3). It is not established how sperm trigger the increases in InsP3 and Ca2+ at fertilization.

One theory suggests that sperm initiate signals to activate the egg by introducing a specific factor into the egg cytoplasm after membrane fusion. This theory has been mainly based upon the observation that injecting a cytosolic sperm protein factor into eggs can trigger the same pattern of Ca2+ oscillations induced by the sperm. The soluble sperm factor protein is a novel form of phospholipase C (PLC), and it is referred to as PLC (zeta).

                                                                                          

      

                                             

                                                                                                

         

                                                      

Slide 24 of 40

                                                                                                

         

                                                      

                                                                                                

         

                                                      

                                                                                                

         

                                                      

                                                                                                

         

                                                      

Embryonic development (1)

Germinal period (movement of zygote and implantation in uterus) lasts two weeksCleavage occurs - 1 cell to 2 daughter cells after 36 hours post fertilizationDaughter cells called blastomeresZygote still covered by ZPZP inhibits blastomeres from falling apartIf this happens, two possibilities occur

1. Monozygotic twins2. Chimaeras

Chimaeras is the fusion of two different zygotes from two fertilized ovum – two sets of two different genotype

Fertilized egg 2 cell stage

4 cell stage 8 cell stage

Embryonic development (2)

Blastomeres becomes morula on day 3Progesterone from functioning CL will stimulate the release of glycogen from endometrium for energy to developing embryo (histiotropic nutrition)High levels of progesterone also inhibit oviductal constriction to enable morula to move towards the uterus by peristaltic contraction and cilia movementBecomes blastocyst on day 4 - 5

Blastocyst

Embryonic development (3)

Blastocysyt has fluid-filled cavity (blastocoele)Has inner cell mass (ICM) surrounded by trophodectum (trophoblast)ICM will form extra embryonic membranes (amnion, yolk sac etc) and fetusTrophoblast forms chorion Blastocyst floats in uterine cavity for 1 – 2 daysPrior to implantation, will shed ZP by enzymatic digestion

Maternal Recognition of Pregnancy

Embryo sends signal along ovarian-pituitary pathwayStop FSH release but maintain LHImportant because luteal regression will occur if the mother does not recognise the pregnancyDrop in progesterone will cause abortionSignal to mother is to change cyclic pattern of oscillating progesterone and estrogen to non-oscillating pattern with only progesterone as dominating hormone to maintain pregnancyProstaglandin F2 secretion are also inhibited throughout pregnancy

Implantation (1)

A nutritional and physical contact between fetus and motherBlastocyst surface becomes stickyTrophoblastic cells (cytotrophoblast) releases enzymes to digest proteins on endometriumSyncytiotrophoblast enters endometrium to suck up metabolic fuel and nutrientsDeep invasion into endometrium occursChange to endometrium occurs (stromal reaction/primary decidualization reaction)Endometrium releases prostaglandins to stimulate vascularization causing edema and increasing nutrient stores

Implantation (2)

The invaded part of the endometrium is called decidua2 –3 days post invasion, decidua enlarges to become secondary decidua Blastocyst enters this deciduaAfter entry, a layer of endometrial cells will cover and bury the blastocystSyncytiotrophoblast on the other hand keep on digesting endometrial cells to get nutrients until the placenta is formed