lecture 11: fertilisation

Question 1

What is the male reproductive tract?

Answer 1

• sperm travels from the testis → epididymus → vas deferens → penis
• long way that it has got to go
• testis = site of sperm synthesis
• epididymus = site of sperm storage

Question 2

What is the female reproductive tract?

Answer 2

• egg has to make its own journey to the site of fertilisation in the ampulla of the fallopian tube
• female reproductive tract is a reservoir for sperm after sex
• sperm can survive for quite some time in the reproductive tract (day or so)
• lurk waiting for egg
• may have sex at night but you probably get pregnant in the tram on the way to work the next day

Question 3

What is the spermatozoa?

Answer 3

• sperm are highly differentiated, designed for a sole purpose
• designed for one thing and one thing only: to find and fertilise the egg
• the DNA in the sperm head is much more highly condensed than in the sperm cell
• use protamines instead of histones
• across the top of the sperm head is a patch called the acrosome
• acrosome is full of enzymes that are involved in breaking down the investments of the egg and getting through the glycoprotein coat around the egg (zona pellucida)
• midpiece contains the mitochondria
• the mitochondria are almost helical and wrap around the actual inner machinery of the sperm
• tail is the largest part / flagellum for movement
• ‘absolutely exquisite cell’
• like lots of things in reproduction it has lots of idiosyncracies, things that are unique to that cell
• unique biochemistry e.g. isozymes ( LDH) that are only found in sperm
• questions about role, function, etc

Question 4

What are the different cells called along the path of spermatogenesis?

Answer 4

• male germ cell in embryo (2n) → mitosis → spermatogonium (2n) in adult → mitosis → primary spermatocyte (2n) →→ first meiotic division
• these cells are diploid
• stem cells undergo rounds of meiosis
• → secondary spermatocytes (n) → spermatids with cytoplasmic bridge → sperm cells (n)
• cytoplasmic bridges are formed due to certain genes that are only found on the X chromosome that produce proteins required for sperm differentiation
• these cells are haploid

Question 5

What is the site of spermatogenesis?

Answer 5

• seminiferous tubules
  
  • lots and lots of tightly coiled tubules that make up the testis
• process of spermatogenesis and spermeogenesis can take about 60 days

Question 6

Where are sperm stored?

Answer 6

• within the seminferous tubules going into the epididymis

Question 7

Are all sperm the same size?

Answer 7

• no
• vertebrate sperm vary in size

Question 8

What is unique about human sperm?

Answer 8

• can use fructose as an energy source so seminal fluid is very high in fructose
• not many cells use fructose as an energy source
• cells lining the female reproductive tract can’t use fructose
• this means the sperm don’t have to compete for energy sources once they reach the female reproductive tract

Question 9

How big is the human oocyte? What does it look like?

Answer 9

• ~110 µm in diameter
• post-ovulation is immediately surrounded by the zona pellucida
  
  • glycoprotein coat laid down in the ovary around the egg
  • contains specific receptors that the sperm need to bind
• surroundind this a cloud of cells called the cumulus ooferous
• these cells are in direct communication with the egg
• prior to ovulation there are gap junction feeding directly from those cells to the egg
• bi-directional communication - dialogue
• these cells around the time of ovulation start to produce hyaluronic acid
  
  • glycosaminoglycan - but no amino groups attached so technically a polysaccharide
  • cells don’t grow
  • get dispersed into huge matrix
  • actually only a few hundred cells even though it looks like several thousand
  • inflated due to hyaluronic acid matrix
• one of the reasons for doing this is to ensure its pick up by the fimbrae/oviduct at the appropriate time

Question 10

are all eggs the same size?

Answer 10

• no vertebrate eggs vary in size and shape
• e.g. xenopus, chick, mouse, zebrafish
• lobe-finned fish eggs are 9cm big
• size is reflective of whether the egg will develop in vivo or externally
• eggs that are layed need to be provided with lots of nutrients and protection cf eggs that develop internally and have access to maternal nutrients

Question 11

What is interesting about the ovulated egg?

Answer 11

• it has not completed meiosis
• the human and the mouse egg do not complete the second meiotic division until the sperm enter
• embryo → oogonia → primary oocyte → meiosis I arrest → adult → secondary oocyte → meiosis II arrest → fertilised egg
• only one egg is produced by meisosis → rest of the genetic material is shunted off in polar bodies

Question 12

What are different mammalian GV stage oocytes?

Answer 12

• mouse: 80µm
• cow: 110 µm
• pig: 125 µm
• cat: 110 µm
• human: 110 µm
• mouse and human eggs contain very little lipid/endogenous nutrients
• whereas eggs from domestic species such as cow, pig and even cats contain a lot of lipid and this reflects differences in maternal physiology

Question 13

What is the ovulated oocyte?

Answer 13

• in most mammals, the oocyte does not complete meiosis until after fertilisation

Question 14

What is gametogenesis in mammals?

Answer 14

• in males, each spermatogonium gives rise to: four hapoloid sperm
• mitosis ceases in the embryo
• meiosis begins at puberty through to end of life
• in females, each oogonium gives rise to ONE haploid egg and 2 polar bodies
• meiosis begins in the embryo
• meiosis I arrested around birth
• a cohort of primary oocytes arrested at Meiosis I resume meiosis at puberty, and at each cycle but ends at menopause

Question 15

What is really important?

Answer 15

• that only one sperm gets through to the egg → otherwise polyspermy → dire consequences

Question 16

What is the egg’s journey?

Answer 16

• egg collected by fimbrium
  
  • passes through infundimbulum
  • ampulla of oviduct
  • fimbriae waft across the surface of the ovary around the time of ovulation
• egg ovulates
  
  • oocyte surrounded by
    
    • zona pellucida
    • cumulus (corona radiata)
• for a successful pregnancy to occur, mature, functional oocyte(s) must be available for fertilisation (in human < 300 / lifetime)
• ovulaton releases one oocyte per follicle
• these must enter the oviduct via the fimbrium

Question 17

What maintains the 3D structure of the cumulus oocyte complex?

Answer 17

• hyaluronic acid
• this is something that the sperm has to battle

Question 18

What is sperm formation, maturation and storage?

Answer 18

• spermatogenesis in testis
  
  • passive transport to rete
  • sperm entering the vasa are incapable of movement
• rete-testis
• caput epididymis
  
  • fluid resporption
  • secretions
  • sperm are immotile and incapable of fertilising egg
  • peristaltic movements along epididymis
  • maturation – morphology, biochem, physiology
• cauda epididymis
  
  • sperm storage
  • immotile but capable of motility (activation)
• epididymis
  
  • androgen dependent
  • regionally differentiated
  • additin of cartinine (profound antioxidant - sperm are very sensitive to oxidation, possibly there to protect mitochondrial function) , fructose and glycoproteins which coat the sperm
  • journey through the epididymis takes 5 to 11 days (species dependent)
  • sperm are said to mature in the epididymis
  • gain motility

Question 19

What is semen?

Answer 19

• sperm and seminal plasma
• consists of sperm suspended in a fluid that nourishes them and facilitates fertilisation
• components of seminal fluid produced in seminal vesicles, prostate gland and bulbourethral glands
• all components of semen join in the urethra and ejaculated through the penis by muscle contractions
• sperm are less than 5% of the volume of semen
• 5 - 10ml of semen at ejaculation
• sperm count anywhere from 20 - 200 million / ml → so a single ejaculate can contain as many as a billion sperm
• seminal fluid is thick and contains mucus
• semen is alkaline to protect against the acidity of the vagina

Question 20

How is sperm transported along the male tract?

Answer 20

• cauda epididymis and vas deferens → contractions
• smooth muscle in walls of seminal vesicle and prostate contract → seminal fluid
• sperm transferred to vagina via penile urethra

Question 21

What is sperm transport in the female tract?

Answer 21

• semen deposited
  
  • in vagina (e.g. human) (pH 5.7, i.e. acidic)
  • in uterus (e.g. pig)
  • in vagina, but carried into uterus by muscular (e.g. mouse)
  • copulatory plug (in humans, a very fine jelly)
• cervix
  
  • cervical mucus is barrier to sperm (varies with cycle)
  • filters out sperm with poor motility or abnormal morphology
  • ~99% of human sperm do not get past the cervix
• uterus
  
  • uterine contractions speed sperm transport
• utero-tubal junction
  
  • barrier to sperm transport
  • sperm need certain proteins on head to pass
• oviduct (fallopian tube)
  
  • storage of sperm in crypts or mucosal folds
  • binding to epithelium preserves sperm fertility and slow release reduces polyspermy
  • capacitation and hyperactivation
• ampulla
  
  • sperm meets egg
  • fertilisation
• typically only a few thousand sperm make it anywhere close to the site of implantation

Question 22

So what is the overall marathon journey of the sperm to the oocyte?

Answer 22

• seminiferous tubules → epididymis → vas deferens → ampullar and ejaculatory duct → prostatic urethra → penile urethra → vagina → uterus → oviduct → fertilisation
• motivated by: secretory pressure (cilia), ejaculation, sperm flaggellum
• 40 cm in total
• relative size makes this about 26 miles
• 40% cases of infertility are due to sperm unable to make this journey

Question 23

What is the longevity of gametes in the female tract?

Answer 23

• human
  
  • sperm: 24 - 48 h
  • oocytes: 6 - 24 h
• mouse
  
  • sperm: 6 - 12 h
  • oocytes: 6 - 15 h
• horse
  
  • sperm: 75 - 120 h
  • oocytes: 6 - 8 h
• extremes:
  
  • sperm greater than 10 years in species with sperm storage
  • sperm less than 1 minute in some species with external fertilisation

Question 24

What is capacitation?

Answer 24

• stripping of much of the glycoprotein coat on the sperm that was acquired during passage along the epididymis
• sperm exhibit a change in the surface membrane properties
• this prepares sperm to undergo acrosome reaction

Question 25

What is hyperactivation?

Answer 25

• changed motility → penetration of cumulus and zona
• change in flagellar beating pattern - increased flagellar bend angle
• triggered by influx of Ca²⁺
• triggered near egg/binding to zona

Question 26

How does the sperm find the egg?

Answer 26

• thermotaxis: temperature gradient
  
  • ampulla 2°C warmer than isthmus (rabbit)
• chemotaxis: chemical gradient
  
  • resact – 14 aa peptide from sea-urchin egg guides sperm
  • formyl peptide/odourant receptors at base of flagellum in human, dog and rat
• don’t know the method in the human

Question 27

What must oocytes have completed to be fertilisable?

Answer 27

• cytoplasmic and membrane maturation
  
  • accumulated abundat reserves (enzymes, lipids, histones, mRNA… energy stores (species dependent))
  • cortical granules in sub-cortical location
  • increased endoplasmic reticulum (reserves of Ca²⁺)
  • increased IP3 receptors
  • increased sensitivity to IP3
• nuclear maturation including:
  
  • oocyte specific DNA imprinting
  • meiosis
  • entered arrest (in human this is MII)
• other
  
  • zona pellucida formation

Question 28

How does sperm get to the egg?

Answer 28

• transit through
  
  • the cumulus
  • zona pellucida
  • egg/vitelline membrane
• involves Many different egg and sperm receptors, enzymes and factors
  
  • including ones acquired en route

Question 29

What is the acrosome reaction?

Answer 29

• triggered when sperm contact cumulus/zona
• facilitated by progesterone
• breakdown of outer acrosomal membrane
• release of beta-hexosamine which digests local zp3 receptor
• release of hyaluronidase from acrosome
  
  • digestion of hyaluronic acid that sticks cumulus together
• exposure of inner acrosomal membrane
• sperm then binds and fuses to the oocyte

Question 30

How long does the acrosome reaction take to complete?

Answer 30

• measured by monitoring entry of dyes into human sperm
• takes a matter of minutes

Question 31

What is the sequence of movements leading up to fertilisation?

Answer 31

• binding of sperm to zona pellucida acrosomal reaction
• penetration through zona pellucida
• fusion of plasma membranes
• sperm nucleus enters egg cytoplasm
• only the sperm head and one organelle enters the egg
• don’t contribute a single mitochondrion

Question 32

What is sperm-egg adhesion?

Answer 32

• at the egg surface
• involves:
  
  • membrane proteins e.g. integrins, fertilins (ADAM1, ADAM2), ADAM3, CRISP1
  • these molecules help ensure species specificity
• animal models which have proved particularly informative are:
  
  • mutants
  • knockout (genes removed)
  • transgenic (genes added)
  • and combinations of those

Question 33

Which organelles does the sperm contribute to the offspring?

Answer 33

• we know that it does not contribute mitochondria
• centriole: as well as providing the DNA, the sperm passes on its centriole
• centriole is involved in cell division
• really important

Question 34

What is Kartagener’s syndrome?

Answer 34

• hereditary defect in ciliary dynein
• defect is manifest by a high susceptibility to lung infections owing to the paralysed cilia in the respiratory tract that fail to clear debris and bacteria
• males are sterile due to immotile sperm
• centriole plays an important role in motility of sperm/cilia

Question 35

What are barriers to sperm?

Answer 35

• several barriers to sperm on its journey
• there is species specificity once it arrives at the egg

Question 36

What is the egg cortical reaction?

Answer 36

• triggered by sperm contact – sperm enters with sperm activating factor
• calcium waves
• cortical granules exocytose
• the contents of these granules block and change the zona pellucida, stop binding of the inner acrosome to the egg
• hydrolytic enzymes modify zona; zp2 and zp3 are targeted
• block to polyspermy

Question 37

What is the role of calcium in fertilisation?

Answer 37

• sperm provides the activation factor
• it does not provide the calcium
• calcium is derived from intracellular stores e.g. mitochondria, endoplasmic reticulum

Question 38

What is the sperm activating factor?

Answer 38

• phospholipase C-ζ (PLC-ζ)
• PLC-ζ exerts its effects by stimulating the release of the second messengers inositol triophosphate (IP3) and diaglycerol (DAG)
• IP3 activates calcium release
• DAF activates protein kinase C (PKC), which is essential for the development of the conceptus

Question 39

What is seen following sperm binding in regards to calcium release?

Answer 39

• when the sperm binds you see a spike - very rapid release of calcium
• this is the release that blocks the polyspermy
• following this you see pulses of caclium every 10 minutes
• these oscillations are very important for programming the fertilised egg into becoming an embryo

Question 40

How were calcium waves after fertilisation visualised?

Answer 40

• looking at mice eggs/zygotes that had a dye that changed fluoresence with different concentrations of calcium

Question 41

What are stages of fertilisation?

Answer 41

• sperm activated by female reproductive tract
• sperm binds to zona pellicida
• acrosome reaction
• sperm lyses hole in zona
• cortical granule reaction
• sperm and egg membranes fuse (triggers egg activation, egg completes meiosis)
• calcium waves (PLC-ζ)
• cortical granule release
• zona hardening
• sperm decondensation
• delivery of male centrioles
• polar body extrusion
• pronuclei x 2
• syngamy
• cleavage
• fertilisation is a very active process

Question 42

What do I expect you to learn from this lecture?

Answer 42

Be able to describe:

• the epic journey of the sperm, from testis to the oocyte
• sperm maturation, capacitation and acrosome reaction
• functions of the epididymis
• the differences in meiosis between male and female gametogenesis
• the steps involved in fertilisation
• how the oocyte prevents more than one sperm entering