lecture 11: fertilisation Flashcards
1
Q
What is the male reproductive tract?
A
- 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
2
Q
What is the female reproductive tract?
A
- 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
3
Q
What is the spermatozoa?
A
- 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
4
Q
What are the different cells called along the path of spermatogenesis?
A
- 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
5
Q
What is the site of spermatogenesis?
A
- seminiferous tubules
- lots and lots of tightly coiled tubules that make up the testis
- process of spermatogenesis and spermeogenesis can take about 60 days
6
Q
Where are sperm stored?
A
- within the seminferous tubules going into the epididymis
7
Q
Are all sperm the same size?
A
- no
- vertebrate sperm vary in size
8
Q
What is unique about human sperm?
A
- 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
9
Q
How big is the human oocyte? What does it look like?
A
- ~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
10
Q
are all eggs the same size?
A
- 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
11
Q
What is interesting about the ovulated egg?
A
- 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
12
Q
What are different mammalian GV stage oocytes?
A
- 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
13
Q
What is the ovulated oocyte?
A
- in most mammals, the oocyte does not complete meiosis until after fertilisation
14
Q
What is gametogenesis in mammals?
A
- 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
15
Q
What is really important?
A
- that only one sperm gets through to the egg → otherwise polyspermy → dire consequences
16
Q
What is the egg’s journey?
A
- 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)
- oocyte surrounded by
- 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
17
Q
What maintains the 3D structure of the cumulus oocyte complex?
A
- hyaluronic acid
- this is something that the sperm has to battle
18
Q
What is sperm formation, maturation and storage?
A
- 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
19
Q
What is semen?
A
- 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
20
Q
How is sperm transported along the male tract?
A
- 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
21
Q
What is sperm transport in the female tract?
A
- 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
22
Q
So what is the overall marathon journey of the sperm to the oocyte?
A
- 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
23
Q
What is the longevity of gametes in the female tract?
A
- 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
24
Q
What is capacitation?
A
- 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
25
Q
What is hyperactivation?
A
- 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
26
Q
How does the sperm find the egg?
A
- 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
27
Q
What must oocytes have completed to be fertilisable?
A
- 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
28
Q
How does sperm get to the egg?
A
- transit through
- the cumulus
- zona pellucida
- egg/vitelline membrane
- involves Many different egg and sperm receptors, enzymes and factors
- including ones acquired en route
29
Q
What is the acrosome reaction?
A
- 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
30
Q
How long does the acrosome reaction take to complete?
A
- measured by monitoring entry of dyes into human sperm
- takes a matter of minutes
31
Q
What is the sequence of movements leading up to fertilisation?
A
- 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
32
Q
What is sperm-egg adhesion?
A
- 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
33
Q
Which organelles does the sperm contribute to the offspring?
A
- 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
34
Q
What is Kartagener’s syndrome?
A
- 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
35
Q
What are barriers to sperm?
A
- several barriers to sperm on its journey
- there is species specificity once it arrives at the egg
36
Q
What is the egg cortical reaction?
A
- 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
37
Q
What is the role of calcium in fertilisation?
A
- sperm provides the activation factor
- it does not provide the calcium
- calcium is derived from intracellular stores e.g. mitochondria, endoplasmic reticulum
38
Q
What is the sperm activating factor?
A
- 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
39
Q
What is seen following sperm binding in regards to calcium release?
A
- 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
40
Q
How were calcium waves after fertilisation visualised?
A
- looking at mice eggs/zygotes that had a dye that changed fluoresence with different concentrations of calcium
41
Q
What are stages of fertilisation?
A
- 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
42
Q
What do I expect you to learn from this lecture?
A
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
