Lecture 2: Germ cells and fertilisation

Question 1

sex cells origin

Answer 1

eukaryote multicellularity is linked to embryonic developments at complex body parts at 4 lineages: animals plants brown algae and red algae

Question 2

multicellular animals metazoans from choano flagellates

Answer 2

Bilaterians from metazoan..
1. deuterostomes: chordata
opening becomes the anus
nervous system becomes dorsal
2. protostomes:
Ecdysozoa + spiralia
arthropoda + lophotrochozoa + Mollusca

opening in embryo becomes the mouth
nervous system always vengen - on the side of the abdomen

choanoflagualtes are unicellular + characterised by a motile flagellum surrounded by a collar of microvilli these features associated by cell types in metazoans i.e. sensory cells

cell differentiation - subdivision of labour

ribosome biogenesis
flagellar
contractibility
microvilli

genes that make the choano flagulate become motile available in contractile cells in us:
muscle-cilia motor cells with flagellum or cilia
ciliated cells in the nephron - sensory motor neurones relying on stimuli to a contractile cell

Question 3

single cells to multicellularity

Answer 3

1. aggregative multicellularity
   aggregation in response to environmental conditions

i.e. starvation, failure to complete cytokinesis or interaction between the extracellular matrix and phylopodia cels becomes so robust that they come together as a clump

to form spore or cysts - there is no cell division division or physical labour

multicellularity is a result of embryogenesis

2. clonal likely path to metazoans

Question 4

why is the sperm cells so small and the egg cell is so big

Answer 4

Anisogamy - Oogamy
look and work differently
egg cell with nutrients
sperm carries not much more than its own DNA
isogamy - germ cells of same size and content

gametes evolved but do not have any somatic function but propagate the species

Question 5

gametogenesis

Answer 5

1. Primordial germ cells multiply via mitosis and migrate to the gonads (derives from the mesoderm) where meiosis and maturation occurs

Question 6

mitosis

Answer 6

start at 2n
first DNA duplicated = 4n
chromatids split and are pulled apart into 2 daughter cells in cytokinesis = 2n

Question 7

meiosis

Answer 7

gametes are haploid 1n dna replication and crossing over
1st meiotic div = chromatids remain together
2nd pulled apart
in mammals oocytes produces and dormant since birth till puberty at 2nd meiotic div completed at fertilisation
1 oocyte + 2 polar bodies
or 4 haploid sperm

Question 8

modes of primordial cell formation

Answer 8

in gonad organ of somatic cells
A mouse
B Axolotl
C Xenopus

in all animals = original mode - induction
mouse and axolotl mesoderm sends signal BMP cell-cell com cells that receive signal suppress somatic cell formation and induces germ cell formation

derived mode - inheritance cells inheriting the germ plasm maternally specified germ line determinants become germ cells = cell autonomous as doesnt require signalling

outer ectoderm
middle mesoderm and inner endoderm

both
leads to somatic pathway suppression
retention of pluripotent potency
capacity to undertake meiosis

can be stored safely in a extra embryonic location

Question 9

polar body can..

Answer 9

In scale insects
complete second meiosis fuse together = 5n - form an organ that will house endosymbiotic bacteria - sticky sap

form tissue to support developing embryo after fertilisation

may replace sperm and sperm the egg in parthenogenesis

plants: form endosperm is fertilised and supports the embryo

Question 10

egg and sperm fusion

Answer 10

occurs in fallopian tube

fusion of egg and sperm plasma membrane
chemotaxis is factor attracting sperm to oocyte and cumulous cells which produce progesterone rich in hylaronic acid - only capable sperm able chemotaxis

capacitated sperm acrosomal sperm head membrane destabilised to allow it to penetrate egg due to hyluronidase enzyme in sperm head and chemical changes in tail allows greater mobility
facilitated by removal of sterols i.e. cholesterol sterols = more fluid membrane + increase in calcium = increase motility

acrosome reaction = exocytosis of actosomal vesicle at tip of sperm digests path through zona pelucida

capacitation is controlled by FPP produced in prostrate gland as a component of seminal fluid
high levels prevents capacitation however once sperm reaches female reproductive tract
sperm become chemotactic once in their lifetime and lasts 1-4hrs
sperm heads bind to integrins in the cell membrane
adhesion triggers fusion = egg activation and exocytosis of cortical granules (which only occurs after fertilisation) translocate to the plasma membranes or to the surface of the egg during meiosis.

Question 11

what distinguishes the zygote

Answer 11

it is a cell with no phenotype as no genes are in use to give it distinctive features = triggers end of meiosis and activates cell

Question 12

zona pellucida function

Answer 12

extracellular coat supports communication between oocytes and follicle cells
protects egg through development and prevents polyspermy

Question 13

implantation in uterus

Answer 13

the first cell divisions all occur in the oviduct and the fallopian tubes, and it is a compacted Morila. lump of cells eventually reaches uterus hollows out and hatches from zona pellucida and can implant in the uterus

seperation of outer ectoderm and inner cell mass

trough ectoderm is required for implantation and to form the embryonic part of the placenta

Question 14

prevention of polyspermy

Answer 14

Fast block
depolarisation of cell membrane by Na+ influx
changes in membrane potential from 70mV to + 20 mV
sperm cannot fuse with membrane that have a positive potential
Slow block cortical granules reaction breakdown of cortical granules - release of contents into space between cell membrane and zona pellucida
forms the fertilisation envelope

Question 15

the first cell division

Answer 15

cleavage pattern is controlled by amount of yolk a,micro,oligo,meso,acro,mega lechithal eggs and localisation iso vd antsolecithal telo and centrolechithal

holoblastic -
little or evenly distributed yolk = isolechithal or mesolechithal eggs
divide yolk completely
mammals echnioderms - sea urchins, amphibians -xenopus

meroblastic
yolk concentrated at one pole - telolechithal egg
cleavage furrows are incomplete -
zebrafish and birds

yolk in centre - centrolechithal
superficial
initially only the nuceli divide - synytiym
insecs - drosophila and crustaceans

orientation of mitotic spindles - radial cleave pattern
initial cleavage planes at right angles to each other
along or perpendicular to egg axis radial + rotational
typical for deuterostomes

spiral cleavage
oblique angle to egg axis
Spiralia: annelids/ molluscs and flatworms - lophotrophs

Question 16

how can early cell divisions be so quick

Answer 16

early cleavages vs somatic cell proliferation

somatic cycle 
G1 5hrs
S 7hrs
G2 3hrs
M 1hr
cleavage 
S and M
rapid increase in cell o
no increase in cell vol
no transcription so rep can proceed unhindered
cell size get smaller

Question 17

the importance to maternal contribution

Answer 17

cell division without cell growth occurs
zygotic gens are transcriptionally silences
transcription starts:
in mice @ 2 cell stage
in xenopus after 12 cell cycles - 4k cells
humans between 4-16 cell stage
after embryonic genome activation cell cycle lengthens

all mRNA and proteins required for early cleavage stages must be loaded into the oocyte during oogenesis

maternal factors regulate complex control systems

Proteins must be loaded into the egg to make fertilisation and early certain divisions work. The genes encoding for these mrna proteins are called motile effect genes because these genes, when dysfunction affect the phenotype of the offspring regardless of their own genotype.
This is because the embryo cannot use its own genes to combat maternal mutations

mitochondria
in the fruitfly, paternal mitochondria are actively destroyed by enocytosis and then autophagy and invertebrates main contribution to the cells pool of mitochondria is barely, barely detectable.

female mitochondrial disease likely to pass them to their children i.e, obesity induced diabetes, atherosclerosis

Question 18

what is the blastula
what are esc
how are they different from totipotent cells of the early cleavage stages

Answer 18

early cell divisions lead to the compact morula 8 cell
to
hollow blastocyst
more cells
zona pellucida
trophoblast/trophectoderm - outer layer - embryonic aspect of placenta
embryoblast = inner cell mass - embryo + extraembryonic tissues such as amnion + germ cells
pluripotent
zygote = totipotent
inner cell mass provides embryonic stem cells
cellular layer blastoderm
fluid filled hollow blastocoel

Question 19

IVF

Answer 19

3-6 weeks

1. suppression of normal menstrual cycle
2. ovarian hyperstimulation - 14 ays of GnRH hormone to block LH from pituitary
   risk life threatening ovarian hyper stimulation syndrome
3. egg retrieval
   transvaginal oocyte retrieval 10-30eggs
4. oocyte and sperm preparation
5. fertilisation
   mixing of sperm and egg 75000:1
6. embryo culture 6-8 cleavage stage/ 3 days or after retrieval
   blastocyst stage
   5 days after or option for autologous endometrial co-culture
7. embryo selection based on morphological criteria
   presence of soluble HLA-G
   option for preimplantation genetic screening and female treated with further hormones
8. embryo transfer
9. cryopreservation of remaining embryos

Question 20

Preimplantation diagnostics and genetic screening

Answer 20

1. screening for monogenic disorder - autosomal recessive, autosomal dominant, x-linked, b mitochondrial disorder
2. minor disabilities
3. sex selection
4. screening for predisposition syndromes mutation predisposing to breast cancer
5. human leukocyte antigen typing to create a saviour sibling
   disordered recessive disorders are cystic fibrosis,
   13:00
   better sardis, severe sickle cell disease and spidered muscular atrophy.
   biopsy
   polar body biopsy -no need to disturb the embryo but limited diagnostic value due to genetic variation of PB and oocyte
   Cleavage-stage biopsy - finish analysis before embryo transfer but limited diagnostic value due to genetic mosaicism (error rate in first cell devision in mitosis is quite high can leave 2 cell lineages within the embryo this problem is eliminated because later cells abberant cells may not continue to proliferate and then are eliminated or the extra chromosomes which don’t aline during mitosis are lost or if aberrant cells aren’t eliminated the whole cell will not live anyways)
   blastocyst biopsy (Takes cells from the trophectoderm) -less mosaicism but time pressure

genetic analysis techniques

1. FISH chromosomal aberrations
2. PCR monogenic disorder high throughput sequencing
3. Preimplantation genetic haplotyping using DNA fingerprinting
4. Transcriptome analysis