Gametogenesis (Smyth) Flashcards
What are germ cells?
They are cells in the body that form gametes (sperm and oocytes)
These are the most important cells in your body, all your other (somatic) cells are there to keep your germ cells alive long enough to make more germ cells (in children)
Biologically, you have no greater function than to keep your germ cells alive
When does Separation of Germ Cells from
Somatic Cells occur?
Occurs at different times in different organisms
Plants, and animals (Tunicates, Cnidaria and Planeria):
- Somatic cells turn in to Germ Cells throughout life
Most animals (Vertebrates, Nematodes, Insects)
- Germ cells formed at a specific and early points in development but not in the gonads they migrate there later
What molecular components define germ cells?
Seems to be highly conserved in animals
- Vasa proteins - bind mRNA and increase translation efficiency of germ cells specific messages
- Nanos proteins - bind mRNA and decreases translation efficiency specific messages present in somatic cells (mesoderm/ ectoderm/ endoderm)
- Tudor and Piwi proteins - silence gene transcription
But how the germ cells become defined differs
What are the 2 ways to form a germ cell?
Pre-determined (Autonomous) Formation:
- Seen in many animals (Nematodes, Insects, Fish, Frogs)
- Here the germ cells are defined by the segregation of Vasa/ Nanos/Tudor and Piwi which become concentrated in a cytoplasmic region of the egg -the germ plasm- cells forming from this area become the germ cell
- Pre-determined/ autonomous - there is no external signal
Induced formation
- Seen in Mammals
- Here the of the germ cell formation occurs later and Vasa/ Nanos/Tudor and Piwi are not present in the egg but their expression is induced by signalling molecules (paracrine molecules) from neighbouring cells and so position
Pre-determined germ cell creation mechanism
- Egg gets fertilised by the sperm.
- Newly-created embryo starts dividing: 1-cell, 2-cell 4-cell etc…
- At some point in that early development a small group of cells becomes ‘earmarked’ (i.e pre-determined) to become germ cells (and make sperm or eggs in the adult animal).
- So removal of the these cells from that embryo would result in a sterile adult animal
Here Vasa/ Nanos/ Tudor and Piwi are already present in the egg and become segregated to specific regions of the cell - the germ plasm - and hence as the egg divides into specific cells of the developing embryo
Pre-determined germ cell creation in worms (C. Elegans)
In the C. elegans, Vasa etc are concentrated into P granules
These are retained in one cell only - the P cells P1 to P4
If you destroy any of these P cells the animal will form no germ cells and be infertile
Cell fate is restricted by “germ plasm” (granule containing cytoplasm)
The P granules markers for the germ plasm segregate to the P4 cell
They contain inhibitors of gene transcription and prevent the germ cells differentiation into somatic cells
Induced germ cell creation mechanism?
In mammals germ cell formation is INDUCTIVE- there is no germ plasm
Human embryo (~100 cells) has NO pre-determined germ cells
Germ cells are made after embryo implants into uterus
Mammalian germ cells form as major body plan develop and become identifiable shortly after gastrulation
The inner cell mast has a group of undifferentiated central cells - the epiblast
What is gastrulation?
Gastrulation - series of cell migrations and differentiation events of the epiblast cells bringing them to positions where they will form the three primary cell layers:
- Ectoderm forms the outer layer (skin, nervous system).
- Endoderm forms the inner layer (digestive and respiratory systems).
- Mesoderm forms the middle layer (muscles, bone, reproductive
What are primordial germ cells and the genital ridge?
Germ cells referred to as: Primordial Germ Cells (PGCs)
In embryos the structure of the embryo which eventually forms the testis or the ovary is called the Genital Ridge
PCs are NOT formed in genital ridges
In all vertebrates PGCs migrate to the genital ridge
How are PGC created in mammals?
- PGCs start life from PLURIPOTENT EPIBLAST CELLS (as do all cells in our body)
- Epiblast cells form the mesoderm, ectoderm and endoderm at gastrulation
They are PLURIPOTENT because they have the ability to form all the 3 primary cell layers and all the somatic cells
They also have to ability to form PGCs
- BONE MORPHOGENETIC PROTEIN (BMP) produced in neighbouring cells causes some posterior epiblast cells to become PGCs
- ie BMP induces Vasa/ Nanos /Tudor and Piwi proteins - it also retains the expression of pluripotency markers (Nano and Sox2) in PGCs
- PGCs migrate in many species to areas outside the embryo proper - In humans/mice to the yolk sac (an extra embryonic membrane) to separate these cells from paracrine differentiative signals in the rapidly forming embryo
Embryology timing
E0.5 Fertilization: This is the day when fertilization is presumed to occur
E1.5 Division (2 cells): The zygote has typically divided to form a 2-cell stage embryo
E2.0 Division (4 cells): Further division results in a 4-cell stage embryo
E3.0 Compaction: The cells of the embryo begin to compact, the cells tightly associate to form a more defined structure
E4.5 Blastocyst created (Embryonic stem cells generated): The embryo has developed into a blastocyst, which is a structure consisting of an inner cell mass (from which embryonic stem cells are derived) and an outer trophoblast layer
E5.0 Creation of egg cylinder: The blastocyst undergoes changes to form the egg cylinder, a structure important for further differentiation and development
E5.5 Epiblastic stem cells created: Differentiation within the inner cell mass leads to the formation of epiblastic stem cells, which are crucial for the development of the embryo proper
E6.5 Gastrulation begins: Single-layered blastocyst transforms into a multilayered structure, forming the 3 primary germ layers. Formation of primitive streak also occurs
E7.5 Development of germ layers: The ectoderm starts to form the nervous system and skin, the mesoderm progresses to develop muscles, the circulatory system, and bones, and the endoderm forms the gut and associated structures. The neural plate also forms, heart also begins development
Where are PGCs located on day 24 of human embryo development?
The PGCs will have left the embryo proper and will be located in the wall of the yolk sack
There will be around 50-100 PGCs
They now need to migrate to the correct site, increase in number and avoid apoptosis
What are the 4 Fundamental Processes in Embryology?
Induction:
- A process where one group of cells influences the development of another group through chemical signals, leading to the differentiation of cells into specific tissues and organs.
- E.g. Bone Morphogenetic Proteins (BMPs) signaling epiblast cells to prevent germ cell differentiation into somatic cells.
Specification:
- An early stage of cell differentiation in which cells are reversibly designated to a specific cell fate.
- E.g. BLIMP1 in primordial germ cells ensuring they maintain a germ cell identity and do not turn into somatic cells.
Migration:
- The movement of cells from their origin to their ultimate location where they will differentiate further and perform specific functions.
- E.g. Neural crest cells migrating to different parts of the embryo to form diverse structures including parts of the skull, sensory ganglia, and nerves.
How do BMPs act in the induction stage of embryology?
Where do BMPs act?
- BMPs act within extraembryonic cells.
What cells do BMPs target?
- BMPs target epiblast cells, crucial for early embryonic layers
Primary Function of BMPs:
- Prevents differentiation - BMPs prevent germ cell differentiation into somatic cells, thus maintaining pluripotency in early developmental stages
Mechanism of BMP Action:
- Signal Transduction Pathway: Utilizes Smad signaling to convey information from the cell membrane to the nucleus
- Protein Production: Leads to the synthesis of Fragilis and Blimp1 proteins
- Fragilis Function: Serves as a plasma membrane receptor, critical for cell adhesion with E-cadherin
Where is BLIMP1 and what does it do?
Where does BLIMP1 act?
- Within Primordial Germ Cells (PGCs).
Target Cells:
- Primordial Germ Cells.
Primary Function of BLIMP1:
- Inhibits Differentiation: BLIMP1 prevents germ cell differentiation into somatic cells, crucial for maintaining the germ line.
Expression in Humans:
- Approximately 100-200 cells express BLIMP1.
Mechanism of BLIMP1 Action:
- BLIMP1 is a transcription factor.
- Activates the expression of Nanos, important for PGC development.
- Maintains the expression of Nanog, pivotal for pluripotency in germ cells.
What happens after Specification in embryology?
Migration
The PGCs must now move back into the embryo proper
This involves passage through the hindgut and eventually to the gonads (called ‘genital ridges’ at this time)
What does NANOS3, KIT and DEAD END1 do in migration?
NANOS3 & DEAD END1:
- Prevent apoptosis during migration
KIT:
- PGCs express receptor for Kit-ligand. Prevents apoptosis, stimulates proliferation, and may help line path to gonad.
Loss of Kit leads to sterile animals
During the migration the PGC increase in numbers to ~5000 cells
Why is the Y chromosome important in sex determination?
For many years a region of the Y chromosome was known to be important in testis development in mammals
This region was called the TDF (Testis Determining Factor)
The TDF was known to be at the very end of the short arm (‘p’) of Y chromosome
Later, the TDF was found to be due to a single gene called
‘SRY’ = Sex-determining Region on the Y Chromosome and is a transcription factor
This was proven to be enough to generate a male phenotype after injecting a female mouse egg nucleus with SRY gene, it will go on to form a male mouse
What is SRY gene?
SRY = “Sex-determing Region on the Y”
A single gene, SRY, is needed to make the genital ridge become a testis
It is located at the end of the ‘p’ (short) arm of the Y chromosome
It is a member of the Sox family of transcription factors
Only males have a copy of the Y chromosome. So only males have SRY.
What happens when female germ cells are placed into a male embryo, and vica verca?
The germ cells will be driven to adapt and differentiate into cells that are useful to the gender of the embryo
The gender of the embryo will not change
What happens when the SRY gene is implanted into a female embryo?
The embryo will develop into a male, due to the SRY gene directing the genital ridge to form into testis
How long does a male and female embryo develop the same?
The development of a male and female embryo will be nearly identical up until around the 6 weeks mark in humans, E6.5 in mice
The only difference at this point, is distinct genital ridges
Where is the SRY gene expressed?
SRY is expressed in the somatic cells of the male genital ridge first at ~day 11 in mice
- It is needed for only 6-12 hrs (in mice) as it turns on expression of Sox9, which is another transcription factor
What is SOX9?
SOX 9 is the shared evolutionary male gene in vertebrates not Sry
- In mammals, Sry- drives Sox9 expression
- In birds (and some reptiles e.g. some snakes), the homogametic sex is male (ZZ) and the female (ZW) - high levels of DMRT1 present on the Z chromosome drive expression of Sox9
- In other reptiles expression of Sox9 is related to temperature:
- Alligator eggs incubated at
33°C 100% male
30°C 100% female
How does SOX9 work?
It is an autosomal transcription factor-expressed in a positive feedback loop with its own gene
It blocks ovary formation by the genital ridge (stops function of the paracrine Wnt/B-catenin pathway)
It activates expression of the paracrine signal Anti-Mullerian Hormone in the genital ridge
It activates expression of FGF9 (in another positive feedback loop with Sox9) in the genital ridge
What is FGF9?
Paracrine signalling molecule formed by the somatic cells of the genital ridge
Causes proliferation and differentiation of some genital ridge cells to form Sertoli cells and formation of chords of cells - gives the tubular structure of the testis later
Coordinates differentiation of PCs (Cyp26b1)
Represses (with Sox9) the Wnt/B-catenin path so blocks ovary formation
The path of the male genital ridge overview
Where is testosterone produced and what does it do?
Produced by the Intestinal cells of the testes
Drives growth of the Wolffian duct to form the epididymis and the vas deferens
Drives the secondary sex determinants
Loss of the androgen (testosterone) receptor produces female external appearance but with retained testes, resulting in an Intersex- failure in genetic and anatomic correlation
What is the path of the female genital ridge?
Wnt 4(paracrine signal) expression remains high in the female genital ridge (reduced/lost in males)
Signals through the second messenger Beta-Catenin (over-expression in males causes an ovary to form eg TG mice)
Induces ovary specific transcription factors and paracrine signals - including the secreted signal Follistatin- which causes the genital ridge to form an epithelium (granulosa cells) surrounding the PGCs, forming follicles
In the absence of testosterone the Wolffian duct is lost to degeneration
The mullerian duct grows (no Sox-9, no AMH) to develop the ovarian ducts, uterus, cervix and top of the vagina
What are the gender differences in gametes?
Women:
- Born with a FINITE number of eggs
- With age these eggs are used up (they mostly die, but some get ovulated)
- By age 51 years (on average) women reach MENOPAUSE
Men:
- Born with a capacity to continuously produce mature sperm from PUBERTY throughout life.
Why?
1. Eggs are costly to make, women make enough to last their entire life (true for most of our evolutionarv past).
- Older women undergo menopause so as to be able to nurture existing children, and not leave very young children motherless.
Fate of PGCs in males?
Initially grow in numbers.
Then PGCs enter a quiescent state.
At puberty restart mitosis and SOME PGCs commit to the process of meiosis.
MEIOSIS is the specialised cell division that leads to gametes (sperm).
Once entering meiosis the stem cell is totally committed to form a mature sperm
At birth male PGCs are still able to divide by mitosis (and will do from puberty)
Fate of PGCs in females?
Initially grow in numbers
Then all PGCs dividing
In utero (3/4 MONTHS) -ALL PGCs commit to enter meiosis
MEIOSIS is the specialised cell division that leads to gametes (eggs)
Once committed to meiosis the egg cannot form any more cells. It is now totally committed to form a mature egg
At birth female PGCs are all committed to meiosis, no new PGCs can be formed
What determines the fate of PGCs?
The fate of the PGCs depends on the gender of the genital ridge they are entering, and not the PGCs themselves
Why do female PGCs enter meiosis?
Retinoic Acid (RA) induces meiosis in females. (RA - the active metabolite of vitamin A)
There are very high levels of RA about the genital ridge
RA diffuses into the PCs and causes the expression of Stra8 (Stimulated by Retinoic Acid = Stra8)
Stra8 is a transcriptional regulator, modifying the gene expression pattern in the cell
Stra8 is ‘the master switch’ for meiosis. It causes PGCs in female embryos to stop dividing mitotically and enter meiosis
Where is retinoic acid made?
RA is made not in the genital ridge (G), but in the Mesonephros (M)
The Mesonephros is next to the genital ridge
Mesonephric tubules (T) and ducts (D- Wolffian duct) physically connect with the anterior end of the gonad
RA diffuses from the Mesonephros into the genital ridge and induces transcription factor signals (Stra8) in PGCs driving them in meiosis
What stops PGCs in male fetal testis entering meiosis?
Stra8 levels do not rise in male PGCs
This is because fibroblast growth factor 9 (FGF9) produced by male gonadal ridge cells:
A) induces Cyp26b1 which degrades retinoic acid before it reaches the PGCs
B) prevents Stra8 expression so inhibits meiosis entry
In absence of retinoic acid -meiosis doesn’t begin /FGF9 maintains expression of pluripotency genes e.g. NANOG/SOX2 so PGCS remain proliferating stem cells
Cyp26b1 is only present in male gonads
How do cells become haploid?
- Commitment to differentiation:
- Primary spermatogonia/oogonia enter a pre-meiotic S-phase (all chromosomes replicated, 4n)
- MALES after puberty
- FEMALES in utero(arrest) - Stra8 driven - Primary spermatocyte/ oocyte (4n) undergo a first meiotic division to form…
- Secondary spermatocytes/ oocyte (2n) undergo a second meiotic division
- in FEMALES occurs at ovulation (arrest) to form - Spermatid or ovum (=n, haploid)- (in female this meiotic division completes at fertilization)
Major differences in male and female - female needs to produce an egg with sufficient nutrients and organelles for later development - hence 4 sperm: 1 egg
Female meiotic division sizes
Female meiotic divisions are asymmetric
Two meiotic divisions (unequal sizes) to become haploid
PB1 (first polar body); PB2 (second polar body).
What mineral is a conserved trigger of fertilisation across the animal kingdom?
Calcium is a conserved fertilisation trigger across the animal kingdom
How does PLC zeta release calcium?
- PIP2 is hydrolysed by PLC zeta
- Generates IP3 and DAG
- P3 diffuses into cytoplasm
- Binds its receptor on endoplasmic reticulum
- Releases calcium ions into cytoplasm
What do cortical granules do at fertilisation?
- Cortical granules (CGs) underneath oocyte plasma membrane released at fertilization.
- Calcium dependent fusion with plasma membrane
- CGs block entry of other sperm.
What is the mechanism of blocking polyspermy at the plasma membrane?
- Sperm protein Izumo1, tethered to sperm membrane
- Forms adhesion complex with its receptor protein, Juno
- Fertilisation does not take place in the absence of this complex
- After fertilisation, Juno is lost from the egg’s membrane, due too fusion of cortical granules exiting in extracellular vesicles
What is the first phase of embryonic development?
Cleavage is the first phase
Each of the identical cells formed by a cleavage division is a blastomere
What affect does yolk content have on cleavage?
The effects of this division vary depending on the yolk content ……………….
Mammals (little yolk- isolecithal) show holoblastic cleavages- ie complete division of the whole cell
Amphibians (moderate yolk - mesolethicial) - show holoblastic cleavages too, however the yolk is unevenly distributed and gives an uneven division rate and cell size
Fish, reptiles or birds where there is more yolk (Telolecithal) show meroblastic or incomplete division where the area of yolk is not divided
Are cleavage patterns random?
In most animals cleavage patterns are not random
The plane of cleavage is always perpendicular to the spindle.
Therefore, the orientation of the spindle determines the pattern of cleavage
What type of cleavage do mammalian eggs show?
Mammalian eggs have rotational cleavage that is holoblastic
The mammalian egg begins to cleave in the oviduct and continues until it enters the uterus (1 cleavage / 14 hr).
Asynchronous cleavage:
- Mammalian embryos are unusual
- Not all blastomeres divide at the same time- hence you may have odd numbers of cells
The first cleavage is meridional, and the second cleavage is rotated 90 degrees. Then 2nd blastomere divide in a different plane – equatorial (amphibians differ)
What controls are lost during cleavage?
During cleavage many epigenetic controls lost
Maternal (pre-fertilisation) control molecules remain present through cleavage however gene activation (embryonic gene expression) occurs early in mammals (later high yolk embryos)
For embryonic gene activity the genome must be remodelled–changes in the epigenome
- New histones are placed on the DNA
- New DNA methylation patterns occur (generally unmethylated)
Gamete (maternal and paternal) patterns are lost and by 8/16 cells a pluripotent pattern seen
What is compaction?
Mammalian embryo compaction occurs at the 8 cell stage
Initial blastomeres, of mammalian embryos are identical and have a loose arrangement, touch only at the basal surfaces.
After compaction, blastomeres adhere tightly, maximizing the area of contact.
During compaction, each blastomere undergoes polarization.
This is initiated by E-cadherin expression, forming adherence junctions
Actin rearrangement occurs
Tight junctions develop on peripheral contact surface, ZO proteins/ Occludins/ Claudins
Apical and basolateral membranes specialise
Gap junctions form on outer cells intercellular communication.
Cell polarity during compaction
Cell polarity at compaction discriminates outer and inner cells of the morula
By 16 cells there are
- Internal cells and external cells
- The external cells seal off the inside of the sphere
The inside cells are non polar
The outside cells are
Two cell groups changed structurally and differential gene expression
Differentiation has started, resulting in different cell types
Trophoblast vs inner cell mast fate theory
Theory depends on the Kinase Hippo
Yap is a cofactor for the TF Tead4
Together they cause Cdx2 expression to be upregulated in outer cells
- Hence reducing Oct 4 here.
Phosphorylation of Yap in the inner cells prevents its entry into the nucleus
So Cdx4 levels reduce in inner cells
- Hence increasing Oct 4 here
1) Outer layer
- Due to a free outer membrane we block Hippo signals here (this increases the TF – Yap in the nucleus) which drives Cdx2 expression
And/or
2) Inner layer
- Due to an increase cells – cell contact enhance Hippo signals (blocks nuclear Yap)
What does Oct4 do?
In the inner cells – Oct4 is part of a mutual positive feedback loop – maintaining the two other inner cell TFs Sox2 and Nanog
Together they drive the machinery for pluripotency
Where Oct 4 expression is lost (due to high Cdx2 in the outer cells) – these cells loose pluripotency
What does Cdx2 do?
Cdx2 drives expression of Na+ channels (on the apical surface) and the Na/K ATPase pump (on the basal inner surface) of the outer cells
The presence of tight junctions means these are retained in the different regions of the membrane
A functioning polarised epithelium which differentially brings ions into the centre of the morula and with the 3/2 – Na/K bias
This:
- Brings water as well via aquaporins (like the kidney tubule)
- The ionic gradient at the cell surface allows uptake of glucose /amino acids via Na co-transporters, nutrient transfer from the outside environment (like the gut)
This causes cavitation to occur with a group of Sox2+ pluripotent cells (ICM) on one side
The surrounding Cdx2+ cells form the trophectoderm
Cell fate overview
What is MUC1?
MUC1 (a mucin) is expressed highly on the uterine
epithelium is inhibitory for attachment of the blastocyst
This is lost in the region where the Blastocyst is during ovulation
Loss of MUC1 allows the glycosylated
transmembrane proteins/proteoglycans in the
uterine epithelium to interact with lectins on the
embryo trophectoderm
This then allows the binding of the blastocyst to the uterine wall
What happens to the human embryo at implantation?
At implantation changes start in the inner cell mass
A second cavity, the amnion, appears within the inner cell mass, leading to the separation of the amnioblasts from the embryonic disk
The amniotic ectoderm forms the upper layer, while the embryonic disk, which will develop into the embryo, forms the lower layer
Primitive endoderm line the embryonic disc and yolk sac
What happens to the mice embryo at implantation?
In mice
- Inner cell mass grows down
- Forms a cylinder
The primitive ectoderm is lined by endoderm
(visceral on embryo, parietal on trophoblasts)
Cleavage in amphibians
In Amphibia – we get uneven cleavage – the formation of cells in the vegetal pole is slower and fewer cells form here due to the presence of yolk
Amphibia have mesolecithal eggs
Amphibian eggs have lots of yolk, but still undergo holoblastic (full) cleavage
The 1st and 2nd cleavage is meridional - 3rd cleavage is equatorial
The cleavage is displaced toward the animal pole due to the yolk
This results in 4 large vegetal blastomeres and 4 small animal blastomeres
What is discoidal cleavage?
DISCOIDAL:
- Birds, reptiles, and fishes
- Telolecithal eggs only, much yoke completely supports embryogenesis
- The 1st cleavage is meridional
- Starts at the animal pole but does not progress
- The 2nd and 3rd cleavages are also meridional
- The 4th cleavage is equatorial, and it creates a layer of small cells on top of the huge uncleaved yolk
- The blastoderm: in birds a disc of ~ 60,000 cells formed by cleavage at the time of lay
What is the next step in telolecithal eggs developmental after blastoderm formation?
The next step in development of telolecithal eggs is formation two layers in the blastoderm
Epiblast: (epi = upon) the upper layer and it forms the embryo proper
Hypoblast: (hypo = under) the bottom layer that surrounds the yolk
Blastocoel: lies between the 2 layers.
