lecture 9: primordial germ cells and sex stem cells Flashcards
1
Q
From where do germline cells originate?
A
- germline cells are segregated from somatic cells early in development
- germ cells split off before formation of ectoderm, mesoderm or endoderm - so really aren’t derived from any of these three germ layers
- in many species primordial germ cells are specified in a region distant to the developing gonads
- compare insect (fruitfly, Drosophila) and mammal (mouse)
2
Q
What are germ cells and what are their key functions?
A
- germ cells (cells that give rise to eggs and sperm) are distinct from somatic cells (that do not make a genetic contribution to the next generation)
- germ cells have three key functions:
- preservation of genetic integrity (prevention of ageing)
- generation of genetic diversity
- transmission of genetic information to the next generation
3
Q
How do germ cells develop in Drosophila? What is one of the critical roles of pole plasm?
A
- in insects, nematodes, fish and frogs components of specialised cytoplasm specify formation of primordial germ cells, or PGCs
- this cytoplasm, or germ plasm, is rich in germ cell-specific RNA-protein particles and is called pole plasm in Drosophila
- to repress transcription in new PGCs
- a 71 amino acid peptide (named polar granule component) prevents phosphorylation of regions in the carboxyl-terminal domain of RNA-polymerase II that are critical for transcriptional elongation
- this repression only occurs prior to migration to the developing gonad but if it is absent (e.g. polar granule component mutants) the germ cells begin to express genes found in neighbouring somatic cells
- transcription needs to be repressed early so that these cells don’t respond to somatic signals and express somatic genes
4
Q
From where do PGCs arise in mice?
A
- in mice, a BMP signal from the extraembryonic ectoderm initiates PGC formation by activating the repressor protein, Blimp1/Prdm1 and stellar, just prior to gastrulation in about 6 epiblast cells
- this increases to about 45 cells (6.5 - 7.2 dpc) and no further cells enter the germline lineage
- PGCs reactivate the pluripotency genes Nanog, Sox2, and Oct4
- PGCs are not pluripotent stem cells – they can only differentiate into germ cells and they have limited division potential in vivo
- at about 8dpc the PGCs migrate into the definitive endoderm, which carries them along as it forms the hindgut they then actively migrate into the genital ridges (gonad primordia)
- need to receive signals to actively migrate
5
Q
What regulates the directional migration of PGCs?
A
- mediated by gradients of secreted molecules received by the migrating cells
- mouse:
- SDF1 is secreted by the genital ridges and acts as an attractant to PGCs that express the receptor CXCR4
- Steel (Kit ligand) is expressed by the somatic cells that surround migrating PGCs and is received by c-Kit on the germ cells
- this signal is required for PGC migration and survival
6
Q
How do male and female germ cells behave when they reach the genital ridges?
A
- differently
- studies in mouse embryos have shown that at 13.5dpc
- female germ cells differentiate into oogonia, enter meiosis and arrest in prophase I
- male germ cells down-regulate meiotic genes and become gonocytes which enter mitotic arrest
- this difference in sexual behaviour is regulated by retinoic acid
- all female embryonic germ cells enter meiosis
- male embryonic germ cells (gonocytes) remain arrested until puberty when:
- seminiferous tubules form
- the epithelium of the tubules differentiate into Sertoli cells
- gonocytes become undifferentiated type A spermatonia or spermatogenic stem cells that can renew themselves and produce differentiated progeny
7
Q
What does retinoic acid (RA) do?
A
- determines the timing of meiosis and sexual differentiation of mammalian germ cells
- In females – RA induces expression of Stra8 which initiates meiosis
- in males 0 Cyp26b1 is a cytochrome p450 enzyme that degrades RA prior to 13.5 dpc
- after 13.5dpc Nanos2 blocks Stra8 expression
8
Q
What is the structure of the seminiferous tubule?
A
- undifferentiated cells found around the edge of the tubule
- progressively differentiate as they move towards the centre
- spermatozoa are shed into the lumen
- sertoli cells act as support cells
9
Q
Where are spermatogenic stem cells (SSCs) found?
A
- In niches created at the junction of Sertoli cells, interstitial (testosterone-producing) cells and blood vessels
- SSCs are also called As spermatogonia
- The niche provides signals to maintain the SSC population as well as direct differentiation to produce gametes
- blood testis barrier - tight junctions
- immune function (protecting spermatocytes/zoa, immune privileged compartment)
- during the first round of spermatogenesis Sertoli cells form tight junctions with each other – the SSCs and spermatogonia are therefore in a separate compartment to more differentiated germ cells and have access to niche signals
10
Q
What factors are secreted by the niche?
A
- GDNF (a TGFbeta family molecule secreted by sertoli cells) is required for survival and proliferation of undifferentiated spermatogonia
- mice that overproduce GDNF have excess undifferentiated spermatogonia and lack later stages
- mice deficient in GDNF become bepleted of germ cells as they age
- As normal males age they become infertile and this corresponds with a decrease in GDNF expression – the niche regulates stem cell maintenance
- CSF-1 (colony stimulating factor 1) is secreted by Leydig cells and promotes SSC renewal as opposed to differentiation
11
Q
How can SSC function be determined?
A
- in transplantation assays, using a donor animal that expresses lacZ in all tissues (ROSA-lacZ)
- the recipient animal can have germ cells depleted via chemical means or carry a mutation that causes loss of germ cells
- take germ cells from blue mouse, transplant them into a mouse that has no germ cells, wait, see that the blue stem cells are able to colonise available niches and start to produce blue progeny
- can count the number of tubules
- an approximation of the number of stem cells based on the number of blue seminiferous tubules
12
Q
Do SSCs have the capacity for infinite renewal?
A
- A: SSC numbers decline with age but…
- B: if SSCs are serially transplanted into testes of young mice they expand in number at similar rates, even when over 2 years of age – the nich regulates their ability to survive and proliferate
- suggests that it is the niche that is ageing rather than the diminishing ability of the stem cells themselves
- niche is losing capacity to support the stem cells
13
Q
What are sertoli cells?
A
- sertoli cells play an important role in regulating niche function
- A recipient of lacZ expressing germ cells with ~50% increase in Sertoli cell numbers will have 3x more accessible niches for SSC colonisation vs a recipient with normal sertoli cell numbers
14
Q
What are some technologies that have come out of these sorts of experiments?
A
- SSC transplantation could be a fast method of genetic manipulation of large, slow growing farm animals
- imagine the mouse was a bovine – SSCs could be manipulated in culture and transplanted into the testis of a recipient bull
15
Q
What is a clinical application of SSCs?
A
- SSCs could be harvested and preserved prior to chemotherapy/irradiation and re-transplanted to restore fertility
16
Q
Can gametes be produced from ES cells?
A
- Toyooka and colleagues in 2003:
- culture ES cells from Vasa-GFP mice (Vasa is expressed in germ cells)
- aggregate into embryoid bodies
- select vasa (GFP) expressing cells
- co-culture with gonadal tissue
- transplant under a host mouse testis capsule to mature
