Saturday, April 27, 2013

More Research Paper Sumups!

I will be focusing on the study guide from here on out.  If I am done that early and have extra time, I will continue the research paper sum-ups.  Until then, good luck with studying!

Savitt J, Singh D, Zhang C, Chen L, Folmer J, Shokat K, and W Wright.  "The In Vivo  Response of Stem and Other Undifferentiated Spermatogonia to the Reversible Inhibition of GDNF Signaling in the Adult."  Stem Cells 4, 2012: 732-740.


There's evidence that GDNF is important for regulating population size of spermatogonial stem cells.

What is the evidence? For a long term culture, you need GDNF to maintain and expand stem spermatogonia (how do you know they are stem spermatogonia? by putting them in a testis w/o germ cells and restoring spermatogenesis).

But wait, is GDNF just a survival factor or does it actually promote the self-renewing kind of replication for stem cells?  Let's see.  Mouse stem spermatogonia were deprived of GDNF for 3 or 6 days, but the population still grew as if it had GDNF.  One would infer from this that GDNF does not promote self-renewal over differentiation.  So that's the in vitro study.

What about in vivo?  We are kinda limited to knocking out or messing with GDNF expression from time of birth or earlier, but not in adult tissue, nor can we try to restore it after knocking it out to see what happens.  Overexpression of GDNF -> lots of undifferentiated spermatogonia that just died by apoptosis and led to infertile adult.  Knockout of GDNF -> lost all germ cells in a week and the few left did not replicate.

Lots of questions, but how to answer them?  We made a new system where we could inhibit GDNF and also reverse the inhibition.  Let's look at the signaling pathway.  GDNF binds Gfr-alpha1 which activates Ret and phosphorylates Ret (also part of the same receptor).  Our transgenic mice had 1 mutation in Ret but it doesn't affect Ret activity.  BUT it increases affinity for a competitive inhibitor NA-PP1.  So by treating mice with NA-PP1, it competes with ATP and prevents the binding of GDNF from phosphorylating RET.  When you take away NA-PP1, things go back to normal.

hypothesis: GDNF is essential for maintaining spermatogonial stem cell pool and also stimulates self-renewing replication of the stem cells.  prediction if hypothesis is true: stem spermatogonia will be lost if GDNF signaling is inhibited for duration of 1 cell cycle (2 days).


the mutation in Ret works: baby mice with underdeveloped kidneys die with the mutation (proves mutation works), but it doesn't affect adults (proves it's usable for studying adult tissue)

ok, so treatment with NA-PP1 for 5, 11, 20 days and looking at Gfr-alpha1 positive cells and Zbtb16+ cells (these are regular spermatogonia, not stem spermatogonia).  the Gfra1+ cells are less and less by 5 and 11 days and basically gone by 20.  The Zbtb16+ aren't changed at 5 but are reduced by 11 and gone by 20 as well.  This doesn't happen in mice w/o the Ret mutation.  similar results seen with mRNA levels of Ret, Gfra1, and Zbtb16.

Still, after 20 days, those transcripts are still detectable (just reduced).  Did we lose functional stem cells or did we just reduce them to numbers so low they're hardly detectable?  Let's do 30 days treatment and collect the tissue either then or 35 days later.  Histology sections show Ret-mutated mice lost ALL their spermatogonial stem cells from 30 days of treatment with NA-PP1.  Further testing showed this happened over a course of several days (not all at once)

After the 11 day treatment though, some still remain!  35 days after the 11 day NA-PP1 trtmt, testes were back to normal.  What?  We saw 3% of tubules active in spermatogenesis and there were dense clusters of Gfra1+ cells.  We also saw that even with a short 2 day NA-PP1 trtmt, some stem spermatogonia are gone.


This is the first use of a chemical-genetic approach to study adult stem cell regulation via growth factor.  This method has 3 advantages:
1) you start with totally normal pool of stem cells (existing in normal organism, not in weird imitation culture, etc)
2) you get normal in vivo response to whatever you are doing
3) you can reverse what you just did and go back to normal conditions

We showed GDNF is required for maintaining stem cells in adult testis.  Also we saw stem cell loss with absence of GDNF, which is opposite to the stem cell growth mentioned earlier in that in vitro study.  But some survive even past 11 days, so there are definitely other factors involved.

This may be due to a) varying normal concentrations of GDNF for various stages of the tubules, b) the cells normally do not replicate at the same time all together, c) it has been hypothesized that there are quiescent ones also who enter the cell cycle only when tissue damage has occurred.

When NA-PP1 treatment ceases, cells migrate and refill empty areas, and new refilled niches will start producing spermatogonia again with time.

Stanley E, Lin C, Jin S, Liu J, Sottas C M, Ge R, Zirkin B, and H Chen.  "Identification, Proliferation, and Differentiation of Adult Leydig Stem Cells."  Endocrinology 153, 2012: 5002-5010

When testes get depleted of their Leydig cells (testes interstitial compartment) with treatment of EDS, they come right back!  There must be cells that make Leydig cells then, but very little is known about them.

A previous study isolated cells from new baby rat testes that could divide w/o differentiating or divide and produce testosterone (normal Leydig cell function).  And when they were put into normal testis, the cells also could differentiate -> evidence that these baby rat cells were Leydig stem cells.

This study looks for stem Leydig cells in the adult testis.  Since it is possible to separate seminiferous tubules and interstitial compartment of a testis, it made it feasible to go looking for these stem cells and their physical niche.


Testicular cells were isolated from Leydig-depleted testis and grown in culture.  Without LH (luteinizing hormone), cells kept growing and growing for about 1.5 yr (325 population doublings).  They didn't produce testosterone.  When cultured with LH, they produced testosterone.

Seminiferous tubules and interstitial compartment were separated from Leydig-depleted testis and cultured separately.  Cells appeared on surface of cultured tubules but not cultured interstitial compartment.  These cells stained for 3B-HSD (enzyme in pathway of making testosterone) and the tubule culture, when treated with LH, started producing testosterone (neither tubule culture w/o LH nor interstitial culture w/ or w/o LH made testosterone).  Therefore, the Leydig precursor cells came from the tubules.

Are they stem cells?  These cells were selectively labeled with EdU, and then given EDS, which stripped the 3B-HSD plus label cells.  Some time later, they came back.  If you measured testosterone, its presence and absence correlated with the presence/absence of the 3B-HSD cells.


Had been known previously that Leydig cells came back after depletion and it was the point of this study to see if there were stem cells ( as opposed to quiescent progenitor cells ) and where they are and how they are regulated.

If they were stem cells, we should have been able to isolate them and have them grow for a long time without trying to repress any Leydig cell markers and be able to differentiate into T-producing cells.  We accomplished this.

There have not been any Leydig stem cell markers identified, so we had to go a different way about locating them.  Our results with the separation of tissue in culture experiment demonstrated they were the cells growing on the surface of the tubules.

If they were stem cells, they should be able to come back again if they suffered another depletion by EDS.  The EDS in the tissue culture experiment showed that.  Also we had an unpublished experiment where we treated the tubules with collagenase and dispase to cut anything attached to surface of tubules off, and any stem-activity was lost (couldn't make cells that produce T).

In terms of regulation, more research is needed.  But considering that they could grow on tubule without the interstitial compartment present suggests the blood vessels associated with interstitial compartment are definitely not important, but they might still have a function in vivo.

Also the tubule culture method is a great way to keep answering questions about niche components in regulating adult stem cells.

Two more paper sumups: Germline Stem Cells

Eun S H, Gan Q, and X Chen.  "Epigenetic Regulation of Germ Cell Differentiation."  COCeBi 22, 2010: 1-7.

Epigenetics modify chromatin w/o affecting DNA sequence.  There are proteins who "write" on the modifications, "read" and act on the modifications, or "erase" the modifications.

Germ cells are "immortal" b/c they produce the next generation.  Epigenetics in germ cells might be involved in
1) meiosis and terminal differentiation
2) keeping the genome information good
3) erasing bad information

GSCs (germline stem cells) initiate gametogenesis.  Their chromatin structure maintains self-renewing and blocks differentiation.  Evidence: ISWI "Imitation Switch" is a chromatin remodeling factor.  it is essential for preventing differentiation (in mutants for iswi, the bag gene is expressed and induces differentiation).  One example is the NURF complex that contains the ISWI factor in male drosophila.

histone modifications also functions in maintaining stem-cellness.  Evidence: scny gene removes ubiquitin from histone H2B.  scny mutant leads to super acetylated H3 and super ubiquitinated U2B => open or loose chromatin -> active transcription of differentiation genes.

also, this chromatin landscape isn't permanently blocked from differentiation but described as "poised" to start differentiation upon stimuli.  in mammalian: have both activating H3K4methyl3 and repressive H3K27methyl3 going on and a paused RNA Pol II.  it prevents DNA methylation, which is a more stable block and would be difficult to remove for differentiation to happen at a snap.  in drosophila: not bivalent (i.e. not an active and a repressive histone modification at the same time) and not associated with paused RNA Pol II, but probably b/c drosophila doesn't have methylase anyway.

only germ cells do meiosis.  with meiosis, chromatin regulators and opposite-fxn histone modifying enzymes are downregulated.  at same time, celltype specific factors are unregulated (i.e. the tTAFs testis-specific TBP-associated facotrs in drosophila spermatocytes)

same/similar-fxn histone modifying enzymes work together though, maybe separated by time (i.e. in drosophila ovary, one isoform is in early germ cells and GSCs while the similar isoform is needed in later stage germ cells).  but in mice, meiosis was found to have both functional enzymes that are opposite fxn.

also during mammalian gametogenesis is when imprinting (DNA methylation) happens.  This might happen via a sort of weird transcription process that goes over modified genes in order to "read" and match it to the new DNA strands.  This happens while oocytes are arrested in meiosis but we're not sure about spermatocytes.  More research needs to be done.

in spermiogenesis in drosophila and mammals, histones are kicked out and replace with Tnps and Prms to super condense the DNA in super nuclei.  This process is also regulated by epigenetics.  Some examples include:
1) H4 S1A substitution/mutation messes up phosphorylation of H4S1 and results in failed sporulation in yeast
2) demethylation of H3K9me2/1 is necessary to increase expression of the Tnps and Prms in mice.
3) (see more in the paragraph)

in human sperm, about 4% of the genome still has regular histone nucleosomes, no Tnps or Prms or anything.  Specially, the Hox genes and imprinted genes that are necessary for early embryonic development.  but things like Nanog that are necessary for ESC pluripotency are off.

after fertilization, Prms get kicked out and given maternal histones.  some chromatin regulating proteins are involved in helping out.  the cells that will be the germ cells for this zygote's babies are the PGCs (primordial germ cells).  They get a genome-wide erasure of DNA methylation so as to get rid of any epimutations that happened between their ancestor's PGCs and this fertilization.

Proper histone modification control takes care of differentiation genes and fertility, but they also have been observed to ensure proper lifespan.

During the journey of germ cells, they need to maintain a correct epigenome, which is ensured by dynamic control, erasure, and re-establishment of the epigenome.  More needs to be done in the field, except it is hard to get a good amount of these GCs and PGCs (ethics!) so if we can has new technology that don't need as many cells, we will be good to go.  Also this will be useful for diseases due to germ cell differentiation fail (infertility, germ cell tumors, etc) but also regenerative medicine.

Tran V, Lim C, Xie J, and X Chen.  "Asymmetric Division of Drosophila Male Germline Stem Cell Shows Asymmetric Histone Distribution."  Science 338: 2012: 679-682.

question: stem cell activity is regulated by epigenetic, but do stem cells retain their epigenetic info?

male drosophila GSCs have been well characterizes and are easily identified.  they divide asymmetrically and can be examined at the single cell level.

histones are major carriers of the epigenome.  we created this switchable dual-color method to label and identify what is a new histone versus and old histone.  can be controlled spatially or temporally (by heat shock!).  labeled histones can get switched from green to red.

how? heat shock causes the recombinant gene to irreversibly shut down GFP-labeled old histone expression and initiate expression of new mKO-labeled histones.  if old histones are distributed btw daughter cells equally, over time any green color will be replaced by red color.  if old histones are kept in the GSC and new histones are kept in the daughter fated cell, then green will be specifically retained.

before heat shock: testes with transgenes showed green nucleus but almost no red.  after heat shock, red started appearing.  of all GSCs, about 70% of them will be in G2 phase, 21% in s phase, and <2% in mitosis.  entire cycle length is 16 hrs.  plus the two daughters will be connected after incomplete cytokinesis and are called "spectrosomes."  examine testes 16-20 hrs after heat shock, specifically the spectrosomes.

results: green preferentially held in daughter GSC, while red was preferentially delivered to fated daughter cell.  in contrast, for symmetrically dividing GSCs, both daughters had equal red and green.  also in contrast, this was observed with canonical H3 transgene, but not with variant H3.3 transgene.  also looking at during mitosis, anaphase pulled green chromatids toward future daughter GSC while the spindle pulled red chromatids towards future fated daughter cell.

change it up: ectopic activation of JAK-STAT signaling pathway.  previously shown that this causes overpopulation of GSCs.  they did it and found that the asymmetric green-red distribution didn't happen.  so equal distribution of old and new histones is also associated with this forced symmetrical divisions.

proposed mechanism: old and new histones are incorporated into separate chromatids during S, and are preferentially segregated at the spindle.

this may go towards understanding how epigenetic is maintained in SCs and reset in sibling cells that will differentiate.

Prepping for Final Exam of Stem Cells Biology

Ok, so the doc has been updated with ALL the lectures of this semester now, hopefully typo free.  Again, comment or email me if you have questions or something looks funny.

Sumups of the other research papers (16 more to do, homg) and a study guide for the exam coming either later today or tomorrow.