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.
Intro: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).
Results:
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.
Discussion:
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.
Results:
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.
Discussion:
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.
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