FUDR – exploding gonad/ protruding vulva in N2

[Terter]

Hi worm experimenters,
It would be great if someone already came across/ around the problem described below and can share their experience.
We are doing some lifespans in presence of FUDR - 50 uM in NGM agar, on the FUDR plates we put carefully staged young adults (formed vulvas, few embryos). We get very high rates of gonadal explosions (around 50%) in our N2 control. These happen from day 5 on. The explosions are temperature dependent since they happen at 20oC and not at 25oC.
Has anyone observed something similar for N2 in lifespan experiments with FUDR? Thank you for any suggestion/comment.

[AVSamuelson]

I have done a lot of lifespan experiments using N2 at 25C versus 20C using FUDR and RNAi to a host of lifespan genes. We occasionally see a few bursting/Rup or Pvl animals after 1-2 weeks adulthood, but this is much less of the population then 50% (more like 5%). However, if FUDR is added too early, we get many more bursting animals. So our interpretation was if animals were not L4 at the addition of FUDR the FUDR inhibited cellular proliferation around the developing vulva, thereby producing an malformed vulva that would eventually produce a pvl/rup phenotype. Since synchronized L1s (through bleach prep) exit L1 arrest at slightly different times, we believed that the tail of of that distribution is the subpopulation that are rup/pvl.

Typically, we grow animals at 15C from L1 to L4 prior to the addition of FUDR. Most of our work centers on using daf-2(e1370) animals so we maintain them at 15C prior to FUDR addition to prevent dauer formation. To be internally consistent, we do this with all our strains.

The N2 stock we use for all our backcrossing and lifespan experiments is the early passage CGCM strain, or N2 male stock that was defined by David Gems and Don Riddle as most closely resembling true “wild type” lifespan here:

http://biomedgerontology.oxfordjournals.org/content/55/5/B215.full.pdf+html

If you would like a more detailed protocol send me an email. I hope this helps.

Happy worming,
Andy

[ryndahl]

@Terter post:1

Hi worm experimenters,
It would be great if someone already came across/ around the problem described below and can share their experience.
We are doing some lifespans in presence of FUDR - 50 uM in NGM agar, on the FUDR plates we put carefully staged young adults (formed vulvas, few embryos). We get very high rates of gonadal explosions (around 50%) in our N2 control. These happen from day 5 on. The explosions are temperature dependent since they happen at 20oC and not at 25oC.
Has anyone observed something similar for N2 in lifespan experiments with FUDR? Thank you for any suggestion/comment.

Hi Terter,
I have seen this same problem. I don’t know why it occurs. However, if you add 1.5% DMSO into your NGM (ie, 7.5 ml DMSO into 500 ml NGM), the bursting no longer occurs at 20C

[eanderson]

I also see rup or pvl N2 animals during aging experiments with FUDR. Usually it’s just a small fraction (5-10%) that just gets censored, but for some unknown reason I’ve had a few experiments where it’s closer to 50%.

@AVSamuelson post:2

I have done a lot of lifespan experiments using N2 at 25C versus 20C using FUDR and RNAi to a host of lifespan genes. We occasionally see a few bursting/Rup or Pvl animals after 1-2 weeks adulthood, but this is much less of the population then 50% (more like 5%). However, if FUDR is added too early, we get many more bursting animals. So our interpretation was if animals were not L4 at the addition of FUDR the FUDR inhibited cellular proliferation around the developing vulva, thereby producing an malformed vulva that would eventually produce a pvl/rup phenotype. Since synchronized L1s (through bleach prep) exit L1 arrest at slightly different times, we believed that the tail of of that distribution is the subpopulation that are rup/pvl.

Oddly though, I’ve done some aging experiments where I started the FUDR treatment on one-day-old adults (where they all have a row or two of eggs), and I still get animals that rupture at roughly similar rates. So I think it’s not due to abnormal vulval development, but I don’t have any better idea about what the mechanism might be.