Kiss meiosis; when good sperm goes bad

Hi guys,

Over the past three weeks, we’ve been making fairly good progress in Dr. Shakes’ lab. I have been brushing up on some of the techniques and protocols used in cellular biology, I’ve been growing up a healthy strain of worms, and have been reading some of the scientific literature related to my topic.

It’s been a while since the introductory post, so we could use a quick recap of the study. C. elegans, our coolest model organism, is used in the study of genetics and cellular biology because of its super fast reproduction time, its easiness to handle in the lab, and its striking temporal progression that enables us to see events in the gonad (where sex cells are made) in great detail. The two sexes, a self-fertilizing hermaphrodite and a male, live on agar plates seeded with bacteria and take about three days to go from embryos to reproductive-age young adults. Our lab’s main focus is spermatogenesis, an intricately complex process that starts with two uncommitted germ cells following a path through the germline filled with mitotic and meiotic divisions eventually differentiating into sperm or oocyte. There’s also a great amount of genetic regulation; genes need to be turned on or off after a certain stage of development, and if this doesn’t happen, well, things go awry.

Newer models include another layer of complexity in which the subsequent stability of these mRNAs is also regulated. RNA interference (RNAi), using many pathways and factors involved in them, is a method to target genes for regulation and silencing. The rrf-3 gene product, which we are currently studying, is one of the factors involved in RNAi pathways. It is an RNA-dependent RNA polymerase, meaning it adds base pairs to single-stranded RNA to make it double stranded (dsRNA). This dsRNA is cleaved at a certain point by an enzyme, conveniently called Dicer, and the products, now short-interference RNA, siRNA, are loaded into an Argonaute protein complex that recognizes and binds to a target RNA and silences it. The mutants make spermatids but are sterile, clearly indicating there’s trouble in the germline development.

To see the problem, we turn to microscopy, which gives us all those pretty cool pictures. There are some previous steps though; the worm’s gonad needs to be dissected, fixed in cold methanol, and then stained with a specific antibody against a cellular structure or protein. I’ve been working to tune up my skills and techniques in these protocols; and it’s amazing how seemingly trivial steps influence greatly the end quality of the picture.

In terms of data, so far we have replicated the results of a previous study done on the rrf-3 mutant. Using the DAPI/tubulin protocol to fluorescently tag DNA and microtubules, we have been seeing the same rrf-3 phenotype described by Jonathan Ghent. Spermatids only carry essential things, and tubulin is not one of them given that they’re not going to reproduce and their motility is done by another protein. However, this mutant shows microtubule wreaths surrounding the DNA in the spermatids, so something is definitely wrong and involved in the sterility. We plan to keep studying several other cellular structures with the aim of gaining a clearer picture of what’s going on with rrf-3, so stay tuned.

I hope you guys are having a great summer, enjoying the warm weather, and that your investigations got off to a good start.

André

Reference:

Gent, J., et. al (2009). A Caenorhabditis elegans RNA-Directed RNA Polymerase in Sperm Development and Endogenous RNA Interference. Genetic Society of America. 183, 1297-1314.

Comments

  1. Suzan Ok says:

    This research sounds very interesting, Andre! So, do you think the rrf-3 phenotype might be these microtubule wreaths? Also, I was wondering about the specific proteins involved in spermatid motility. Do mature C. elegans spermatids have flagella? Because then wouldn’t they have microtubules and thus tubulin in those structures? I don’t know much about these roundworms, so any information you can provide would be helpful. Good luck these next few weeks – I look forward to reading about your results.

  2. andrenaldo says:

    Thanks Suzan! Sorry for the long reply time. I’m working on another blog that talks about MSP (major sperm protein) which is what C.elegans nematodes use for sperm motility and pseudopod formation. There is no tubulin or actin involved; that’s why the tubulin should be discarding into the residual body (along with other nonessentials). I really want to know what are the molecular mechanisms as to why the tubulin is not getting discarded. Since RRF-3 is just one of the many players in competing RNAi pathways it’s complicated to explain the phenotype in terms of molecular dynamics when just one component has been removed, and for all we know, other components might be taking over since RNAi pathways are known to be redundant. I know this is not super insightful but we have other directions that we can pursue and I talk about some of them on the last blog, hopefully one of them works out. =]