Fourth Annual DNA Grantees' Workshop
Wednesday, June 25, 2003
MORNING SESSION
Question-and-Answer Session
MR. STAPLES: Do we have any questions for any of our speakers? Yes, sir?
MR. SLIDER: Hi, I'm Tim Slider. This is a question for John [Herr]. Those of us from casework labs are familiar with the fact that if you want to recover DNA from spermatozoa you have to use reducing agents. In explaining this to attorneys or new trainees, we usually talk about it in terms of crosslinking cell surface proteins, but it clearly can't be that if the spermatozoa don't have plasma membranes.
So do you have a feeling or do you have a thought about the targets of reducing agents and where they would be localized within what remains in the spermatozoa? Would it be the condensed chromatin or would it be some protein associated with the nuclear membrane?
DR. HERR: There is pretty extensive literature on sulphur-sulphur bonding that occurs during epididymal maturation, and there is quite a number of proteins, including nuclear proteins, that are forming sulphur-sulphur crosslinks. Bedford is among the authors that originally showed that this phenomenon occurs.
It seems to be a general phenomenon that epididymal maturation prepares the sperm nucleus for its life outside the body. That's about as much as I can tell you right now. But if someone were to ask you why this happens, I would say that it's all part of the packaging of the genome in an attempt to protect it in a time capsule.
MR. STAPLES: Any more questions? Yes, sir? Go, Jack [Ballantyne].
DR. BALLANTYNE: I also have a question for John [Herr]. Have you noticed any difference in the structural integrity of sperm as the postcoital interval is increased? We have done quite a lot of work with postcoital time interval. We find that sperm appear to be more labile in some manner as time progresses after intercourse.
Do you have any evidence from your ultrastructural studies that there's some structural integrity loss over time in the postcoital vaginal tract?
DR. HERR: The only phenomenon that I'm clear on is the increased separation of heads and tails. We haven't looked at the enzymes that are involved, but you can predict that at least the intra-acrosomal hydrolases are probably going to be playing a role in digesting some of the substrates associated with the sperm as long as they remain in a hydrated state and remain active around the cells.
Similarly, most of you know that prostate-specific antigen (PSA), which is the basis of the prostate cancer test and is secreted by the prostate gland, is a serine protease and its role is to primarily act on the seminal gel—to liquefy the soft gel that forms at the time of ejaculation and to liquefy it so that the sperm are released in a periodic way from the seminal coagulum. The PSA enzyme also acts on nonseminal gel and substrate, so that is just some of the proteases that are involved in semen that degrade the proteins that are there. That's about as good as I can do with what we know from our end.
DR. BALLANTYNE: With regard to the postcoital sperm having lost its membrane, what's actually holding it together, then, because what you have is just a nuclear membrane? It's just the nuclear membrane holding, basically a naked nuclear membrane, so to speak. What's holding it?
DR. HERR: The way it looks to us is that the nucleus and nuclear envelope are intact. The subacrosomal cement that runs from the inner acrosomal membrane to the nuclear envelope is intact, and the inner acrosomal membrane is intact. Therefore, targets associated with the inner acrosomal membrane and with the nuclear envelope seem to be the ones that we need to look at in the context of long-term postcoital samples.
Some of the acrosomal matrix proteins do cling to the inner acrosomal membrane, but I think in most cases, with the processing that's going on, many of the acrosomal matrix proteins, as well as the plasma membrane, will be eluted and solubilized and lost.
DR. BALLANTYNE: The reason I ask is that we can detect the presence of messenger RNA in sperm, and I was just wondering where perhaps if it could be from then nucleus—where you normally don't find them.
DR. HERR: Well, no, no. This is an extremely important point. There has been a lot of literature suggesting that the sperm is transcriptionally silent and translationally silent, but I don't think that's true at all. If you look at the neck of the sperm there's almost always a few cisternae of endoplasmic reticulum that are left. There's always a small droplet that clings there, and I think your RNA is both going to be nuclear stuff that's packaged in the region. The crater looks like it's actually the nucoilus, and we're trying to prove that actually that structure is the remnant of the nucoilus, so some of it's going to be in that domain.
Then I think the rest of it is going to be in the remnants of the cytoplasmic droplet that exists in the neck of the sperm. I no doubt think that there's going to be a set of messages that persist in the sperm, and you're going to be able to PCR amplify those RNAs up, and I think it's going to be an important source of material for the community.
DR. BALLANTYNE: Thank you.
MR. STAPLES: Anything else?
(No response.)
Lisa.
DR. FORMAN: I again want to thank this wonderful panel. It's more about the Y chromosome than I'd ever heard before. It was really wonderful and informative, and Ted did a wonderful job moderating. I'd like to thank you all again.
Before I release you for lunch, I just want to tell you a little bit about the role that NIJ has played in some of this. What you don't know is how we get grants through our system. Our reviewers review the grants, we look at the relationship of the grants to the entire portfolio, and then we go up the chain telling each person a little bit about the project.
When it came time to fund Dr. [John] Herr's project, we had an Acting Director at NIJ and she was very, very cautious not to rock the boat. So Lois went in to pitch the program and got smiles, nods, and pleased looks all the way up to the time when she started talking about postcoital samples. Then this look of horror started to spread across our Acting Director's face, and she leaned over and she said in a very quiet voice: Do you mean we're paying for sex? Lois, however, managed to convince her of the scientific validity of the project and we were able to go on. I just wanted to share that with you.
Enjoy your lunch and I'll see you back here afterwards.