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Why Sex? Researcher Studies Proteins in Sperm for Clues About Need for Intercourse

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Timothy Karr
research associate professor
Syracuse University
Name: Timothy Karr
 
Position: research associate professor (adjunct), dept. of biology,
Syracuse University, 2006 to present; reader of biology, dept. of biology
and biochemistry, University of Bath, UK, 2002 to present.
 
Background: PhD in chemistry, University of California, Santa Barbara; postdoc work at the University of California, San Francisco, 1981-1987; assistant professor, University of Illinois, Urbana, 1987-1994; research associate, University of Chicago, 1994-1996; assistant professor, University of Chicago, 1996-2003; visiting professor, Kyoto Institute of Technology, Drosophila Genetics Resource Center, 2000-2001.
 

 
In work published in the November edition of Nature Genetics, Karr and his colleagues identify and describe 381 proteins found in the sperm of the fruit fly. The study, they said, is the first substantial “whole cell” characterization of the protein components of a higher eukaryotic cell and could lead to further research that will provide greater insight into the role of sex, the process of fertilization, and possible therapies for infertility. 
 
ProteoMonitor recently spoke with Karr about his work, how he got interested in studying sperm, and what he hopes to accomplish with his continuing research.
 
Tell me what you did and how you did it.
 
Basically, the idea was to identify the proteins present in sperm, in pure sperm. It turns out that sperm when they’re made in the testes and when they’re ejaculated, they combine with thousands of other proteins in the semen. And so if you wanted to ask what’s in sperm, you’d have to first get rid of this large number of proteins that stick to the side of sperm or otherwise interact with it. So the first thing was to get pure sperm. And for that insects were very useful because they store their sperm in a sort of sack of water, and they’re not anywhere near their seminal fluid.
 
And to find out what actually was in them in terms of the different kinds of proteins in the sperm, we used two techniques. One was mass spec and 2D gel electrophoresis in conjunction with mass spec. The first thing I did was take pure sperm, trypsinize them so that you cleave the proteins, dissociate the sperm so all the proteins are solubilized. You then add an enzyme that chops the proteins up at one amino acid.
 
If you have a long polypeptide chain in a protein, every now and then there will be a lysine or an arginine amino in this chain of amino acids. Trypsin will cut at that point generating two peptides, one which has added in a lysine or an arginine, often a lysine.
 
So you then chop up the protein at these lysines, and you end up with what was once a bunch of big proteins [but is] now a bunch of tiny, little peptides. And those are what you put into the mass spec machines. And the mass spec machine very accurately is able to determine the composition of those peptides. Once you know the composition and the sequence of those peptides, you can then query using bioinformatics and computer-based systems, where is that peptide, [where is that], say, 15 amino acids that we found in this particular mass spec spectrum.
 
Where is it in the genome? That is, back-translate the amino acids into nucleotide sequences, the DNA sequence that would have been coded for that protein. Now you have, let’s say for those 15 amino acids, 45 nucleic acids, a sequence of 45. And when you ask where that is in the genome, you very often, 95 percent of the time, get just one hit back to the genome. And that one hit, of course, is in the gene that encoded that protein. So now you know the whole gene. That’s gene discovery by basically determining small pieces of proteins.
 
How did you get interested in studying sperm?
 
I showed a long time ago that total sperm entrance into the egg is a common occurrence in animal fertilization. And that was not known nor appreciated at that time. And that got me going in knowing what is in a sperm, why are all those sperm proteins in that egg. Is there a reason that giant thing goes into the egg?
 
So subsequent to this discovery, numerous other people went back and examined or reexamined fertilization in other organisms, like humans and mammals. And total sperm entrance is the rule in animal fertilization, not the exception. Now we can say that this is an ancestral trait in fertilization.
 
We can start asking questions about what happens to those proteins, how does the egg accommodate what one of my colleagues calls an insult to the egg. In the process of me trying to understand that for the last 10 years, it became clear that it would really help if I actually knew what was in sperm.
 
Everybody wants to know what’s in sperm because they’re targets for study of human infertility, of zygote health and viability, etc., etc. Now that we know the whole thing goes into the egg, if those proteins are doing anything in the egg, then they’re important.
 
So you’re looking at the first 381 proteins. There are going to be more — and other sperm from other organisms may have a different set of proteins, we don’t know yet — but at least in Drosophila, where you can do genetics and actually test function in a very straightforward way, it’s the ideal organism to have a proteome like this in order to study sperm function.
 
Do you have any idea how many proteins in total there are in the sperm of the fruit fly?
 
We’ve been able to identify 381. Our best estimate is that we’ve identified approximately 90 percent of the proteins in the sperm. That is a rough estimate, I have to emphasize, based on looking at the overlap between determining the proteome using one technique versus another. It looks like we’ve gotten about 90 percent, say 80 to 90 percent. In Drosophila sperm, there may be 400 or 500 proteins.
 
Put into the context that this is the first [study] that’s ever been done [and] things always change, especially in biology, we don’t expect this to be the last number, and the number can grow to anything. We don’t really know.
 
Is there an estimate for how many proteins there are in human sperm?
 
No one knows since nobody’s studied it.
 
Aside from the amount of proteins in sperm, what else did you learn?
 
We learned that the nature of the kinds of proteins that are in sperm have been quite surprising. About one-third of all the proteins are proteins for which we don’t have any idea what they do. Over 100 are proteins which have never been studied and of whose function we have no idea what it’s doing.
 
And they’re specific to sperm?
 
We don’t know yet. Some are, some are not. They’ve not been identified in other parts of the fly. The transcription of the genes, which is a different way of looking at expression, making RNA from the DNA, you can look at that, and in other organs of the fly, in other parts of the fly body, they are expressed. But we don’t know anything about what they do, or how it would relate to sperm. Many of them are sperm specific, but we don’t know what they are doing.
 
Half of the proteins that we identified have human homologs, so we’re guessing — and a lot more work needs to be done on this — that there are a lot of similarities between the proteins in the sperm of fruit flies and the proteins in human sperm.
 
We’ve also identified a protein that is a known protein involved in male infertility in mammals. It’s been shown that mutations of that gene in mice result in infertile, sterile mice. And the sperm that they make can’t move, apparently. There’s one clear indication that there’s a gene that has an analog or homolog in humans and mice.
 
My assumption is that now that you’ve identified some of these proteins, the next step is to characterize them and determine their functions.
 
That’s right, and to also ask questions about the ones that we do know their functions. Many of the genes in Drosophila have been studied for so long, they were discovered because they made funny-looking eyes, or their wings weren’t right, or their nervous system didn’t work right. So why would a gene that’s known to be involved in the functioning of nerve cells be in a sperm?
 
There are a lot of really interesting questions about the use of proteins for these two seemingly different functions. And there’s a class of those in our sperm proteome. Many of these genes are quite interesting in and of themselves as studied in other parts of the animal. But now we know they’re in sperm also, there’s a whole new avenue to approach the function of those proteins and the evolution of them.
 
What do you mean when you say that these findings can help us get a better understanding of sex?
 
That can be answered at different levels. One is just what we’ve been talking about — if you don’t even know what’s in a sperm, how can you understand sex? And by sex I mean the cell biology of fertilization, how it happens, what proteins are important, what these proteins do. We don’t even know what’s in sperm, so if you really want to know how something works, you’ve got to tear it apart. It’s like if you want to know how an automobile works, you have disassemble the engine and put it back together to figure out how all the parts work.
 
It’s no different here. You’ve got to know what’s in it. How does sex work, how does fertilization work, why did sex evolve, how is it maintained? Those big questions have been asked for a long, long time.
 
So you’re trying to understand it as a mechanical process on the cellular level?
 
That’s one of them, but also at the evolutionary level. If you have 300, 400, 500 genes that you know encode proteins that are all in sperm, then you can ask, ‘Where did they come from evolutionarily?’ And you can do that by studying other sperm proteomes and comparing all the genes in those sperm to try to work your way back to the origins of where sperm came from.
 
Are you trying to find out the biological imperative of sex as well?
 
Yeah, why do we have sex, why sperm, why two sexes, why not five? When did it evolve, and why is it so widespread and so common?
 
We don’t need males. Males are just a burden on the population because if you replaced every male with a female, you’d have twice as many reproductive units. It’s the female that makes the egg and if you could just get rid of the male and didn’t need the sperm — and many organisms do that, it’s a small percentage, but they still do it and they can reproduce genetically. And on paper that should be the better way to go.
 
There are many animals that reproduce asexually. You don’t have to have sex in every species that we know of. There are what were called ancient asexual species — they’re complex eukaryotes just like us, and they don’t have sex. So there are some big, big evolutionary questions about sex that have never been answered.
 
I’m not saying we’ve answered them. I’m just saying that by knowing what’s in a sperm, and ultimately by knowing what’s in eggs and understanding how the sperm and egg interact [we can answer some of these questions]. We know nothing about how that happens.

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