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SPEAKER: This is a production of Cornell University.
MARIANA WOLFNER: Today, we're going to continue our discussion of how sperm find and bind to eggs. And as you remember, when we finished this discussion, we were sort of in the middle of invertebrate sperm finding and binding to eggs. We're going to finish that off in the first part of the lecture, and then we're going to transition over to mammals and talk about the same situation in mammals.
One of the things I think you're going to see is that at the moment, the situation in mammals is much more complex, both in terms of the molecules and in terms of the controversy and agreement or lack thereof about models. So we'll be talking a lot about the nitty gritty details of that.
But let's go back to invertebrates for the moment-- marine invertebrates. And I'll just remind you where we were at the end of that discussion. We had been talking about how the sperm of sea urchins and starfish and abalone, which are the three best model systems, find and bind to the eggs. And what we saw was that the first cue was the release by the egg of a species-specific peptide.
This had to be very species specific because these things are released all into common tide pools. So you only want your own species of sperm to find you. We saw that these peptides, like resact and speract and [? astrozap ?] bound to receptors on the tail of the sperm. We saw that those receptors were guanylate cyclases, and that when they bound their ligands, that caused a transient calcium rise in the tail the sperm, and that changed the swimming pattern of the sperm. So that instead of swimming in tight circles, it started swimming in ovals that moved it towards the source of the peptide.
Then focusing on sea urchins and talking about what happens when the sperm reaches the egg, we saw that the sperm reaches first the outer jelly coat of the egg, which is very thick, and that this jelly is primarily composed of a sulfated polysacchride polymer that's also species specific. So in sea urchins, it's called FSP, fucose sulfated polysacchride. And that the sperm with a specific receptor for egg jelly, or REJ, bound to this fucose sulfated polysacchride.
This binding then pulled together the REJ molecules on the surface of the sperm, and that caused them to trigger a calcium influx because they're related to calcium channels. The calcium influx then caused the fusion of the acrosomal vesicle with the plasma membrane of the sperm, and the acrosome reaction occurred. We saw that that happened because calcium entered the sperm, and that helped to trigger the acrosome reaction.
And also hydrogen ions went out of the sperm, and we saw that that caused an increased pH in the sperm, which caused an explosive polymerization of actin. Remember, that pushed out that membrane of the sperm coated with another protein called bindin, which is what bound the sperm to the vitelline envelope of the egg. And that's where we left it.
And-- oh, no. We saw that it bound to its receptor on the vitelline envelope of the urchin egg called EBR1, a large protein with repeated sequences. And interestingly, all of the proteins I've mentioned and that polysacchride are all highly species specific. So we saw that at each step, there were species-specific barriers.
And so we left the sperm sitting, touching the vitelline envelope and bound to it by EBR1. But it still had to get through that envelope, and that's what we're going to talk about today. But we're going to switch marine organisms. We're going to talk about abalone just because it's better understood in abalone at this point.
So before I talk about that, are there any questions on this sort of whirlwind summary of what we covered or anything that came up as you thought about it or read about it? OK. So the abalone sperm goes also through a kind of jelly coat and reaches the vitelline envelope. And now it needs to get through the vitelline envelope in order to be able to reach the plasma membrane of the egg.
And I went through this very quickly last time and then promised to do it slowly the next time. So that's what we're doing right now. And so we need to talk about the proteins that are involved in this. So if you have a substance on the sperm that's necessary to dissolve this vitelline envelope, considering what I described of the sea urchin sperm and analogous things in the abalone sperm, where do you think-- if you were a biochemist, where should you look for that substance?
Would it be on the sperm plasma membrane? Would it be inside the acrosome? Would it be inside the rest of the sperm? Where would you look if you were a biochemist? Remembering what I just described, OK? That the sperm gets through the jelly coat, undergoes the acrosome reaction, gets pushed up to the vitelline envelope. Where should the thing that will help dissolve the vitelline envelope be?
AUDIENCE: In the acrosome.
MARIANA WOLFNER: In the acrosome. Excellent. Inside the acrosome should get exposed when the acrosome reacts. And Victor Vacquier and his colleagues reasoned in exactly that same way. And so what they did is they got a lot of abalone, which was easy to do at the time. Now they're much harder to find.
And they've got a lot of sperm from the abalone, which is very easy to do even if you only have one abalone because they make so many. And then they isolated acrosomes biochemically. They basically isolated sperm heads, lysed them very gently so the plasma membrane lysed but not the acrosomal membrane. And then they could spin them through solutions in a gradient so that the large acrosome separated from everything else in the sperm.
So they had a very pure preparation of acrosomes. And then they simply lysed that preparation, the membranes of those acrosomes, and ran the proteins on a gel. And the most abundant protein that they found was a 16 kilodalton protein that they named lysin. And they named it lysin because its function is to lyse the vitelline envelope.
And so then the question became, how does lysin lyse the vitelline envelope? And I think I mentioned last time that you probably wouldn't want a protease to be lysing the vitelline envelope because that could really cause some problems. That could make too-- that could destroy too much of the vitelline envelope protein-- bless you-- and make a large hole, which would be a problem since the vitelline envelope needs to convert to the fertilization envelope after the egg is fertilized. So you want something that's going to make a very tiny and precise hole.
When they looked at the sequence of lysin, they found that it didn't look at all like a protease. And, in fact, it has no protease activity at all in vitro. So this suggested that, in fact, lysin was not a protease. Yet it dissolves the vitelline envelope. And here's a photograph of it dissolving the vitelline envelope.
What you can see, here's the vitelline envelope, and you can see that these fibers are sort of being unspooled and released. And here's a biochemical picture of basically the same thing. Focus, if you will, on this graph here. So what this graph shows is they take vitelline envelopes, and they add more and more and more lysin. And they measure how much protein becomes soluble.
So the vitelline envelope, as you saw here, is a very solid matrix. And what they're asking is, when it's dissolved here, these fibers and units of them will become more soluble. And so they're asking, how much protein becomes soluble, and can we see it in solution? Can precipitate out the envelope and see how much remains.
And what they see, again focusing on here, is when you add lysin from the abalone of a species called red-- because the abalone are red-- the more lysin you add, the more the vitelline envelope gets solubilized. Whereas if you add the same lysin from red abalone to the vitelline envelope of a pink abalone, it doesn't do that. So it seems to be species specific in dissolving its own vitelline envelope. So lysin is able to dissolve the vitelline envelope in a species-specific way, but it's not a protease.
Before I tell you how it does it, I want to tell you one other thing about lysin. Up to now, every protein we've mentioned in the egg-sperm interaction between marine invertebrates has been highly species specific. And so the question is, how about lysin? Is lysin highly species specific? Lysin is the most rapidly evolving protein known. So it's very species specific.
Between two particular abalone species whose average genes are 1%-- or average protein coding sequences are 1% different, lysin is 15% different. So it's extremely different. Its sequence is changing very, very rapidly. And so just like some of the proteins that we talked about last time and we'll continue with next time, this is one of the proteins that evolutionary biologists have been paying a lot of attention to in terms of the pressures that select for species differences in speciation.
But right now, we want to know, how does lysin dissolve the vitelline envelope? And to do that, we need to identify the receptor for lysin. In other words, we need to figure out biochemically what is this protein that we're seeing meshed here and falling apart here.
So Willie Swanson, a graduate student with Vic Vacquier, took this on as his thesis project. And what he did was he purified vitelline envelopes from abalone, and he solubilized their proteins. And then he ran the solubilized proteins on a column to which lysin was bound, thinking that if lysin is responsible for breaking up the protein, it must bind it. And therefore, the protein should bind to a column with lysin-- so an affinity column. Is that OK?
All right, so he did this experiment. So the next slide will show you a gel. And it's actually two different purifications from two abalone. So you can pick whichever pair of lanes you want to look at. So in lane one, you can see what happens if you dissolve vitelline envelopes and just run the proteins on the gel. And you see a whole bunch of nice little bands, and then this crud up here. And that's a very pretty looking gel.
So then Swanson ran those proteins over a lysin affinity column and looked for what bound lysin. And when he first looked at the gel, this was very depressing because, as you can see, it's completely blank here. None of these proteins bind. But when you look at the whole gel, up here in the stacking gel where proteins are too big to even enter the gel, notice that there's protein up here.
And so there were two possibilities. One is that this is just some kind of garbage that just happens to stick to everything, and it happens to be there for that reason and doesn't load on the gel. And the other was that this really is the protein to which lysin binds. It's just huge, so huge-- molecular weight of about 1,000 kilodaltons, as opposed to most lysin is 16. OK, it's a huge protein, just too big to get in a gel, but maybe it really is the lysin receptor.
So Willie is a very optimistic guy, and he decided to name this stuff up here the vitelline envelope receptor for lysin, or VERL. And to test if, in fact, this stuff was really the receptor for VERL-- I'm sorry, for lysin-- he did the following experiment. If VERL is really the lysin receptor, then if you take that assay that I showed you before where you add lysin to vitelline envelopes and you see them getting dissolved-- if you add a huge amount of VERL in solution, then that VERL will probably bind to the lysin in solution.
So there'll be less lysin around to break the vitelline envelopes. Does that make sense? So that's what he did. So this is a competition assays done in solution, and we're going to come back to this theme again when we talk about mammals.
So Willie did that experiment. And so he took a constant amount of lysin and a constant amount of vitelline envelopes, and he added more and more VERL in the solution. And what he found was when he added no VERL, 80% of the envelopes-- 90% of the envelopes were dissolved. But when he added a lot of VERL, none were dissolved, and there was a beautiful dose response curve in between.
So, in fact, this thing up here is not crud. It really is the receptor for lysin, and it's behaving biochemically that way. It's just really big and therefore really hard to work with biochemically or to get in a gel. Part of the reason this protein is so big is that analogous to EBR1, the receptor for bindin, it's got a lot of repetitive units, and they form a huge part of its central structure.
So now that we know what VERL is, he wanted to do one more experiment to see if it had anything to do with the biological structure that you could see. Remember you could see these filaments splaying out when the sperm was going through. And so he took his purified VERL, and he let it sit in a test tube with buffer and then looked at it under EM to see if it formed filaments.
And I hope you can see this. I'll trace them. Here's an electron micrograph, and there's a filament of VERL. And there's another one. And there's another one. And these filaments have the same dimension as the filaments that you see splaying apart in the vitelline envelope.
So putting all these data together, it looks like we have the ligand, lysin, and the receptor VERL for the sperm vitelline envelope interaction that allows the sperm to bore its way through the vitelline envelope in abalone. So before I go on about how it actually does that, are there any questions about this, about the experiments or other things they did or anything like that? Yep.
AUDIENCE: He never identified a protein specifically, just the whole conglomeration of protein?
MARIANA WOLFNER: So the question is, did he identify a specific protein or the whole conglomeration? So it turns out that VERL, like many of these other extracellular proteins, is highly glycosylated. And that makes it run as a smear on a gel, and that's even worse because it's so big it can't get in the gel.
So he actually did identify a protein that was just really big and easy to break and high up on the gel and glycosylated, so it didn't look nice and sharp. But he was able to show by sequencing that it appears to be a single protein, and there's a single gene that codes for it. But I agree. I mean, if I had seen that gel in my lab, I would have had the same question because it's this wide thing. Other questions?
OK. Let's talk about how lysin breaks VERL because I think this is particularly cool. But the one thing we know is it's not a protease. But how can it break VERL? So the Vacquier lab continued to study that, and they decided to do that by crystallizing VERL since it's-- sorry, crystallizing lysin since it's a little tiny protein, easy to get a ton of it, easy to purify. And it turned out reasonably easy to crystallize.
So they crystallized it and with their collaborators solved its crystal structure. And that told us how it worked. Because when you look at the structure of VERL, it turns out-- so here are two faces of VERL. Let's call this the back and this the front. One face of VERL, which we'll call the back--
AUDIENCE: Is it VERL or lysin?
MARIANA WOLFNER: I'm sorry, lysin. Thank you. This is lysin. Let me try this again. Lysin from the acrosome was purified and crystallized because it's small and they could get a structure. Thank you, Stacey. And so when you look at the back of lysin, the residues on the back face are very hydrophobic. So they like to associate away from water.
And when you look at the front face of lysin, the residues shown here in blue are very hydrophilic. They're actually-- if you were standing up here, you would see they were all arginine, arginine, arginine, arginine, lysin, arginine, arginine, lysin, lysin-- very positively charged. That's interesting because VERL-- and I mean VERL-- is negatively charged.
So lysin has a positively charged face and a hydrophobic face. VERL is negatively charged. They discovered-- they did some more biochemistry on lysin and discovered something else important, which is when you put lysin an aqueous solution, it forms dimers, presumably with the two hydrophobic faces sitting touching each other and the two hydrophilic positively charged faces pointing out. OK?
So imagine you have a dimer now with positive charges on either side, and VERL has negative charges all over. OK? So they made the following hypothesis. And I will try to illustrate it for you also. Their hypothesis was-- so if you imagine this side of my hand being positively charged and this side being the hydrophobic, their suggestion was that this is what lysin looks like.
And when it binds the two VERL, the positive charges here will bind to the negative charges there on VERL and cause a conformational change, which will split the dimer. And that's what's shown here. So here's a lysin dimer moving up to VERL, binding by charge-charge interaction. That causes a breaking-- a change in confirmation of the dimer, which breaks it in half. And so now each half is associated with part of VERL, and that opens the VERL molecule.
The way that Vacquier explains it and shows it when he gives talks is he suggests that this opens up, which then makes a space where more lysins can get in and do the same thing, and it's like unzippering. So I brought a zipper with me just because. And so what's happening here is the first lysin comes in and pulls it open.
That makes space for the next lysin to come in, which makes it open more, which makes space for the next lysin to come in, which opens it more. And the next thing you know, there's enough room for a sperm to come in. So this is the model that the Vacquier lab has proposed, something you can think about when you go outside and put on or take off your parka. Just keep thinking about lysin.
And it's, in other words, a non-enzymatic stoichiometric dissolution of VERL. And what's so nice about it is this keeps the dissolution of VERL very, very local-- only where that sperm was. It can't spread beyond that. It can't destroy the vitelline envelope. You will make a hole in the vitelline envelope just big enough to get that sperm in.
So that's the story of lysin and VERL and how lysin unzips the VERL to get through the vitelline envelope. We don't yet know how other molecules unzip the vitelline envelopes that they need their sperm to get through. But lysin is a perfect example of one way. So we're going to transition in a minute to mammals. But before we do that, are there any questions?
AUDIENCE: [INAUDIBLE]
MARIANA WOLFNER: It's thought that VERL is most of these large fibers that you can see all the way throughout the vitelline envelope-- everywhere. There obviously are other smaller proteins in there given those gels that we saw. But VERL is by far the most-- oops, wrong way, sorry. VERL is by far the most abundant component if you compare the staining here to the amount here. So it's mostly VERL. Yeah. Mm-hmm?
AUDIENCE: Isn't that hole fairly big anyway, though?
MARIANA WOLFNER: Well it's got to be big enough for a sperm. So it's not tiny, but there's no way that it can diffuse further and destroy any further than the region of the sperm. There will always be a small hole because you have to get the sperm in. Yep.
AUDIENCE: So there's nothing that tries to like zip it back up afterwards at all?
MARIANA WOLFNER: Well, that's a very good question. The question is, does something try to zip it back up? I don't know the answer to that. And I don't know that-- I wouldn't say I don't know that Vacquier has ever looked because he's a really good biochemist, and he might have. But he's never said in a talk anything about after opening it up. But that would actually deal with Stacey's question because you'd still leave a little hole in there. It's possible. I don't know. It's also possible that just having all these fibers, they could just kind of start to mesh around each other and close it up a little bit.
AUDIENCE: Is there like other things getting in?
MARIANA WOLFNER: Yes.
AUDIENCE: Like proteins-- [INAUDIBLE]
MARIANA WOLFNER: Yes. Yes. I mean, the sperm itself is going through there. So there's a tail in there, at least for a while. But you're right. You could maybe-- just the proteins that are right there could form a meshwork. Maybe those other small proteins we see on the gel could participate in helping to seal it. I don't know anything about this, and I'm sure nothing's been written or said about it, at least until a few months for now. So yeah.
AUDIENCE: What happens to the hole--
MARIANA WOLFNER: Good question.
AUDIENCE: [INAUDIBLE]. Does it go away?
MARIANA WOLFNER: So what happens to the hole? I don't know. We don't know what happens to the hole. Nobody really talks about it. I mean, we're so--
[LAUGHTER]
Yeah, what it is is that everyone's focused on getting the sperm in, and then fertilization happens and we say a fertilization envelope is raised. Now one of the things that happens when an envelope is raised is what raises it is the release of the contents of the cortical granules that are underneath the plasma membrane of the egg. And they've got all kinds of proteins and other things in them.
My guess is that they will just fill the space, and that hole will probably still be a hole in the envelope. But there'll be so much other stuff there that it'll basically be closed. But no one has ever considered that question that I know. Other questions? Yep.
AUDIENCE: So how do you maintain species specificity when it's really kind of just overall charges that are--
MARIANA WOLFNER: So how do you maintain species specificity when it's only overall charge that's doing it? I don't know the answer to that. There is species specificity in a lot-- in lysin. And VERL has also got some regions that appear to be under positive selection. So it may be more than simply charge.
It's presented as charge, but it may be that the shape of the charged region on VERL will only fit the face of lysin. It's possible that they have not proceeded to that point yet. That would be my guess. And also, this is the third or fourth species barrier. So it may not need to be quite as tight. But I bet it is, since lysin is changing so quickly.
Any more questions? It's going to get complicated now because-- I mean, this was easy-- because we're going to mammals. So please ask away. All righty. Let's switch to mammals.
So I think five or 10 years from now, whoever is speaking-- hopefully me-- about this in mammals will say, oh, this is easy, just like I just did. But to be honest with you, I've taught this quite a few times, and every single time I teach it, it's different because this field keeps changing and changing, and new proteins appear and old ones get discredited. And so I will do my best.
I'm going to tell it to you-- since this is 600 level course, I'm going to tell you some of the nitty gritty about how things were found and how they fell by the wayside. So in mammals, though, the sperm still has to find the egg. It still has to penetrate a thick layer of something that's outside of the vitelline envelope.
In the case of mammalian eggs, we saw this was a layer of cumulus cells connected by hyaluronic acid by an extracellular matrix. But still, it's a thick layer that's not part of the eggs just around the outside. Instead of the jelly, it's these cells. Inside that is the vitelline envelope.
In mammalian eggs, it's called the zona pellucida, which is, I believe, Latin for the pellucid or almost transparent zone. And you'll see a picture of a mouse oocyte in a little while. You'll see why they call it transparent. But it's basically a vitelline envelope, also a meshwork of particular proteins.
The sperm has to bind the zona pellucida. It has to undergo the acrosome reaction. And in contrast to the marine invertebrates, that happens at the vitelline envelope or zona pellucida. That doesn't happen before.
And then, of course, it has to cross the zona pellucida and reach and fuse with the plasma membrane. Today, we're going to get it up to and maybe bound to the zona pellucida, maybe acrosome reacted. But we're not going to fuse until next week or so, OK? So we're just going to go the first few steps.
So there are some differences between mammals and marine invertebrates in terms of fertilization, along with everything else. First of all, mammalian fertilization is internal, which means that you don't have quite the large volume that a marine invertebrate sperm has to find the egg in. And you shouldn't have competition of gametes from other species.
So for various reasons, you don't need as many sperm in mammals as you do in marine invertebrates. So whereas we talked about how sea urchins can release 10 to the 12th sperm, in mammals, it's more like 10 to the 7th or 10 to the 8th per ejaculation. So it's still a lot of sperm, but it's not anywhere near as many as the marine invertebrates.
Interestingly, only a few of these sperm actually reached the site where the egg is going to be fertilized. They have a long way to traverse through the female reproductive tract up into the fallopian tubes where the eggs are being released by the ovary into the fallopian tube. So they have quite a distance to go.
And depending on the organism and whom you read, only a few reach the region where they'll fertilize the egg-- 2,200 in mouse, about 250 or somewhere between 80 and 1,400 in people. So the dynamics are very different. And given the way I've been praising these marine invertebrates for giving you bucket loads of pure gametes, you can see that the technical issues in working with mammalian gametes, especially eggs, are much more complex and complicated.
Secondly, as you heard from Professor Travis, sperm are not fully mature in mammals when they leave the male, in contrast to the situation in marine invertebrates where they are mature or immediately become mature when they hit the seawater. They require a process called capacitation where their membrane fluidity changes. Their protein phosphorylation change.
Their carbohydrate distribution can change. Their intracellular pH and ionic balance can change. And protein localization on their surface can change. And you heard all about this from him a while ago-- I'm just reminding you about this-- and in one of the papers.
You might expect less need for species specificity since there are some barriers prior to mating that would prevent, for example, a chihuahua sperm from encountering an elephant egg or something like that. So you expect a little bit more potential for relaxation of these intensely tight species-specific barriers maybe.
And finally, the female reproductive tract itself provides not only an environment to mature the sperm, but a somewhat selective environment for the sperm. The pH is low in the female reproductive tract. That's not great for sperm. On the other hand, sperm gets stored in the reproductive tract.
in mammalian females, they get stored here at the uterotubal junction. In insects, like I work on, they get stored in specific storage organs as well. And what's nice about having sperm get stored in the female is several things. First of all, storing the sperm in organisms that can storage them for a long time, like insects and birds and some bats, it means that a female can mate but continue to have progeny from that mating for a long time.
Most mammals store sperm much less time, like a couple of days. So that doesn't make such a difference. But it also allows the continual release of sperm if the female has sperm stored in her body. And it allows for some powerful evolutionary forces like sperm competition, where if the female's mated with two males, one male sperm may win and fertilize more eggs or fertilize eggs more frequently. Or its counterpart, female choice-- where if the female has mated with two males, she may choose one male sperm by methods that are still mysterious so that she has more progeny with that male.
Here at the wall of the uterotubal junction where sperm are stored, in mammals, you can see [? crifts ?] in the epithelium where the sperm tend to nestle. Susan Suarez here at Cornell and the vet school has done some beautiful studies that show that the sperm-- a protein, a seminal protein actually, bound to the surface of the sperm binds to sugar residues on proteins in this region of the female reproductive tract, and it helps keep the sperm in place and stored.
While the sperm are stored there-- and I'm not sure if Professor Travis mentioned this-- they can, as part of their maturation process, take on a new form of motion called hyperactivation. Did he mentioned that term? So if you take hyperactivated sperm and put them in a regular buffer, it looks like they're swimming like this, which seems like a completely pointless thing to do.
But if you put them in the viscous environment of a reproductive tract, normal sperm, which are not hyperactivated, have trouble swimming. The hyperactivated ones are now able to swim. Hyperactivation is thought to also take them off the wall off the storage site. So the female reproductive tract allows her to store the sperm, possibly to select against poor sperm, allows the sperm to mature, and allows for things like sperm competition.
But they still have to find the egg. So are there any questions on storage at all? OK, so they still have to find the egg. So mammalian sperm actually use two different kinds of mechanisms to find the egg, at least two. And I'm going to tell you what both of them are, but the first one is going to take me about a minute to tell you because we don't know anything about it except that it happens.
And this mechanism is called thermotaxis, which means movement towards temperature or temperature biased movement. It turns out that the region where the sperm are stored is slightly, slightly-- like 0.8 degrees-- cooler than where the egg will be. And upon ovulation, the region around the egg, for reasons, again, that are not known, gets even warmer. So it's like about a degree and a half difference between where the sperm is stored and where the egg will be.
And sperm appear to have-- mammalian, sorry, sperm appear to have the ability to swim towards higher temperatures. They seek higher temperatures. How they do this, we don't know. But one thing that can happen is that the sperm can leave storage and start to move this way simply because they're moving towards higher temperatures. And that may help them get towards the egg.
But in addition, mammalian sperm, analogous to marine organism sperm, can chemotax towards the egg, and I'd like to tell you a little bit about the chemotaxis. First, I'll tell you a little bit about just some straightforward experiments looking for chemoattractants and what they found.
And then I'm going to tell you about some experiments that are real. The data are published and look really good. But we don't quite understand what they mean, but they're really intriguing. And I'll come to those in a little while.
So the first experiment that suggested that there might actually be some chemotaxis going on was to consider the fact that when mammalian females ovulate, often the ovary on one side will ovulate and the ovary on this side won't ovulate until the next cycle. And when a female has mated, sperm will, of course, be stored on both sides.
But if you look at the number of sperm in the fallopian tubes on the two sides, researchers noticed that there were more sperm in the fallopian tube with the mature oocyte than in the other one. So that suggested that the oocyte or the follicle during ovulation must have released some molecules that attracted the sperm.
And so there have been experiments testing oviductal fluid and follicular fluid and the secretions of the cumulus cells to see if any of them attract sperm. The way these experiments were done is exactly the same as the way the experiments were done in sea urchin.
You put your fluid that you're interested in into a pipette. You drop from the pipette a drop into a dish of sperm. And you see if the sperm go towards the pipette tip or whatever you dripped in, OK? Are we OK so far? All right.
So that was done. And a number of molecules were found. First of all, they don't seem to be as tightly species specific as the marine organism ones. And for example, the follicular fluid contains-- of humans-- contains a chemokine called RANTES. It's a small eight kildalton protein. And this molecule appears to be a chemoattractant for sperm.
So if you remember, the oocyte develops within a follicle that then ruptures and releases the oocyte with its cumulus cells around it. So the follicular fluid is only going to be released at that one point. It's not going to be a continuous source. So although there could be something there that attracted sperm, it wouldn't keep sperm coming towards the egg the whole time.
So other researchers have looked to see whether the egg itself puts out attractants. And they have found that the eggs of some mammals put out progesterone from the cumulus cells, and sperm respond to progesterone by going towards it. If you put progesterone antagonists in, you prevent the sperm from going towards it. So it really does suggest it's a chemoattractant. And they appear, therefore, to be modulating their behavior in response to progesterone.
So I've now told you about at least three things that can move the sperm up to this region. One is a temperature gradient. The second is something from follicular fluid, and I gave you the example of RANTES. And the third is something released by the egg, and I gave you the example-- or the complex that contains the egg, and I gave you the example of progesterone.
In fact, if you read the literature, there are probably three or four additional chemoattractants that have all been shown to attract sperm in different systems and by different labs. It is possible that-- I mean, this is a serious distance for a sperm. This picture is not to scale. Sperm are much smaller than that. And so it's conceivable that you need multiple attractants to get them to the egg.
Another hypothesis that's been put forth is that different attractants may act at different times to get the sperm partway there and then a little bit more and then a little bit more. So for example, the example I gave you of the follicular fluid, which is released once and goes away, would be a good example of that. It might get the sperm started, but then they need something to help them get any further.
So clearly, there's a lot more to do. We don't know the-- we do know the receptor for RANTES, but we don't know what it does inside the sperm. And for some of these other things, we don't know the receptors. We don't quite know how they work in the sperm, except that they change the swimming behavior of sperm.
But clearly, analogous to what we've seen with the marine organisms, there are chemoattractants released by and near the egg that call the sperm in. OK? Any questions about this before we go on? It's all clear? Yeah.
AUDIENCE: I understand how there's interspecies competition, like, within intraspecies competition since it's only like 20 or 200 sperm reach the site. And there's intracelluar [INAUDIBLE]. How do the sperm become different from each other?
MARIANA WOLFNER: So that's a very good question. And the competition that I was talking about would be between the sperm of two different males of the same species. And so that kind of competition, that's straightforward, right? Somebody may have sperm that swim better or fertilize better or whatever.
Between the sperm of a particular species--
AUDIENCE: [INAUDIBLE].
MARIANA WOLFNER: I don't know. And I honestly-- in contrast to lysin and VERL where I really did know, here I don't know whether it is known. Everything I've read has not discussed differences between the sperm of a single male, except perhaps the speed and stability of Y-bearing versus X-bearing sperm. But taking two X-bearing sperm from the same male, which may nevertheless have some differences, and seeing which one of them is better, I haven't seen anything about this. I've only seen it between males of the same species.
AUDIENCE: Why would so few make it?
MARIANA WOLFNER: OK. So why so few can make it partly can be answered by-- this is a big distance to go. There is a region here at the cervix that's very difficult chemically for the sperm to get through. It's hard for them to find the right place, and then they have to be at the right stage.
They can't have gotten too far in capacitation. They have to have gotten far enough. They have to adhere to the walls. It's just a long and complicated trek for them to get there.
Why those 20 or 100 make it? And it may be 20 or 100 now and then maybe another 20 or 100 released tomorrow or something like that. So it's not that there are only 20 up here. It's that they get here when the egg is there. I think it's stochastic, but I don't know. I will see if I can find that out. Fortunately, Dr. Suarez is right up the road, so I will ask her by next week.
AUDIENCE: Isn't it to prevent polyspermy?
MARIANA WOLFNER: To have very few sperm could prevent polyspermy? It could well prevent polyspermy. That's absolutely possible. Yeah.
AUDIENCE: And also, I had the impression that the muscle contractions of the fallopian tube played a very important role.
MARIANA WOLFNER: They also help move things.
AUDIENCE: Right.
MARIANA WOLFNER: Right.
AUDIENCE: I thought that was more important than-- I mean, I know chemotaxis was also there. But I had the impression that it was more important than chemotaxis. Is that-- I don't know.
MARIANA WOLFNER: You could be right. I don't know of anyone who has been able to separate the two in vivo. But, yes, you're right that there are muscle contractions that also help to move things along. Yeah. I will see if I can find out about within male sperm competition.
AUDIENCE: Oh. And another thing regarding that is that even in a single ejaculate from mammals, I think that a lot of sperm are just defective. They've just--
MARIANA WOLFNER: That's correct.
AUDIENCE: --fluid with defective [INAUDIBLE]
MARIANA WOLFNER: That's correct. In mammals and in flies of certain species, there are sperm ejaculated that are not going to be functional.
AUDIENCE: But why would they be different from each other if they're sharing all the same [INAUDIBLE]?
MARIANA WOLFNER: So it can be things like when-- so I'm going to now do flies because I know flies better than mammals. But in flies, the sperm develop as a 64 cell cyst where they're all initially connected together. And then at the very end of spermatogenesis, the membranes tighten around each sperm and push the cytoplasm down the tail and off. So if the membranes don't individualize correctly around the sperm, you can end up with a sperm with two heads or two tails, which can be a problem.
So that's one particular case. If there's any structural problems in the nuclei or the acrosome, then even though they share the cytoplasm, they still might not be the same. And I think it's those types of things. Otherwise, they do share the same proteins, and so they should be pretty much the same. So both of you are right. I mean, you'd think they were the same. But at the same time, there are sperm in there that are not the same. OK. Other questions? Yeah.
AUDIENCE: So people had to look at [INAUDIBLE]
MARIANA WOLFNER: So there have been experiments done where oviductal fluid at different stages in the cycle have been used. And I don't know the answers to that. All I remember is that at the time of ovulation, the oviductal fluid has chemoattractants, but I don't know whether they change. I just don't know that. That's something's, again, I need to check that. OK? Other questions?
All right. So all of this makes sense, right? I think we just need to get it down to the timing of these molecules and the receptors for these molecules. But from a completely different-- from left field basically came a completely different way to look at sperm chemotaxis, and this came out about five years ago.
So we'll get back to sperm in a moment. But your nose has a number of odorant receptors, which are G protein-coupled receptors in the membranes of your olfactory neurons that sense odorants by binding to them. And each one will recognize a particular suite of utterance with a particular structure. Animals have many odorant receptor genes because they need to be able to recognize many odorants.
So for example, mice have about 500 to 1,000 different odorant receptors. In fact, a few years ago, the Nobel Prize to Axel and Buck went for the discovery of odorant receptors for this class of G protein-coupled receptors. And people, of course, study these odorant receptors in the nose and in terms of the sense of smell and how it's all hooked up and does a particular neuron express more than one or not and where do they project in the brain.
But surprisingly, when people started looking at odorant receptors, they discovered that sometimes certain odorant receptor genes are expressed in the testis, which is very strange. So here is a reverse-- whoops. Here's a reverse transcriptase PCR of testis RNA with primers for a known gene expressed in the testis. This is just a housekeeping gene, just a control.
And then primers for these particular human odorant receptors tested on human testis RNA, and this is just genomic DNA as a control to prove that the primers are working. And what you can see is shockingly two odorant receptors shown here is called OR17-4, which is one we're going to talk about, and OR17-2 are expressed in the testis. They make RNAs in the testis.
So most of the odorant receptor labs said, OK, they're expressed in the testis. And they went back to studying the nose. But the lab of Hanns Hatt decided to focus on the testis and to ask what these things could possibly be doing. So I will tell you about the data that they have. But as I tell you about, and its published-- the papers as beautiful, as you'll see-- bear in mind that this is somewhat controversial.
Nobody understands exactly why they're finding what they're finding, but nobody disputes what they're finding. OK? They're seeing this very clearly. So they said, well, maybe there are odorant receptors expressed in the testes. Maybe these odorant receptors are expressed on sperm, and maybe the sperm are, in a sense, smelling their way to the egg. OK?
So they decided to test that. So having learned that these two-- and I should say in mice where there are about 1,000 odorant receptors, studies have shown that about 50 of these 1,000 genes are expressed in the sperm. So it's about 5%. It's not negligible.
So the lab of Hanns Hatt decided to see what these things might be doing in the sperm. And the first thing they had to know is, what is the odorant that these things recognize? Because maybe they're non-functional, or maybe we can't identify it. Alternatively, maybe it is an odorant that you might expect sperm to be associated with.
So what they did is they took these human odorant receptor genes, and they focused, again, on 17.4. So it's abbreviated like this-- human odorant receptor 17-4. And they expressed it in tissue culture cells. And now most G protein-coupled receptors work by one of a few downstream pathways.
And odorant receptors in particular often work by acyclicing a P-dependent system that raises the calcium level in the cell. And so what Hatt decided to do was to use a standard assay for what the ligand of an odorant receptor was, which is to express the odorant receptor in tissue culture cells and have those cells contain a molecule that would fluoresce if calcium levels went up in the cells.
So I'll take you through what they did. And just bear with me through this slide. We're going to focus here on this lower panel here. So these round things are tissue culture cells, and what they did is they treated those cells with DNA that expressed OR 17.4 The way they did this, not every cell picked up the DNA. So only some of them did.
The cells contained a chemical that would fluoresce when calcium was present. And this is just a control showing that when they treated them with ATP, which would trigger a rise in calcium in those cells, they would indeed fluoresce. So that just proves that they contain this probe.
So normally, you can see that the cells wouldn't fluoresce. But what these investigators did was they took a mixture of small chemical compounds. The mixture's named Henkel 100. I have no idea why. It's just a mixture of small potential odorants of all different structures, and there are 100 of them in the mixture. And they threw it on those tissue culture cells.
And they asked, do any of these cells show a response to the mixture? And to their amazement, cells did show a response, and that response went away when the mixture was removed. So it looked like there was some odorant in this Henkel 100 system that was responding-- that the odorant receptor they had put into the cells was responding to.
Now remember, this is a receptor that's in the nose as well as the testis. So they're looking for the odor that it's sensing, OK? You with me so far? All righty. So then there are 100 things in that. So they just took all 100 things, and they tested them in groups and then broke down the groups. And ultimately, they found that this molecule cyclamal, which was in that Henkel 100, could trigger a response in cells that had the odorant receptor.
And as you can see, I forgot to tell you that obviously these cells that didn't have the receptor are a control, right? So they have this chemical mix on them, and they're not doing anything. So clearly, we're seeing-- cyclamal is doing something to the cells that have the odorant receptor.
They then took a series of compounds that resembled cyclamal in various ways. And they tested them one by one on these cells to identify-- to see whether any of those molecules actually triggered the receptor even better, to find the perfect ligand.
And the perfect ligand turned out to be this molecule called bourgeonal. And bourgeonal is a molecule that most of us perceive as the odor of the flower lily of the valley. OK, so when you smell lily of the valley, it's your HR017-4 that is firing, among other things.
Interestingly, another molecule called undecanal, which is here, which supposedly smells like green leaves or a green plant, could block the-- if you have someone smell bourgeonal, they say, great, that's lily of the valley. If you add undecanal, it competes, and you don't smell the bourgeonal as well. So it seems to be competing with activation of this receptor. OK?
So this is all very cool, and this is interesting also for the nose. But these guys are interested in sperm. So if sperm are expressing this odorant receptor, and they are, it appears to be located on their flagellum. And if you knew that bourgeonal was the ligand for this odorant receptor, what experiment might you do if you were [INAUDIBLE], who's the first author of this paper?
AUDIENCE: [INAUDIBLE]
MARIANA WOLFNER: That's one thing you would do-- look and see where bourgeonal is located. I'll tell you that's a very important experiment to do-- to look and see whether mammals have bourgeonal anywhere. So far, there is no answer to that question. OK?
But why would you look for where bourgeonal is located? What would even make you look for it? I just told you it's in lily of the valley. So that works for the nose. But why were you going to look for it in people?
AUDIENCE: But what if there's a receptor for bourgeonal on a sperm? Then if bourgeonal's being expressed anywhere in the female reproductive tract, then likely [INAUDIBLE]
MARIANA WOLFNER: So what you're proposing then is if bourgeonal were expressed somewhere in the female reproductive tract, it might be a chemoattractant for the sperm if the sperm are expressing this receptor.
AUDIENCE: Or at the very least, it might increase the movement of the flagellum.
MARIANA WOLFNER: OK. So that's right. So let's do a simpler experiment. Let's just see if bourgeonal can attract sperm, OK? So that's what [INAUDIBLE] et all did. And to everybody's shock, it can. So here are three different concentrations of bourgeonal-- 10 to the minus 6 molar minus seventh molar minus 8.
And so by minus 8, the sperm are probably not really chemotaxing towards the pipette. But by minus 7, they certainly are. OK? And, of course, by minus 6 they are, too. So bourgeonal appears to actually be a chemoattractant for sperm.
Interestingly, if you add undecanal, which the experiments had shown interfered with the ability of the receptor to respond to bourgeonal, now the sperm don't chemotax as well. So it appears that sperm are responding to something that this odorant receptor will sense and something that interferes with the odorant receptor is able to interfere with that. OK?
So right now, it looks like there is an odorant receptor on sperm that at least can potentially function to detect this lily of the valley odor. We see there's is a concentration response. The sperm swim to the odor. A competitor competes for the odor. There's also some data that show that calcium levels increase in the sperm, as you were suggesting, upon exposure to bourgeonal and that cyclic AMP is required for this, as you would expect for a GPCR.
So is bourgeonal important? Is any of this important in sperm chemotaxis to the egg? The answer is we don't know. We don't know if follicular fluid contains bourgeonal. That's just not known. Or if it contains something like bourgeonal that wasn't in their mixture that triggers this. We don't know.
We don't know if this receptor is actually doing the sensing, although the biochemical data certainly suggests that it would be. There's a paper I've put on our website by Leslie [? Vasile, ?] a review article, that raises the intriguing question that you could do an experiment in people this way.
If you had a whole bunch of volunteers come and you test their sense of smell, which is something her lab is doing for studies of another receptor, if you were to find someone-- a man-- who couldn't smell lily of the valley very well, maybe his sperm don't have this receptor on it. And then you can see whether he's fully fertile or not. And that would be an interesting question. And we have no clue, but maybe this will be done.
AUDIENCE: Can't you knock it out and do the same thing?
MARIANA WOLFNER: You could knock it out. But, well, you can't. No, because you can't do that in humans. So I'm-- but wait. But hold your horses, because I'm going to tell you about this. So I'm-- well, I'm not. Leslie [? Vasile ?] was proposing a way to screen for a knockout mutant in humans by looking for a man who can't smell this.
But, in fact, mice have a different receptor. It's called OR 23 that's expressed in their sperm that senses a chemical called [? lirel, ?] and I don't know much about [? lirel ?] except that it's another floral odor. So you could do something like that.
There's also the question of whether something like-- if this hypothesis is correct-- and I will again emphasize that although you see the data and they look good, it is somewhat controversial. But if it is correct, then you could imagine that something like undecanal or an analog to it could actually be a useful contraceptive because it might help the sperm not find their way if you could get it to the right place.
So these are the kinds of wide open questions in the sperm chemotaxis field in mammals. It's fascinating, and the papers are really pretty. But we don't quite understand yet what's going on. It's not quite as clean as the speract and resact where I could tell you that binds here, and it triggers this, and then they change their movement.
It's still a little less clear in mammals, but there are some very fascinating receptors and ligands coming up. So this is probably a good time for us to take the break. We've gotten the sperm to the egg. And in the second half of class, we will get it very quickly to the vitelline envelope. And then we will talk about the vitelline envelope. OK. If you didn't turn in a--
[SIDE CONVERSATION]
Welcome back. Are there any questions about the first part of the lecture? OK, let's continue on. So we have now gotten the sperm to the egg. And we have to get it through that outer layer of cumulus cells and then bound to and then through the zona pellucida or vitelline envelope. And you'll hear me using those words interchangeably.
And then we need it to acrosome react and then reach the sperm plasma membrane so it can fuse. And I'll just remind you then the egg is about 80 microns in diameter. Then there's the zona pellucidas of a few microns thick, and then the cumulus oophorus, which is made of about 3,000 former cumulus granulosa cells, now just called cumulus cells, and an extracellular matrix of hyaluronic acid holding them together.
So the first thing that the sperm has to do is get through this layer of cells stuck together and penetrate the cumulus. And it does so very straightforwardly using an enzyme called hyaluronidase, which basically digests the hyaluronic acid and lets it get right through in between those cells.
I'll tell you how the hyaluronic acid was discovered because some of the techniques will be similar to some of the techniques that we're going to see used when we talk about the vitelline envelope. And actually it's similar to some of the techniques that we saw used for marine invertebrates.
So at first, before anyone knew what any of the molecules were that the sperm used to reach and bind and penetrate the egg, they did the same kind of experiment that Vacquier did with the sea urchin sperm. Where should the enzyme be that helps the sperm penetrate the cumulus? Should it be on the outside of the plasma membrane? Should it be in the acrosome? Should it be inside the sperm?
AUDIENCE: Outside.
MARIANA WOLFNER: Outside, right? Right, exactly, outside. So what they did is they injected sperm, whole sperm, into mice and raised monoclonal antibodies against molecules that were exposed on the surface of the sperm. And then they asked whether adding any of those antibodies to sperm with eggs inhibited anything about the way the sperm interacted with eggs.
And when they added an antibody against a protein that they then called PH-20-- probably that was the name of the antibody, I would guess-- it blocked penetration of the cumulus by the sperm. So what that meant was that when you add eggs with cumuluses around them, sperm and anti-PH-20, the anti-PH-20 must be binding to the PH-20 on the surface of the sperm and, therefore, preventing its activity or its ability to access its target or something. But obviously PH-20 was important in the penetration of the cumulus layer. OK?
Now this was a very important experiment. It led to a very important conclusion. But I will caution you that these experiments with antibodies have a flaw. Because the antibody will stick to the antigen, but its antibody's a big thing, and it could be blocking other things that were near that antigen.
So just keep that in mind whenever people do antibody competition experiments that probably they're interfering with what they think they are. But if that one experiment gives you results that are completely different from any other experiment, then you should start getting a little suspicious, OK? But in this case, it didn't, because then this is in mouse. If you're a geneticist and you don't believe antibody experiments, what would you do?
AUDIENCE: Knock it out.
MARIANA WOLFNER: How could you test that? Knock it out. Make a mouse knockout who doesn't make PH-20. And when you look at the knockout, you would expect if ph20 is important, that the sperm of that mouse would not penetrate the cumulus very well, and that's exactly what they saw. So those two pieces of data suggest that PH-20 is important in the penetration of the cumulus, OK? That's really pretty.
There's one small, as we say in my lab, fly in the ointment, which is that these mice were fertile. And this has been a problem in the mammalian fertilization field a lot. And when I was thinking about this lecture, I started thinking about, how come we-- are mammals really that complicated? How come it's so simple in marine invertebrates? And then I realized, of course, they haven't made mutants in marine invertebrates yet.
So everything I've told you in marine invertebrates has been based on very beautiful biochemistry and interspecies experiments. But beautiful biochemistry in mouse also gave very clean results. And then when they made mutants, some of the proteins that looked really important turned out not to be essential. And so we may see the same thing happen in the marine systems. Or they really may be simpler, and the results I told you may bear up when they knock out the proteins.
The explanation that people give-- well, tell me what-- if you were working on PH-20 and you had done this antibody experiment and it worked as beautifully as it did and you knew there was hyaluronic acid holding those cumulus cells together and you sequenced PH-20 and found it was a hyaluronidase, which should dissolve hyaluronic acid, and you did a knockout and it didn't penetrate the cumulus, but then it turned out that the knockout was fertile, how would you justify this, if you were writing the paper? What might you say?
AUDIENCE: There are other general proteases.
MARIANA WOLFNER: There are other general enzymes, other hyaluronidases, other enzymes with similar activity that are there, too. And that's exactly what they said. So in other words, this is a very important one, and if you inhibit it, it doesn't work. But clearly in vivo, there are other enzymes that assisted or can back it up. And that's what they said.
So in terms of cumulus penetration, all I'm going to really say at this point is that a hyaluronidase is involved and important. It may not be the whole story, but it's certainly important. And one other thing, which is that if you look at the sperm that are penetrating the cumulus and getting through-- so here's your egg. Here's the zona pellucida or the vitelline envelope. And here's the cumulus layer.
If you look at the structure of the sperm that gets stuck in the cumulus layer versus the ones that make it through, 90% of the sperm that make it through have not acrosome reacted. But much less than 90% of the sperm that are stuck here have not acrosome reacts. In other words, the cumulus layer, apart from being around the egg and supporting it, appears to also be selecting the sperm and only letting sperm through that haven't already acrosome reacted.
And that's important because you're going to need sperm that haven't yet acrosome reacted to bind to and penetrate the vitelline envelope. So that cumulus appears to be this selective thing. Now I think Professor Travis mentioned to you, and I will certainly come back to it later today, that the acrosome reaction is not quite as all or none in mammals as it is in marine invertebrates.
Did he mentioned that, how it sort of partially reacts? So what I'm talking about, though, is the visible acrosome reaction, which is the last stage of it where it looks really different. And so when I said that non-acrosome reacted sperm make it through here, I mean the ones that have not reached that last stage. OK, but they may be letting it out and in a little bit. This was all by microscope.
OK, so the sperm have reached the zona pellucida That was very easy. Now let's talk about how it binds and how it penetrates. And this is the part that is constantly in flux at this point and remains somewhat controversial. And so I'm going to present to you the current model, and this is quite current. This is based on recent papers, but also on talks at a meeting on fertilization from last July. So it's pretty, pretty current.
The first thing we'll talk about is the zona pellucida itself and its composition. This is totally not controversial. If you purify eggs and you strip off the zona pellucida and you solubilize it and run it on a gel, you find that it consists of either three or four sulfated glycoproteins. And these proteins have been given the name zona pellucida protein 1, 2, 3, and 4. Or, in the new naming system, they were given letters, something like that. These can vary.
And so you'll see them in the literature, and in the articles I posted, you'll see both sets of names so I'm going to put them up that way. So there's three or four ZP proteins. Mice have three, these first three-- A, B, and C. Humans, rats, and many other animals, [? enopis, ?] and so on, have four.
These proteins are transcribed during oogensis by the oocyte itself. So this is different from Drosophila, for example, where we saw the follicle cells make the vitelline membrane. Here it's the oocyte transcribing the proteins, and the oocyte-- so this is the oocyte here. And the oocyte secretes the protein. Protein has a transmembrane domain, sticks out of the oocyte, and that's how the proteins are made.
The proteins are initially tethered into the oocyte membrane. But they then start to form intermolecular interactions. And at some point, they are cleaved by an extracellular protease that releases them from their membrane tether. So they can spring out from the plasma membrane of the egg because now they're crosslinked together, and so they're not going to just float away.
And so you get this vitelline envelope moving out from the plasma membrane of the egg. But it was assembled initially as its proteins were made and tethered in the plasma membrane. OK? Any questions on this? OK.
We know a little bit about the regulation. There's a basic helix loops-- helix transcription factor called Fig 1 Alpha. Those of you who are mouse people may have heard of this molecule, which regulates the expression of these genes. And I believe their promoters are used pretty commonly in mouse research to drive things in oocyte. So for example, some of the experiments we talked about where Jaffe and colleagues drove G protein agonists or antagonists and oocytes. They were using these promoters.
So they're synthesized. They're assembled. And then they're released. And let's see what they do. So, of course, if you're working on mouse, you can make mutants. And the lab of Jurrien Dean at NIH has done all of what I'm going to tell you and has made a very comprehensive study of this. So they've made mice that have mutations in either ZP1 or ZPB, ZP2 or ZPA, and ZP3 or ZPC.
And they looked at the oocytes produced by those mice. So first, this is just to show you the follicles of the mice. And you should just notice that they all have oocytes in them, and the oocytes look fine. They have nuclei, and there are granulosa cells around them. Everything looks just fine. Even at the pre-ovulatory stage, here are the cells that will become the cumulus layer. There's the oocyte. And these guys also have those cells.
But if you look at the final oocytes that are produced, here's wild type. So here is the egg, and this is the zona pellucida. The cumulus cells have been removed by the investigators treating these things with hyaluronidase to get rid of the cumulus layer. And you can see the zona pellucida is this nice thick layer, kind of clear, around the egg.
But look at what happens in the ZP3 mutant. There is absolutely no obvious zona pellucida at all. And this mutant is, in fact, totally sterile. OK? So ZP3 not only is in the zona pellucida, but it is essential to make a zona pellucida to have ZP3.
ZP2-- sometimes you'll get an egg with kind of a little wimpy, fragile zona pellucida. So it's not totally gone. It's in the ZP3 mutant. Although in these oocytes, it looks pretty much gone. So ZP2 is also very important for the structure of the zona pellucida.
ZP1, on the other hand, is a little different. You can see that in the absence of ZP1, there is a zona pellucida around these oocytes, but it's kind of strange looking. It's also very fragile. It's usually kind of stretched out like this. You can even see it's stained here, sticking out rather than being nice and thick and tight.
These mice, however, are fertile to some extent-- not as fertile as wild type. So of the three ZP proteins, ZP3 is the most essential to make a zona, and ZP1 is probably the least essential, although it's still important. OK. Any questions about this? Pretty straightforward. OK.
But knowing this led to a model for the structure of the zona pellucida. This comes from a paper by Jurrien Dean that I've put on our website. So he proposes that the three proteins-- here they are, 1, 2, and 3-- that a zona pellucida absolutely has to have either ZP1-3 dimers or ZP2-3 dimers. They have to be there.
If you don't have both of these because you don't have ZP3, you're just not going to be able to make the structured meshwork that is bound together that you need to have a zona pellucida. So this is what he's proposing is happening in the ZP3 null. The ZP1 and ZP2 really don't have anything to link them together.
In the ZP2 null, you still can get some kind of structure with ZP3 and ZP1. And in the ZP1 null, you can get a pretty good structure without the ZP1. But he's proposing that the proper ZP3-- sorry, zona pellucida has all three of them together, where ZP3 is really holding the other two together in a structure.
So this is what we know about the mouse zona pellucida structure. We don't know as much about the zona pellucida structures of the other animals because we don't have mutants in all the ZP proteins in the other animals. But it's probably something analogous to this. So the question is, how do sperm bind to the zona? And that's where I'd like to go next.
So the original assays that were done were a type of competition assay analogous to the antibody assay that I told you about a moment ago. So the idea was the following. You take an egg. You strip off the cumulus layer with hyaluronidase. And you have an egg that's surrounded by a zona pellucida. You can see it here with this thick region.
You then toss the egg into a plate with some sperm. This is an unfertilized egg. And the sperm will now bind to the zona pellucida. OK? These are obviously capacitated sperm, and they can bind to the zona. So you can see a whole ton of sperm coming off the egg.
Now you can tell immediately that this is not totally physiological because there are probably more sperm stuck to this one egg than would normally be around the egg in vivo in the female. But for this assay, that's fine. You can see that this is clearly the case.
Initially, when you throw the sperm on the egg like this, you get a very loose binding. It's called attachment, not binding. And it is very non-specific. So for example, there's no species specificity. There's no stage specificity in the loose attachment. They'll attach to an unfertilized egg and to a two cell embryo, which should have a fertilization envelope, not a vitelline envelope or zona pellucida.
And if you take those eggs with sperm attached to them and pipette them, the sperm will just fall off. But if you wait a little bit of time, you get a more specific interaction between the sperm and the eggs, which is called binding. Binding is stronger. If you pipette the sperm and eggs up and down, then the sperm don't come off the egg.
It is more species specific. It's not completely species specific, but it's more species specific. I'll show you later a picture of a mouse egg with human sperm, and there's no binding at all, OK? So it seems to be more species specific.
It's stage specific. So this picture is actually a binding, not attachment. And the reason I can say it's binding is not that it looks any different, but that this is an unfertilized egg. It's got a million sperm around it. Here is a two cell embryo, no sperm. So it's highly stage specific. It will only bind to the vitelline envelope of an unfertilized egg.
And finally, it's region specific in the sense that it's the head of the sperm that binds, and it's, in fact, the side of the head of the sperm that binds, just like the way the sperm, as you know, comes to lie against the vitelline envelope with the side of its head, tip side. So first, you see attachment, very loose. Then you see a stronger binding.
Then you see acrosome reaction, again, by the microscope. So this is not this exchange thing, but you just see that the whole acrosome is gone. And then the sperm still stays associated with the egg. So whatever caused these unacrosome reactive sperm to bind to the eggs, something else must be holding them on to the egg once they have acrosome reacted. OK?
So the question is then, what does the sperm bind to? What causes the sperm to bind? So the initial assays were competition assays. And the idea was you take your sperm. You take your eggs. You mix them together. You should get this. Right?
Now you start adding things to the reaction, and you see if you ever add something that interferes with the ability of the sperm to bind to the egg. OK? If it does, then that something must have bound to whatever on the sperm was binding to the egg and prevented it from being exposed and being able to bind the egg or bound to whatever on the egg was binding the sperm and preventing it from being exposed and binding to the sperm. That OK?
All right. So what you want-- now I'll tell you, this is a picture from a different experiment. But it shows what you want is if you're looking for a particular competitor, instead of seeing this, you'll see this. You'll see no binding. OK?
So a long time ago in the 1980s, late '80s, the lab of Paul Wasserman did a set of absolutely beautiful classic experiments that involved competition assays. These experiments are still in textbooks. The current edition of the Developmental Biotech still has these data. They are beautiful. But we now don't 100% understand exactly what they mean.
So what they did is they wanted to know what on the egg binds to the sperm and what on the sperm binds to the egg. OK? So it's easier to ask what on the egg's vitelline envelope binds to the sperm because there are only three things in the vitelline envelope-- ZP1, ZP2, and ZP3.
So what Wasserman did-- Wasserman and his lab did-- is they purified vitelline envelopes. They dissociated the proteins, and they added the proteins to a mixture of unfertilized eggs and sperm. OK? And so they asked, does exogenously added ZP protein compete with sperm binding so you would get this, or not? So you would get this. OK, and they got this. All right?
So if you add soluble ZP protein, you compete with the binding of sperm to eggs. So what that says is that the sperm seemed to normally be recognizing and binding to something in the ZP proteins on the zona pellucida. OK? All right. So if you have a mixture of three proteins that inhibits the binding of sperm to eggs and you're a biochemist, what would you do next?
AUDIENCE: Test each of those proteins.
MARIANA WOLFNER: You might test each protein, right? You might separate them and test 1, 2, and 3. So that's exactly what Wasserman did and his colleagues did. And this is their experiment as taken from a textbook, and here's what they found.
If you add ZP1 to eggs and sperm, you can add it until you can't add anymore, and the sperm keep binding. There's no inhibition of sperm binding. So by this definition, ZP1 is not something that binds to the sperm. Because if you add it, it's just like you added albumin or something. It isn't interfering. OK, so it couldn't be binding to the sperm. OK?
Same experiment with ZP2. No interference. So it's not ZP2. But when they did ZP3, ZP3 inhibited the binding, so they saw something-- whoops-- that looked like this with ZP3. So that suggested that soluble ZP3 was capable of interfering with the binding of sperm to the zona pellucida.
And the simplest explanation for that is that the soluble ZP3 was binding to the part of the sperm that bound to the egg. And, therefore, that receptor in the sperm was no longer available to bind to the egg. OK? All right. So this is an absolutely gorgeous experiment.
And it was one that was taught all the time and everyone would quote as the most beautiful experiment. And it's still reproducible. You can do this experiment today. You will get the same result. The question is, as you'll see, we don't understand 100% anymore why they got-- what this result is telling us biochemically.
But let's proceed with their experiment a tiny bit more. I mentioned to you before that all the zona pellucida proteins were sulfated glycoproteins. So they are proteins with sugar attached. So Wasserman and colleagues decided to ask whether it was the sugar or the protein that was binding the sperm.
And you can see here they chemically treated ZP3 to remove the carbohydrates. And what this shows is that when you remove the carbohydrates, it doesn't interfere so much with sperm-egg binding. What this picture doesn't show-- but a different figure from one of their papers would-- is that if you take the carbohydrates, the purified carbohydrates from ZP3, and add them, they would behave like this.
So Wasserman's group concluded at the end of these studies that sperm bind to the zona pellucida by binding to ZP3 and specifically by binding to the carbohydrates on ZP3. They had additional data. They and others had additional data that were consistent with this.
For example, if you purify ZP3 and label it up either radioactivity or have an antibody to it and throw it into some sperm, you can see it's binding beautifully to the head of the sperm exactly in the place that the sperm comes to lie on the vitelline envelope. And this is the same thing only with a radioactive label. So everything is pointing to ZP3 being the sperm receptor.
In addition, there was evidence from several labs that ZP3 also could trigger the acrosome reaction, so that this sperm-- sperm like this that had been incubated with ZP3-- would ultimately undergo the acrosome reaction. OK. So it suggested the following models.
Sperm bind to the egg by binding specifically to ZP3 in this case-- and I'll come back to this in a minute-- and probably to the sugars in ZP3. And when the egg is fertilized, something will happen to destroy or remove ZP3 in this case or other ZP proteins, and that prevents further sperm from binding.
Or alternatively, if sperm bind to the sugars, which is what the data really showed, then all you need is to release glycosidase to destroy the sugars, which is released from the cortical granules. Get rid of the sugars, and no more sperm can bind. So this is the model in textbooks. It's a beautiful model.
It explains why sperm bind with a certain specificity. All you need to say is that the sugars that are on a mouse ZP3 are different from the sugars that are on a human ZP3, and you've got your species specificity. And it explains the stage specificity. If you get rid of the sugars or get rid of the proteins, you're then going to fail to bind sperm to an embryo. So this is really beautiful.
I will just mention why this particular figure from the Dean review has [? ors ?] here, which was a mystifying thing, which is that although ZP3 clearly played the role in these type of assays as the sperm binding protein in mice and its counterpart ZPC in people is also thought to play the same role in the same kind of an assay, in other mammals, it's different ZP protein.
So in rabbits, it's ZPB, which is ZP1. In pigs and cows, it's ZPs B and C, which is 1 and 3. You don't need to know the individual ones. I'm just giving you examples. You do need to know mouse, but not the other guys. If you ever want to know, just send me an email. I've got it written down. Xenopus is ZP A and C, which is 2 and 3. OK?
So that was a little confusing. But there's a lot of variation between species. In other organism, may be different AP proteins evolve different functions than these. It wasn't terribly upsetting. And so instead, the model became known as the single zona protein 1-- ZP 3 and [? sum, ?] ZP 2 and [? sum-- ?] but just 1 or the single zona glycan model.
And this model has held up for many, many, many years. Additional experiments towards it were, for example, you could express ZP3 in tissue culture, make a mutant that couldn't be glycosylated, and then throw sperm in with the tissue culture cells. They would bind to the cells with the normal ZP3 and not with the mutant. I mean, just everything was falling into place.
And it was really fun to teach. But it's fun to teach in a different way now. Because five years ago, the lab of Jurrien Dean the one that had done the mutants, decided that now it's 2003, we can do mass spectrometry, which they couldn't do in 1987 or whenever they did these first experiments. Let's look for these sugars, these O-linked sugars that are supposed to bind the sperm.
And this is a lab with a lot of experience working with mouse ZPs. They know how to handle the ZP proteins really well. And they purified them, and they did mass spectrometry, and they didn't find O-linked sugars at the right place. So these sugars that-- in tissue culture cells, the experiment said that these particular sugars were critical. They're not there in vivo. So that was a bit of a problem for the glycan model.
A second problem was what I rattled off and told you not to write down-- was that surprisingly different ZP proteins seemed to be the sperm receptor in different mammalian species. Now you can rationalize this. But it was still a little bit unusual that a completely different protein would carry out the same function when the other one was still there. So that was a little odd.
Third, the lab of Jurrien Dean also began to ask about the species specificity of binding to the ZP proteins. Now remember, again, this is not a marine mammal. So species specificity is not critical. But I told you before that mouse oocytes incubated with mouse sperm. They bind. But if you incubate them with human sperm, they don't bind. And so there is some kind of species specificity.
So they did an experiment that is extremely beautiful and elegant and really, really hard to do. But they did it completely, and they have results that are clean and that were sort of the death of this model, I'm afraid. What they did is they took mice, and they made mutant mice that they knocked out the mouse ZP protein and replaced it with the human ZP protein of the same flavor.
Like, they replaced mouse ZPC or ZP3 with human ZPC or ZP3. And here's a picture of oocytes that just prove that they did-- it's a very difficult experiment because they have to make a knockout and then an ectopic expression in the same mouse. And then they actually have ultimately, although I won't show you this, made a mouse in which all three mouse proteins are knocked out and replaced with all three human proteins. It's a lot of work.
This is just to show you that when they say they make a certain kind of mouse, they do. So the blue antibody is against mouse ZP1. The red is against mouse ZP2. The green is against mouse ZP3. The yellow is against human ZP2, and the pink is against human ZP3. So this is normal mouse oocytes.
This is a mouse oocyte in which human ZP2 is expressed, but mouse ZP2 is not. And here's one that has human ZP2 and 3 and not mouse ZP2 and 3. It's a mouse oocyte but with these two human proteins and mouse ZP1.
And they then tested these oocytes to see whose sperm they bound. And what they found was all of these guys bound mouse sperm. None of them bound human sperm. Now what kind of control would you do to make sure that-- I mean, it could be a trivial reason why none of them bound human sperm.
Right, those sperm could be like defective or duds, OK? And you can't do an experiment where you take human oocytes and just use them to test if human sperm are OK. This would not be ethical. So what you can do instead is use sperm from men who have fathered children. You know that their sperm are capable of binding to a human egg. And so that's what they did.
So they're using sperm from donors that they know would normally be able to bind to a human oocyte. the mouse sperm, you can do whatever you want to test. And so here we see that, of course, mouse binds to mouse, and human doesn't bind to mouse, and that's fine.
But here's mouse with human ZP2. No human binding, but mouse binding. Mouse with human ZP3. And remember I said ZP3 seems to have the same role in humans and mouse. But it binds mouse, not human. And even with both of them, it binds mouse, not human. And even with all three of them, it binds mouse, not human.
So when they saw that result, which admittedly was a shock to them and to everyone else in the field, it clearly meant that the model where it's a single protein that determines the binding can't possibly be right. It has to be more complicated than that.
Finally, there was-- so I'm going to come later in the lecture or next time to tell you about the receptors for the zona pellucida. And I'll just say right now that it's a highly controversial area as well. But there was one receptor which had been identified-- a putative receptor-- which had been identified to bind to the sugars of ZP3 in mouse. So that would fit this model again that it's something special about ZP3 and its sugars.
And just at the same time, the lab of Barry [? Shure ?] which had been studying this thing, made a mutant, and the males were fertile. So that also argued against this model that it's a single zona protein and a single receptor. So what does this all mean?
So Dean and his lab thought hard about the models, and they came up with a new model. And this is the current model. This model has only been out for three years or so, and it is still being tested and played with. And it's called the supramolecular structure of the zona matrix model or the supramolecular structure model.
And the idea is the following. The sperm-- the idea is the following. The sperm is not binding to a single protein type or to the sugars of a single protein type. But rather, this zona pellucida has a structure that's formed by ZP1, ZP2, ZP3 interacting with each other, like I showed you before.
And that structure itself has a shape. It's like a large complex with a shape. And the sperm is recognizing some feature of this shape. So in mouse, the shape may be like this, and the sperm sees this thing, and that's what it likes to bind to. It's not binding to any one protein. It's binding to a structure made of three types of protein.
This is the latest model. It's in some ways satisfying because it explains some things like why mutants and individual proteins don't seem to change the specificity at all. It's unsatisfying at the moment because we have no idea what this structure is. And so it makes it a little hard to figure out how to find receptors, and it makes it a little hard to figure out how to probe the structure.
This is not a criticism of the model. It's just my way of explaining to you why I can't explain much more than that. We can try to explain the competition experiments. If a competitor binds to a region of this structure, it could block that region, just like an antibody might block if it binds to its antigen.
And that could then block sperm-egg binding. So it might not be competing by binding something on the sperm surface. It might block the structure. Or it might bind to the sperm surface for some reason and block the region of the sperm that binds to the structure.
But the model is that you need all three proteins together to make a structure that the sperm can recognize. You can work species specificity into this just fine if you want by proposing that the structure forms in a species-specific way.
And it may have to be with its own proteins in its own oocyte with its own glycosylation machinery to make it completely species specific, which would explain why human sperm don't bind to mouse oocytes expressing human ZP1, ZP2, ZP3. OK? Are there any questions I'm going to take a stop here for a second because I know this is pretty shocking or something. Are there any questions?
AUDIENCE: Why doesn't the 3D structure translate?
MARIANA WOLFNER: Why doesn't the 3D structure translate? I can't answer that question. It's a very valid question. Why doesn't it translate from species to species?
I can't answer that question because we don't yet know what the structure is, and because it could translate, but it apparently doesn't translate. And so that was why I was waving my hands and saying that when you assemble it in its oocyte, for example, maybe the enzymes that cleave it cleave at different places in mouse or human.
The glycosylation pattern may be different. It's not very different, but it could be different in mouse and human. I'm going to come back to the cleavage in a moment and give you an example of that. But it's got to be something like that. Mm-hmm?
AUDIENCE: So when all three mouse proteins were mixed human proteins-- what you just said, it seemed like the complex would be different, yet mouse sperm still bind.
MARIANA WOLFNER: Right.
AUDIENCE: Why is that?
MARIANA WOLFNER: I don't know why it is. That's what they see. So the question is, if you had all three of these were human proteins but made in a mouse oocyte, maybe the complex would have a different shape, and yet it binds to mouse sperm and not human sperm. And so far, no one has speculated on why because we really don't know what features of this complex are being bound.
It could be the way in which the proteins are put together is something that's not-- I'm going to make this up, but you need two ZP3s for every ZP1. That may be the case in mouse. They may assemble it in a certain way that they wouldn't in human. So I don't know. We don't know that yet.
This is still something that-- I mean, it's a 600 level course. If this was a 300 level course, I'd probably stop with the textbook picture. But I'm telling you where it is now, and we don't know. It's like one of these stay tuned. Maybe next week a paper will come out-- I'll bring it in if it does-- but that'll explain it. So, yeah, but, actually, again, as of last summer's meeting, it was still like this.
AUDIENCE: So the sugars-- maybe it's the way they're glycosylated. That's probably species specific.
MARIANA WOLFNER: There is some-- yeah. Some people have that hypothesis that the glycosylation may be species specific. There's some data to say that it isn't. But those data are on amount of glycosylation. They haven't looked at the specific glycosylation yet.
So that's right. That could do it. If the glycosidases are different, you could indeed have a sugar-- clearly, sugars are somehow important, and you could be putting mouse sugars on the human protein, and that may be more important to the mouse sperm in terms of binding. Yeah. That's right. Other questions?
So it is a bit amorphous, but I actually find it fascinating that it's going to be a large 3D structure that's recognized rather than one individual protein. It's also interesting, for those of you who like to think about evolution, to think about that in an evolutionary sense. Because at least ZP2 and ZP3 contain sequences that are evolving rapidly. And so maybe they affect the shape of the vitelline envelope.
So after fertilization, though, we've got to make this structure not bind sperm, right? Because we need to block polyspermy. And the experiments that Jurrien Dean's lab did did suggest a mechanism for this. So I'll just remind you that what they-- I'm showing you here just the controls and-- sorry, just the experiment without the controls that mouse sperm bound to all of these guys even with human proteins, whereas human sperm didn't.
But when they looked at the two cell embryos that should have a fertilization envelope that should not bind sperm, what they found was when they had human ZP2 instead of mouse ZP2, these embryos' zona pellucidas or fertilization envelopes still bound sperm. So this has human ZP2, and this has 2 and 3.
So it looks like under normal circumstances, in order to get rid of the sperm binding buy mouse sperm, you need to have mouse ZP2. So Dean and his group looked at what was different about mouse ZP2 and human ZP2, and they found that there's a region of mouse ZP2 that can be cleaved proteolytically. And although human ZP2 can also be cleaved proteolytically, it isn't proteolytically cleaved at this place.
And so they have come up with the following model. There's this supramolecular structure made of the three ZP proteins. We don't quite know exactly how it looks. But we know it requires all three of them, and it forms a shape that the sperm recognizes. After the egg is fertilized, the cortical granules released their contents into the perivitelline space under the vitelline envelope.
And among the contents of these cortical granules is a protease that cleaves ZP2. And this results in the destruction of the ability of this 3D structure to have a shape that can bind more sperm. So breaking ZP2 in this way must just alter the structure enough that no sperm can bind further.
So there are few questions left in this model. The big one is, what is this supramolecular structure? How can we identify it? What does it look like? What parts of it are important? And what in the sperm binds to it? And we'll talk about what in the sperm binds to it next time. But we still don't know the answers to these, and these are really important and really exciting questions I think right now.
In addition, we need to try and figure out how the old data, which were beautifully done and perfectly controlled, fit into this model. And I suggested to you one way, which is they could still be blocking receptors or blocking structures. And that may well be how it is. But we still need to try and reconcile these two sets of data.
So I think this will probably be a good place to stop. I'll just summarize what I've told you today and then tell you where we're going to go on next week. So I'll tell you first where we'll go on to. What we'll do starting at the start of the next lecture is we'll talk about now that the sperm has bound, what triggers it to undergo the acrosome reaction.
And then we will spend a little time talking about what on the sperm does bind to the structure in the egg. And eventually, we will get to fuse the plasma membrane with the sperm maybe before spring break or else just after spring break.
So to summarize what I told you today then, I showed you that in abalone, there is a non-enzymatic way that lysin breaks apart the crosslinked fibers of VERL so that it makes a little hole for the sperm to get in. That finished up our discussion of the male and female proteins that attract and bind sperm to eggs and get them to the plasma membrane in marine invertebrates.
So then we turned our look to mammals. We saw that mammalian sperm do lots of things in the reproductive tract. They get stored. They thermotax to where the egg is. And they chemotax in response to a number of different cues and possibly using odorant receptors that they express towards the egg.
And then we saw that they go through the cumulus layer with hyaluronidase, they come up against the zona pellucida, and they appear to recognize some feature of this three-dimensional structure caused by the three proteins, ZP proteins, within it. Next week, we will pick up from there. Have a good week.
Sit in on BIOGD 682, Fertilization and the Early Embryo. In this lecture, Mariana Wolfner, professor of developmental biology, explains how invertebrate and mammal sperm find and bind to eggs.
