EP49: Egg Activation: Calcium Dynamics with Dr. Karl Swann

In this episode of The Future Conceived, we delve into the extraordinary moment life begins: egg activation, the precise signal that jolts the egg out of its arrested state and launches embryonic development.

We are joined by Professor Karl Swann (Cardiff University), a world-leading expert who has spent four decades decoding the chemical interactions between sperm and egg.

Dr. Swann explains why the entire process depends on a series of small, rapid bursts of calcium . These repeated calcium oscillations are the vital trigger, lasting for hours, and are necessary to fully commit the egg to development. We discuss the challenge of accurately capturing these minute, long-running chemical signals in the lab.

Central to our conversation is Dr. Swann’s groundbreaking discovery of the sperm-delivered protein that causes these calcium pulses. This protein acts as the 'switch' that the sperm injects into the egg, causing the egg's internal stores to release calcium in a repetitive, rhythmic fashion.

We explore the importance of this specific activation mechanism—including how the egg powers this demanding process using its mitochondria—and address a crucial clinical issue: why current methods used in fertility clinics to artificially activate eggs are often ineffective, relying on outdated technology that only produces a single calcium burst instead of the necessary rhythm.

Transcript

I mean, we're working on trying to get a better way of activating eggs. That is one of the projects in my lab, and it's something that we're trying to put together and hopefully get something out on soon, because we're trying to find a way of of causing more than one large calcium transient in a mammalian. It shouldn't be that difficult. Doesn't mean it's easy. Um, so that's one thing that I, I want to try and fix that problem. Um, so that clinics have a better option than calcium ionophore because I feel like that's nineteen seventies technology. Hello, listeners, and welcome to another episode of The Future Conceived, the official podcast of the society for the Study of Reproduction. I'm Cam Schmidt, assistant professor in the Department of Biology at East Carolina University. In today's episode, we'll continue exploring the mechanisms that control egg activation. I have the pleasure of chatting with Professor Karl Swann, head of the Biomedicine Division in the School of Biosciences at Cardiff University in Wales. Doctor Swann has been studying cell signalling and metabolism during fertilization and early embryo development for nearly forty years. His careful use of biochemical methods has led to key advances in how we understand interactions between sperm and egg, which includes the discovery of sperm derived phospholipase C zeta and its role in the control of egg calcium dynamics. I really enjoyed learning how he thinks about this problem, and I hope you will too. Karl, thanks so much for joining us on the show today. Well, thank you for inviting me to speak. Maybe I can start now by telling you a little bit about the motivation for the series and why I'm asking some of the questions that I'm asking when I look at the journal scope for some of the journals that I publish in, I noticed that they tend to say we publish mechanistic insights into x, y, and z. And when I go to conferences, I've noticed that almost uniformly all the presentations are presenting on some sort of a mechanism of something. And my background and the kind of work that I do in the lab is, is somewhat interdisciplinary. I work with physicists and computer scientists, and I think as you kind of look broadly for definitions of what exactly that means, different people have have different, different takes on it. And so when I started asking people what they thought a mechanism was, or what counted as a satisfying explanation for a biological process, I noticed I was getting really different answers depending on the backgrounds of the people that I asked. So metabolism people tend to think about things differently than cell signaling people. And physicists tend to think about things very differently than cell signaling people and metabolism people. And and so that's kind of the goal is to to pick one kind of unifying theme, something that like where where different researchers are sort of working on the same thing, like egg activation, but then interview people who are coming at it from different perspectives. So that I can try to understand how they think about the problem, because I think how we think about the problem influences how we try to study the problem. When you think about mechanism and biology, what do you think about? Well, I suppose there's different levels. Um, but I normally think about, um, chemistry. I would say that mechanism in biology, the proximate explanation that you want is, is, is the chemistry. So I think biochemically and, and from a signaling point of view there for um, I think you can understand something when you've, when you can write a mathematical model, ultimately, you know, if you can write the equations down and you can simulate the behavior of the thing you're studying and get it to behave in the way that the variation follows the biological variation, then I think you have a good understanding. So I think you need to go all the way to the level of a mathematical model. And in fact, that's something I'm trying to do with the calcium signals at fertilization. I'm working with mathematicians and it's, it's it's quite a lot harder than I thought actually, to get things right. But, you know, that's what ultimately I would like to get is, is, is a mathematical model which captures the essential features of the phenomena you're looking at. What do you think about that? Uh, the modeling problem makes it so challenging. I think it's getting all the. There's so many free parameters that you have to deal with. Um, and also, um, I mean, with calcium oscillations, I mean, most of my attempts at modeling are to do with calcium oscillations. There's there's several different classes of models. And you have to decide which one to go for. And it's it takes quite a long time to explore all the parameters, the parameter range, and decide which ones you're, you're going to keep fixed and which ones you're going to vary? Um, and I think my mathematical colleagues sometimes get a little bit frustrated with the number of things I said, no, no, you've got to change that and change that. Can we look at that? And, and and that's actually just like doing experiments. Those things take quite a bit of time. And I think I hadn't had not anticipated how long some of that stuff can take for, you know, a mathematician. They've got students doing this work. But, you know, it's like it's like a full project to to investigate the parameter ranges. I was listening to an audiobook recently with a physicist who's working in biology problems, and she told this anecdote about a physicist, goes to a neuroscience conference and meets a neuroscientist there and wants to learn how the brain works. So the neuroscience or sorry, the physicist asks the neuroscientist, tell me how the brain works. And, uh, the neuroscientist says, well, it has two hemispheres. And the physicist says, wait, stop. You've told me too much. Yep, I know that. So actually, one of the things about calcium oscillations models is that they are a bit too generic to be useful. So yeah, you could say the phenomena has been explained, but they are very much for sort of some typical cell. And an egg isn't a typical cell. There's things that are distinct. Um, and I think that's why I got interested in doing that, because, you know, I feel like some of the key features I want to see just are not reflected in the models or the model makes assumptions, um, which are just not true in from what we know about eggs and calcium. So that's, that's the feeling that the explanation isn't there because the model doesn't really work. And I think a lot of people don't notice that. Um, and so when you start measuring calcium a lot, you realize that there are features that don't fit with the mathematical models. Is that because of the, the kind of nested or or Our multiscale nature of biological systems. Do you think you mentioned that you know, the the chemical explanation for cellular processes is kind of like where you where you go for mechanistic descriptions. But I think it's a it seems to me a pretty big part of the challenge, being able to describe big, complicated cellular processes all the way up from molecules. Yeah. Although in principle, I mean, explaining calcium oscillations is not necessarily that complicated. So the mathematical models going back to the nineteen nineties, which worked fairly well apparently. But, you know, some of those early models, they relied on the entire calcium store emptying during each cycle so that, you know, the the way the calcium increase in the cytosol is terminated is the stores completely empty, and then they completely refill in time for the next calcium transit. Now that just doesn't happen in mammalian eggs. Eggs are huge spheres, and it's pretty implausible that would happen, that all the calcium in the air would sort of come out of the cell and then all come back in again to trigger the next spike. I think the evidence is against that. And so you look at the old models, you think, well, that doesn't work. And some of the models are the kinetics of the IP3 receptor don't really match what we know about the receptor from single channel studies. And so again you start looking at the detail and thought, well that assumption isn't right. That bit isn't really right. So those generic models from twenty or thirty years ago just don't apply for eggs, because the calcium oscillations in eggs are a little bit different from the ones in somatic cells that everyone has been looking at for quite a few years. Do you think the limitations in the assumptions come from not having enough or the right kind of data? Partly. Um, it's quite hard to do some of the experiments being done in somatic cells. You know, you can transfect cells with various reporters and put them calcium indicators inside the ER and the mitochondria. You can do all those sorts of things. It's quite hard to do those in eggs. It's more challenging. Um, you know, they're out for a limited time. They age. You've got to make sure you can put reporters in by injecting RNA, and there's a limited time to get things targeted. And some of the targeting strategies don't work. So it is more difficult to do that sort of stuff, particularly in mammalian eggs. Um, so you haven't necessarily got all the, the parameters that you have from somatic cell studies. Um, the oscillations are also there are different dynamic range. You know, people that study somatic cells are often look at their traces and think, well, that's fifteen minutes or experiments over. You just throw on the agonist and they get this high frequency oscillation the last fifteen minutes. Well mammalian eggs you know, are recordings are typically five hours. Um, we fertilize the egg. The oscillations can last two or three hours. Uh, they're low frequency spikes. You've got to you know, that's a whole day recording just to get one set of oscillations. Um, and so that's more demanding. So you just can't the turnover is going to be slow for that. And I think a lot of people don't necessarily measure the calcium, uh, effectively because they use dyes which leak out of the eggs or, um, they have some effect. A lot of the small molecular weight dyes don't work very well. Um, we use dextran linked dyes. We always micro inject dextran linked dyes. And with that, you know, you know, your dye is in the cytosolic compartment forever. It's not going anywhere and it's just indicating that one compartment. So I think and that's and there's not many other people do that. And I think you get so I get oscillation patterns that look different from many other people. But I would argue that that's a genuine reflection of the cytosolic compartment oscillating. And you're not seeing the effect of the dyes leaking out or getting compartmentalized, which I think causes, uh, various artifacts in your recordings. Maybe we could, uh, kind of start with a little bit of physiological background into the the egg you mentioned, you know, kind of where the egg was and the meiotic cell cycle. And I think that's kind of important to understand the context of the problem of egg activation. So in nineteen ninety three, you co-authored a review and development called Lighting the Fuse at Fertilization, which I think is a really, really cool metaphor. So can we start with a little introduction into how egg activation fits into the broader context of mammalian reproductive physiology, and maybe address the question of why an egg needs a fuse at all? Well, the thing that the egg needs is a stimulus to activate, um, as the key thing, the sperm fusing with the egg and bringing in the paternal genome, that's that's not going to stimulate development, that's not going to activate. So the there has to be an activation stimulus, which is separate from sperm entry. And there are cases in parthenogenesis where the activation stimulus is there without any sperm at all. So, um, you can separate the two. And I think we, we know from all species that have been studied that that that activation stimulus is an increase in cytosolic calcium. So the calcium signal. I've not come across any example where an egg is activated without a calcium transient. I mean, you can activate eggs artificially without calcium transients, but physiologically it's always a calcium signal. Um, and the fuse refers to this delay because this was actually first done in sea urchin eggs many years ago, where you can measure the electrical changes and the depolarization of the plasma membrane potential. Um, and in fact, a firing of an action potential occurs. And then there's a delay of about fifteen seconds before a calcium wave takes off and crosses the egg. Um, and that delays is what we refer to as a latent period. Um, and we and the idea of lighting a fuse is that the sperm does something. It's clearly, you know, done something with the egg. And in fact, it's we know in the sea urchin eggs that that initial electrical change is actually caused by depolarization through the sperm. So the sperm is fusing and then depolarizing the egg with channels, ions going in through the sperm membrane into the egg. So it's sperm egg fusion effectively. So sperm egg fusion occurs. And then fifteen seconds later, a calcium wave crosses the sea urchin egg. The wave is endogenous because if you inject a little bit of IP3 to trigger calcium, you can trigger that wave without a sperm. So we know that you can trigger the wave endogenously, but the sperm does something to trigger that wave. And the idea is, well, there's a latent period and the fuse has been lit and something is going on, uh, for fifteen seconds. And then the calcium wave kicks off and we in the sea urchin egg, we still don't really know what's going on there. That's the amazing thing. We've done a lot of work on mammals, of course, but in sea urchins it's still not known exactly how the sperm does that. I suspect the sperm has some kind of factor in sea urchins, but there's not really strong evidence for that. But that's that's the fuse. The fuse is that latent period which I think exists in in mammals as well. In the mouse, we think it's about a minute or so that there's slightly different experiments because there aren't the electrical changes in, in, in mouse eggs during fertilization from dye transfer experiments, it looks like the sperm and egg membranes fuse. And then there's about a minute or so before the calcium the first calcium wave, takes off. There's then multiple calcium waves, but the first one is a delay after a minute or so. And, you know, we've got a good idea what might be happening there. But it's and it's related to this protein plc zeta. But it's the same kind of phenomena. There's a latent period and something is diffusing in from the sperm head, we think in mammals into the egg. And that's the fuse that's been lit. So I recently had a conversation with Mariana Wolfner at Cornell University, and she mentioned that Drosophila eggs activate prior to fertilization during while they move through the oviduct. And I think it was kind of a combination of membrane permeability and mechanical forces, but also still stimulated through initiation of ion transients. And and I had asked her about the the relative importance of polyspermy in Drosophila because they I think they kind of have fewer sperm there. And then there's also a micro pile on the egg. So the sperm can't just bind anywhere to fertilize. So what I thought was really interesting about what she said was that egg activation seems to be this kind of underlying process that is very, very broadly conserved. But the control of when that process happens seems to have evolved different stimuli. So I was kind of wondering if you had thoughts about how the mammalian activation and maybe even sea urchin activation for a comparative reference compare to one another? Well, I mean, the thing about sea urchin eggs, which I worked on during my PhD, so I spent a few years working on the you get you get one calcium wave. And then development is launched and it lasts about five or ten minutes. So you've just got that one response to look at. Whereas mammalian eggs you'll get that initial calcium transient about five minutes. And then you get repeated pulses of calcium. And we think there's between ten and twenty pulses physiologically. So there's there's there's a lot more to to work on on mammalian eggs. It's a cooler response, I think, because you've got this long period of beautiful calcium spikes lasting many hours. But in terms of the different strategies, what what's interesting is that calcium is is, is the universal trigger. Um, I tell my students about this that calcium is more important than the sperm at fertilization, because sometimes it's not the sperm that does the activation as in Drosophila. There's other examples, by the way. So in zebrafish the eggs are seem to be activated on hitting the pond water. And there's some receptors on the surface of the egg. So that's not sperm egg fusion triggering anything their that sperm does fuse with the egg, but the trigger for activation is not sperm. Then in the zebrafish. It's also true in a shrimp where I know the again, the sort of the release of the eggs into the seawater. And I think there's evidence for a magnesium activated receptor in that species. So there are species where the act of releasing the eggs in some form into seawater or pond water triggers activation, but it's triggering a calcium increase. It's just just the sperm isn't causing that calcium change. In all mammals then you're seeing the sperm causing the calcium change. And in fact, there's an awful lot of species like the sea urchins and ascidians, frogs that people work on. That's always the sperm causing the calcium change. So it's probably more common. But, you know, again, it's always calcium. And there's this polyspermy is interesting because, um, there are these examples of species where you get physiological polyspermy you actually, you not only have lots of sperm fertilizing the egg ten or twenty, but it's necessary. Uh, so Newt's a good example of this in newt eggs. Um, you have to have ten to twenty sperm to fuse, and each one causes a little calcium transient, and they summate to cause a big enough calcium transient to activate the egg. So you can either have the sperm not causing any transient. It can cause a transient that has to summate, or it can cause the entire thing. It all comes down to giving a calcium transient that is big enough to trigger development. So the strategies vary a great deal, but it's always calcium. So you can bypass a natural stimulus to to trigger calcium waves. And what happens in the egg if you do that artificially. Well it depends on the species. Uh, in mammals you have um, I mean, you can go through preimplantation development. You can certainly get development in mammals up to blastocyst fairly successfully. Um, you have to do some little tricks. You have to make sure that the you suppress the second polar body formation so that you get the the developing pathogenetic embryo is is diploid because haploid embryos don't develop very well. And then after implantation you're going to get the effect of imprinted genes which means it's not going to develop to term. But it's amazing how you can get several days of development even in mammals just with the calcium transients. And there's then the issue with mammalian eggs that you you probably need to have multiple calcium pulses. That single calcium transient doesn't work very well. It doesn't mimic the physiological situation. And the best developments are generally seen with stimuli that cause multiple calcium transients. What are the most relevant physiological constraints in mammals that favor activation occurring in conjunction with sperm binding rather than some other stimulating event? I think in mammals in particular, the females invested quite a lot in producing a viable egg or a small number of eggs, and if you have pathogenetic activation. It's going to fail to develop, but it's got a risk of perhaps even triggering pregnancy that will then fail. So there's I think there's a risk if the sperm isn't there, if the paternal genome is not there, it's not going to develop. So you have to make sure that if you're going to activate the egg, you've definitely got the the male pronucleus in there. Um, so I think that's going to be particularly true in mammals because of the investment. I mean, although I think in many species, the two are clearly tied in the sea urchin doesn't have the same investment there. But, you know, the sperm seems to fuse then cause calcium release. So I think the full reasons why you've got the different mechanisms of activation is not clear. That will require a different kind of explanation from what we're discussing, because clearly there needs to be some evolutionary explanation there. And I haven't really see people explain that. Do you know if there's any research on on activation and parthenogenic species? I know there's some lizards that do that fairly regularly. Yeah, I don't really know. Um, many studies on that. there's insects will be probably the better case to to look at where you've got you know, you can have sexual or pathogenetic, uh, activation and development. So I don't really know that there's people have really looked at that. But the calcium transients you would imagine would be pretty similar if like in Drosophila. I mean, there are other species of fruit fly, I believe, where you can get pathogenetic development. So perhaps those could be looked at because I think the calcium trends, it'd be interesting to see if there's any difference between the pathogenetic and the, um, the fertilized eggs. I suspect there wouldn't be because, you know, it's it's triggered. Activation is triggered by ovulation, so insects would be a good place to look. By the way, there are some fish and I can't remember the names of the species. There are some fish where the sperm does activate the egg. But you then got a genetic development. The paternal nucleus is kicked out. So that's another variation. Sperm can activate the egg but doesn't get incorporated. No one's looked at calcium in those species. It's. I don't know what species it is, but I remember reading that story and I thought that's yet another variation that probably exists. That's really interesting. Yeah. I like a sexual parasitism thing or something where they fool the male into giving them sperm. I'm willing to bet that species still has a calcium transient, though. Even then, you know, the sperm will probably be. It's almost like you're using the sperm to trigger a calcium transient. And then the egg is kind of kicking the sperm out. And I don't need you anymore. I'll just do the calcium, and then we don't need the nucleus. So calcium oscillations are clearly an essential trigger for egg activation in mammals. Can you talk a little bit about why they're important for downstream control of cellular processes? Well, one reason we know is because of the way the arrest at metaphase is maintained and it's maintained through high levels of cyclin dependent kinase and cyclin B, and the activity of that complex has to be taken away. And the evidence is that a large single calcium transient will temporarily bring down the Cdk1 activity. So the MPF activity that keeps the egg arrested, it will come down. But resynthesis of cyclin B can then bring the activity back up again. And if you have a second big calcium transient, it takes it down again. And it seems like you've got to keep pushing it down to make sure it stays down. And at some point then the egg commits to. So exiting meiosis and going into interphase. And so repeated pulses of calcium seem to be a way of keeping the cyclin B levels down over several hours. I mean, one of the things about mammalian eggs is it is quite clear is that the, you know, the first cell cycle is really long. It's going to be about twenty hours compared with a sea urchin egg or a frog egg, and which have about a one hour first cell cycle. So one big calcium transient that lasts sort of five or ten minutes in those species is is a substantial fraction of that first cell cycle. But in mammals Um, it's going to take several hours, maybe four or five, six hours to get Pronuclear formation just to get out of meiosis into the first interphase. You've got several hours. And if you wanted to have a an equivalent calcium rise, it would have to last an hour. And I think the cell would die if you try to maintain high calcium for an hour. So I think one way you can get around that to keep a calcium signal going for several hours, the way you do is you have pulses of calcium, which kind of activates the signaling process. And there's presumably some kind of hysteresis where those that that stimulus stays despite the fact the calcium has come down. So pulses at every ten fifteen minutes, at least thirty minutes seems to work pretty well to to stimulate activation. There's a review article a few years ago that Carmen Williams lab at Niehs here in the US had published where they they made this figure that showed a bunch of different species, and they showed the differences in the kind of qualitative waveforms that you see on egg activation. And they were there were all sorts of different shapes. Some of them were kind of monotonic, increasing and monostable, and some of them were were oscillating. And I don't remember if that's what they discussed in that review as a potential explanation, but do you think that that's a reasonable explanation for seeing different waveforms in different species? Well, it's some of the explanation, but the the time scales in some of those recordings have to be looked at. Because in mammalian, as I say, you've got you've got many hours for the activation process, whereas frogs and sea urchins, you've got most of it's occurring in about the first thirty minutes. But there are some species, um, like the ascidian oocyte that's fertilised, um M1 arrest, they have oscillations in calcium, higher frequency oscillations, um, and they have a short cell cycle. So they're like the sea urchin but they have oscillations. So you know, one thing that's not clear is why is it the sea urchin egg has a single calcium transient, and then the ascidian has oscillation despite having the same kind of cell cycle length. So it's not all about cell cycle length, but those oscillations in ascidians. Um, fertilization are quite high frequency. They're they're not one calcium spike every twenty or thirty minutes like mammals. They're you know, they're one every five or ten minutes. They're much higher frequency oscillation. So to get an oscillation you'd have to have kind of a source and a sink and a time delay. So I guess one possibility would be calcium comes in from outside the cell and then gets pumped back out with a time delay. Or calcium is released from an intracellular store, gets pumped back into the intracellular store with a time delay, um, or uh, some uh, combination of those two things. Calcium comes in and then is taken up into an intracellular store with a time delay. Do you have a sense of which of those scenarios applies to mammalian eggs? Well, the source of calcium in mammalian eggs. It's its intracellular release from the endoplasmic reticulum that's the predominant source. And most of it seems to go back into the ER. I mean, eggs being large cells, plasma membrane flux is going to be relatively small compared with somatic cells. But the flux across the plasma membrane can modulate frequencies and things like that. And exactly how that works is not entirely clear. I think it's due to changes in cytosolic resting calcium. Some people think it's due to calcium stores, but whatever the case, there is a modulatory role of fluxes across the plasma membrane. But the calcium release is nearly all coming from endoplasmic reticulum. And I think a lot of it is going back into the ER and some of it probably going into mitochondria as well. Um, so and then it kind of probably comes out of the mitochondria over time cycling back into the ER. So it's internal membranes. And I think that's true of most eggs and certainly eggs that oscillate. That appears to be true because there are some species where the main source of calcium is across the plasma membrane. Calcium influx can actually provide the main source of calcium increase for some marine worms. That's the case, but they seem to have one single large calcium rise. Um, long as in ten minutes long. Not as in hours long, but, you know, so calcium influx can be the source. But I don't know any species where it's calcium influx and oscillations. You know, when it's oscillations, it's it's internal release as far as I know. So they might have something I guess akin to the, the serca pump in skeletal muscle. Yeah. There's going to be, um, serca pumps. Um, but I've been interested in the positive feedback loops in that oscillation because there's the IP3 receptor has some very interesting kinetics which suggest that it, it has um, calcium and IP3 can synergize to, to cause more calcium release. And there's a positive feedback loop, um, which you can probably come on to discuss in a moment because it's, it involves the sperm factors, which initiates a positive feedback loop which gives you the upstroke of the of the the wave, as it were. The upstroke of the oscillation. The pumps are involved in the downstroke. But exactly. Exactly how calcium comes back down again is is not well understood. I'd say. Sometimes when I see research related to calcium oscillations, the eggs are denuded of the zona pellucida. Do you denude the eggs when you do the research? And does that have any implications for the waveform dynamics? Well, we do take those owners off most of our experiments because we we want the eggs to stick down to a coverslip so we can image them. And particularly when we're doing it at fertilization, because you add the sperm, the sperm start to jiggle the egg around and it moves out of the field of view. So you want the egg to stay in the field of view and stay sort of more or less stuck. And so we literally stick them down to a coverslip a glass coverslip, which you can do if you take the zona off. However, with um, with artificial stimuli that we have for causing oscillations. We've done that either with or without the zona, and the oscillations are essentially the same. So we don't think the zona influences the pattern of oscillations in any way. You've made enormous contributions to our understanding of the events that trigger calcium oscillations. Can you summarize how you're thinking about this control mechanism has evolved since you began studying it back in the nineteen eighties? Yeah. So when I started my career doing a PhD on sea urchins, and then I started to work on mammals, I mean, the fundamental problem was we didn't know how the sperm actually cause the calcium oscillations in mammals or the calcium release in sea urchins. So it's, you know, what's the signal transduction process? I think people began to realize that calcium activated the egg. The question then is how does the sperm cause those changes and the received wisdom at the time, in fact, in the textbooks, was the idea that sperm acts like a giant hormone, works on a surface receptor, triggers calcium release that way, which may turn out to be correct for some species, actually, but I don't think it's true for the sea urchin egg, although that's not clear, but I think it's almost certainly not true. Now, we would say for, for mammalian eggs and in mammalian eggs. Then, um, I did an experiment where I wanted to see if the sperm contained a factor, because actually, in the late eighties, uh, studies were emerging suggesting that sperm egg fusion occurred before calcium release. So that idea became more and more plausible. So it was around eighty nine that I first decided to just to grind up sperm and make an extract and inject that and see if that caused oscillations. And one of the problems with doing that experiment is, is if you've only got one calcium transient and you inject material, um, and if certainly if the material's got any calcium in it, you can cause a calcium transient with that material, with that extract, or possibly just by the act of putting a needle through the plasma membrane, a little bit of calcium will go in. So when you've only got one transient to look at, you never know whether is that transient because you've got some special sperm factor? Or is it because you some artifact of the injection or and that's that's always a problem when you've only got one calcium transient to look at. But I've been working actually, I was working in Japan in my postdoc, and I'd done a lot of experiments where it was quite clear that no matter what you did to a mammalian egg, whether it was a mouse egg or a hamster egg, you know, stabbing the egg or injecting calcium only ever caused one response. You could not get oscillations. It's very, very hard to make those eggs oscillate. Even injecting IP3 doesn't work very well. Certainly one bolus of IP3, you get a few oscillations there damped and they go away. And that's it. So I knew that, you know, you cannot get sustained oscillations over several hours with anything. So when I then ground up sperm extracts, injected them into mammalian eggs, mouse and hamster eggs, I saw these oscillations that lasted for hours. And I thought, that's something special. That means that it must be something in the sperm that's doing that. And you, you use, um, proteases, you destroy that activity. Uh, and perhaps also equally important is if you make extracts from any other tissue sort of brain, liver extracts, lots of cytosolic extracts and other tissues, nothing happens whatsoever. So it seems amazing that the sperm extract alone is able to cause the oscillation response, which is unique to fertilization. So that was the the evidence that convinced me there must be some sperm factor. It then took about ten years to find that factor. That's the slight problem. And we got the wrong protein on the way as well. Which really doesn't help us because when you're going against the grain, a little of opinion. Think getting something wrong on the way. You know, people notice us. So it took quite a while to find it. What protein was that? We had some metabolic enzyme actually involved in glucosamine metabolism, which we thought it was was could be the sperm factor. We only made a correlation. We never we never actually proved it could cause oscillations. And when we cloned it out, we found it didn't cause oscillations. But for a while we thought we'd got the right protein based on correlating with activity, but it didn't turn out to be the right thing. And we eventually published a paper sort of saying this is the wrong protein, and we sort of had to go back to the drawing board. And we were looking at mechanism, and we're doing experiments with actually sea urchin egg homogenates where you can do a cell free assay of calcium release. And we managed to sort of pretty much prove that the, the thing in mammalian sperm extracts is a phospholipase. It's some kind of phospholipase. The problem was that it didn't seem to fit with any of the known phospholipases. Uh, Phospholipase C's. So we thought, well, the sperm injects a protein which generates IP3 directly, but it's not a PLC of the known types, which are the PLC, beta, gamma, and deltas at the time. So we had to go and look for a new kind of PLC sperm specific. And that's how we eventually got to PLC Zeta, which we a great relief. Then we microinjected it and it caused oscillations as we injected the RNA because that's easier to to make and use the protein. As an experiment we did in two thousand and one, I think it was published in two thousand and two, eventually. Do you have a sense of what's different about PLC Zeta compared to the other isoforms? Like does it does it have different binding affinities or activity or something? I mean, since then we've tested the other isoforms and we found that you can get a you can get some responses with PLC delta one, but it takes fifty times more. So PLC Zeta appears to be, you know, orders of magnitude more potent in eggs. It's also interesting PLC two only works in eggs. It doesn't work in other cell types. So. So there are two things about PLC zeta that are special. One is its calcium sensitivity. It's it's it's sensitive to very low levels of calcium. All PLCs in mammalian cells, at least the betas and gammas, they're all sensitive to calcium. But PLC zeta is the one that's sensitive to resting calcium. So you can get about fifty percent activity at resting calcium levels, which is quite surprising because that's not really true of other isoforms. There's a really interesting question there for why it doesn't do anything in the sperm, but the idea is that for some reason is not active in the sperm. As soon as it hits the egg cytosol, it starts hydrolyzing Pip2 to make IP3, and the resting calcium stimulates that. But then there's there's an increase in activity of PLC zeta as calcium starts to go up. And that's part of a positive feedback loop. So the other thing that may be unique about PLC Zeta, which probably relates to the question of why it doesn't work in the sperm, is that PLC zeta binds to intracellular vesicles in the egg. Because, you know, the the usual story is that Pip2, the substrate for PLC, is going to be in the plasma membrane, and that's where you'd expect to find it localized. But PLC zeta doesn't seem to localize to the plasma membrane, at least certainly at physiological levels. It doesn't go to the plasma membrane. It binds to lots of vesicles. And we've used antibodies to Pip2 to show that those vesicles are full of Pip2 and eggs are unusual. This is a really unusual feature about eggs. And it's it actually it's something that only develops during maturation of the egg. They seem to develop a store of Pip2 in little vesicles throughout the cytoplasm. There is Pip2 in the plasma membrane of the egg. But PLC zeta seems to ignore that. It goes for vesicular Pip2, which is very rare. It's not found in other cell types, which is possibly why PLC zeta doesn't do anything in other cell types. It's targeting, uh, intracellular vesicles. We think they might be Golgi because the Golgi is actually fragmented in an egg, like a most, uh, metaphase arrested cells. So we think that's an unusual thing of PLC to it targets these intracellular vesicles and hydrolyzes Pip2 from there, which is why may only work in eggs. And other PLCs can't reach that Pip2. And that may be why they're not effective. Um, and so that's, you know, one of the distinctive features of PLC. It's specifically designed to work on a Pip2 source, which is only found in eggs. So egg activation clearly has these downstream developmental processes that that are our follow on to the stimulus, but also the changes in the zona pellucida are presumably also related to. So are those two processes sort of part and parcel with egg activation and how separable are they? Well, I mean, the calcium increase, the initial calcium increase, um, which lasts five minutes or so in mammalian eggs, that that triggers a substantial amount of exocytosis, which leads to the release of things, proteins like ovastacin things which will modify the zona pellucida. And in fact, it also causes a release of zinc ions as well, which is why that can be detected. But a lot of that is occurring on the first calcium transient. You know, the one one big transient. And the first transient seems to trigger a lot of that, which makes sense, because once the sperm's got in and cause that initial calcium transient, then it's time to stop other sperm coming in. So, you know, the zona pellucida modifications seem to pretty much occur on the first calcium transient at fertilization. And that's part of a polyspermy block, which is found in mammals. And which is why it's odd then in some ways the oscillations then continue for several hours. But that's to do with, I think, the cell cycle machinery and other things inside the egg which need the oscillations. We can sort of think about this as a very specialized, differentiated cell that has all these processes kind of already set up, waiting to be activated. How is the low resting cytoplasmic calcium concentration established? Well, again, I think it's the ER is the main player there. Um serca pumps in the endoplasmic reticulum, um will pump calcium down. There's a calcium pump in the plasma membrane probably. And there's a sodium calcium exchanger in the plasma membrane as well. So there are a few mechanisms. But I think the ER is probably doing the bulk of the work. And certainly, you know, if you put on some of these inhibitors of serca pumps then you you can you can make the basal calcium just shift up a little bit if you use the right concentrations. We've done some work on that. So and it's also, you know, going to be an ATP dependent process because that's one of the interesting things I'm looking at now is the interplay between ATP and calcium. So thinking about it that way with, with the er having these, these fairly important calcium pumps kind of reminds me of skeletal muscle, where skeletal muscle also uses calcium transients to activate myosin actin interactions and motor protein activities. And then in order to control the frequency of contraction, the calcium is taken back up into the endoplasmic reticulum. So the normal resting potential is very low. Um, and that is an incredibly energy dependent process in skeletal muscle. So it actually costs more energy to relax the muscle by taking up the calcium than it does to contract the muscle. So how are eggs set up to support the energy demands of both maintaining a low resting calcium potential and also supporting transients at the right timing and rate. Yeah, that's something I've been interested in. It's one reason we started measuring ATP and calcium in eggs at the same time. So, um, which generally people don't do. Um, we, we inject luciferase into the egg rather than doing a whole cell assay, which destroys the egg. And to measure ATP at one time point with luciferase, we inject the firefly luciferase. And we can do the luminescence imaging of the egg to look at the ATP and then the calcium with calcium dyes. And we can switch between the two. So it's one of the surprising things because I mean, as you're alluding to from muscle, that a lot of ATP is going to get consumed in pumping calcium back into stores. And I'm sure that's true in eggs as well. In fact, I think the major energy requiring processes of fertilization are indeed calcium pumping. I'm sure that's why the energy consumption would go up. And so when we looked at calcium and ATP, you might expect there to be some decrease in ATP, but in fact there's an increase in cytosolic ATP at fertilization. And some of that is almost certainly due to the calcium stimulating the mitochondria. So that's another of the stories which emerges in other cell types, is that calcium goes into mitochondria during the cytosolic transient. And then that'll stimulate mitochondrial. We've looked we've looked at mitochondrial redox changes and they're stimulated by calcium. And that helps the ATP go up. But it's surprising that it's kind of overcompensated. It goes up rather than staying constant. And it goes up substantially. And you know, the story seemed fairly straightforward. Calcium release stimulates ATP production from mitochondria at the same time as consumption goes up. But more recently, we've found that there's there's more to it than that because with PLC Zeta you get the same effect, but it's actually quite modest. The increase in ATP is quite modest. With PLC zeta of fertilization, the increase in ATP is larger. And that's partly because it's twofold. And there's a late kick of ATP that comes in after about an hour, which is not explained by PLC Zeta. And in fact, if we suppress oscillations, we still get that second kick of ATP. So one of the things we're interested in now is the sperm may not only introduce PLC to, but something else which stimulates mitochondrial ATP production, which is I didn't really expect that. And that's kind of an odd thing to think about, is that there's another sperm factor, possibly, that stimulates mitochondria and that allows the ATP to go up at fertilization. It may be involved in the initial changes as well. So something else is stimulating mitochondria and not just calcium. I think one of the big challenges in, in studying energetics is that the ATP concentration or really more appropriately, activity doesn't change that much as a proportion of the total concentration of ATP that's there. What really does change is ATP or free ATP, but it's incredibly difficult to measure for ATP. And I guess maybe for the listeners who aren't super familiar with this, the the energy potential and the ATP hydrolysis reaction, which is what kind of powers all these processes within the cell, is only quantifiable relative to some reference point, which we use the chemical equilibrium for that reaction. And that's not actually different from like when I teach this in biochemistry classes, I show the students, I just hold a ball up, and the energy potential of holding the ball above the floor is the mass times the acceleration due to gravity times the height that it is from the floor. But we're usually on the second floor when we do that. And so if I held the ball out of the window, then the energy potential is actually higher because my reference point has changed. And that's one of the challenges, I think, with using luciferase based assays for ATP because you're you're measuring you don't you don't have a sense of the height, how far it actually is from equilibrium. No one can really measure it well because it's it's like the best way to measure it has really been in skeletal muscle using NMR. So no one has measured ADP in eggs for fertilization. It's hard enough to measure ATP. And actually I should emphasize that the luciferase measures magnesium ATP. Um, strictly speaking as well. So it doesn't it doesn't measure total ATP or ATP for minus. For example, it measures magnesium ATP. And you're right, it doesn't tell you whether you've changed that equilibrium between ATP and ADP because we can't measure ADP. It's going to be a lot lower. And there's no live cell probe for it. So the concentration of magnesium ATP does increase but it's possible. Yeah. But the free energy available doesn't change very much because ADP could still could well be going up at the same time. I mean that reaction is, you know, supposedly nine or ten orders of magnitude away from equilibrium. So the energetics may not be that important actually. Um, I don't I think about the kinetics being important here because because if it's ten orders of magnitude, if ATP changes a little bit, it's not going to change the free energy available in ATP hydrolysis. And this is a massive change in ATP, which is possible, but I'm not sure that's likely. So if it's the free energy that's not really the big thing. It's. But there are kinetic effects of ATP. So there's quite a few processes where if you double the ATP you will double the process. And that might include things like protein synthesis which increases at fertilization, and calcium pumps. Because one of the odd things we see with, with, with fluorescent dextran dyes, we see this. And I think other people don't generally see it with some of the dyes. They're quite good at measuring resting calcium. And at fertilization, the first calcium spikes start to occur. The calcium level between the spikes is actually lower than before fertilization. In other words, you could say that the kind of pseudo resting calcium, the interspike interval calcium goes down at fertilization, not up. So it suddenly it seems like the like the pumps are working harder and they've overcompensated as well. And I've wondered whether that's to do with the fact that the ATP has gone up and the serca pumps may actually be stimulated by higher ATP. So once the calcium spikes over the resting calcium between spikes is lower than it was before fertilization. And we see that all the time with, with particularly with one of the dyes we use, which measures resting calcium rather well. It's a great point about energy potential doesn't matter as much as the kinetics. And we rarely ever actually have kinetic and energetic data at the same time. One way to measure the at least mitochondrial energy turnover kinetics is to measure the rate of respiration. Do you know if anybody's been able to do that in eggs during egg activation? Um, not with sufficient time resolution. Um, it's I mean, we've tried with some oxygen probes. It's one of these experiments we've, we've played around with and not really got very far because, um, there is evidence for an increase in respiration in mammalian eggs. Actually, this classic data on things like eggs show an increase in respiration. But a lot of that is coming from a peroxidase to do with the formation of the extracellular structures and things. But in terms of mitochondrial oxidative phosphorylation and oxygen consumption, they're the probes just don't have the right temporal resolution or spatial resolution. So I haven't been able to get any real data on that, but I would expect oxygen consumption to increase. But I would like to do it alongside calcium transients. I think that's the thing. And that's proved difficult. Have you thought about membrane potential dependent dyes like Tetramethylrhodamine methylester or safranin O. Some of those kinds of things. Yeah, we've used fluorescent dyes for mitochondrial membrane potential quite a lot. And and they do respond to poisons and things like that. And Oligomycin you put the usual things on, on the eggs that will affect mitochondrial potential and they do respond. But at fertilization we can't detect any change. And we don't know if that's because they're not sensitive enough, not fast enough. And can that be the problem. So those dyes are a bit slow in responding. So and also they slowly leak out as well. Um, so it's kind of hard to get a baseline signal that's convincing. But over the years, I've tried three or four at least at fertilization and not really had any joy. So we don't know if the mitochondrial potential changes at fertilization. It may do. It might be quite small. So we talked a little bit earlier about how you use dextrans with fluorophores that are sensitive to calcium concentration. We also talked a little bit about using luminescence to measure ATP concentrations. Can we spend a little bit of time talking about the kind of technical aspects of those measurements and how you interpret them, and are all calcium dyes equal? How do you choose calcium dyes sort of thing? We go for calcium dyes, ones with different affinities for calcium. Um, sometimes when we want to look at those resting changes in calcium, we have a higher affinity dye of one called Oregon green bapta dextran, which is very good. But we also have a little, uh, go with some of the lower affinity dyes. Um, and there's a new range. Things like Cal five twenty, Cal five ninety, and they're becoming available as dextran. So we're using those. They their KD for calcium is around five hundred nanomolar, which is pretty good because that's pretty much halfway between the peak and the baseline. So I think those are looking particularly good now. So you know, we inject them as dextrans. Um, they're single wavelength dyes which people may think is a disadvantage. Um, you know, but it's actually very hard to calibrate any fluorescent dye for calcium in the egg, because normally you get an f min and f max using various techniques, and they don't really work very well in eggs for various reasons. I think it's very hard to get a convincing f min or f max before the egg just falls to pieces and everything's gone. So it turns out it's quite hard to calibrate the dyes, so we don't try. To a large extent. We just we look at um, fluorescence signal divided by the starting fluorescence to get a so we can normalize the signal. And that works quite well. Um, we also prefer to use dyes which are longer wavelengths, sort of fluorescence in the green or red channel because that seems to be less damaging to the egg. And I think then if you do all those things, you will see ten to twenty calcium spikes that last four or five hours. But a lot of people tend to see oscillations that die away in an hour. And I think that's because the dies are getting pumped out, or else the some of the dies are damaging the the egg. I think people I mean, so the one thing I would recommend is don't use fura two to measure calcium. Most people do and it's not the dye to use. It's a UV dye. It's ratiometric. Yes. But your your ratio signal from the wrong compartments, it compartmentalizes a lot. Um, and then gets pumped out. Um, and I think if you use the Am version, there's the acetoxymethyl ester loading technique that people use the the products of that reaction include the dye and formaldehyde. So you are loading the cell with a little bit of formaldehyde. That's in Roger Chen's original papers from the eighties. People tend to forget that. But it's a little bit of fixative in there, which I'm not sure is a good idea. So, um, the main thing with the microinjection technique is this electrical insertion, which we think is, is good. And we know that that doesn't harm the egg, that we can get development to the blastocyst quite successfully having done that. So we don't think that causes any particular harm. Can we also talk a little bit about the, uh, the luminescence measurement of ATP? Yeah. So what we do for, for measuring ATP is a firefly luciferase, which is, you know, very specific to ATP. It's it's major confounding factor is, um, well, it can be sensitive to pH as well. So you have to be a little bit wary if there's a pH change. Thankfully at fertilization in mouse eggs, there's there's no pH change, unlike the sea urchin egg, whereas a mouse egg pH doesn't change. So you're kind of okay on that score. You're going to get much less light out of it because you're not using fluorescent signal. So the signal is going to be very weak. Um, you have to incubate in luciferin. You have to wait for the system to equilibrate. So there are some disadvantages. But if you get all those things settled and obviously you've got to inject we inject the protein. You don't have to inject. I guess you could express from RNA we find the best method is just to, to buy the recombinant protein and inject it, and you let it equilibrate with luciferin then it's it's responsive to ATP changes. Um, the main problem is you've got to get a specialist camera. Your average CCD or CMOs camera is not going to pick up luciferase. We use a particular camera, which actually it's actually a CCD camera, but we use a CCD camera where you can use a lot of pixel binning. Um, it turns out that feature, which is really good about CCD CCD cameras, is you can reduce the noise level. So we set it up so that the pixel binning reduces your spatial resolution. You get better signal to noise, but your spatial resolution goes. But eggs are big. That's you've got you can play that advantage. Eggs are really big cells. So even if your pixel binning sixteen by sixteen pixels, for example, you can still see it's kind of spherical. It's an egg, you know, it's it's a you take the advantage of eggs. They're so big, you can get away with a lot of pixel binning and get the sensitivity into the range where you can actually measure the luminescence signal with a a cooled CCD camera. Um, but it's more like a ten thousand dollars camera. It's not your off the shelf type camera. So that's the one thing to be think about. The other thing is that you have to have a very good dark box, because you won't believe how sensitive these cameras are then. And by the way, you're doing ten second sort of integration bins. You can do longer if you want, but you do some kind of long integration window. You will pick up every bit of light in your lab. So an average dark lab is nowhere near good enough. We have a dark lab with a dark box around it. So that's the other thing. You have to, you know, no photons allowed anywhere near your system. And you can't have those microscopes with all those automated bits in because they tend to have little LEDs measuring things you have to make. It's better to have an old fashioned microscope with no automated bits to avoid any light getting into the dark box. So if you do that, you can then measure a signal. And in both cases, and this is always kind of difficult to avoid. But we're thinking about a little bit adding for example one of these fluorophores that reports on calcium. What it's really reporting on is its binding equilibrium state with the calcium that's there. So it's acting sort of as like a thermodynamic sponge for calcium. So it's lowering free calcium at the same time that it's measuring it. Well that is a big problem. And that's why you you have to get your camera set up to be as sensitive as possible so you can get away with as little dye as possible. So you, you also have to turn the light down because light can be damaging to, to eggs. So you're, you're you're racking up the sensitivity of your camera. So to get away with as little as possible and preferably below one hundred micromolar. I mean, calcium is quite well buffered, so it's not too much of a problem. And it's not a problem with ATP at all because that's even better buffered effectively. So yes, you do have to think a little bit about that. And I think some of the early studies people just put too much dye in, um, too much light, too much dye. Calcium transients look pretty awful. Um, you've got to, you know, rack up the sensitivity of your camera so you can get with a smaller dye as possible. My general rule of thumb is if you can look down the eyepieces at the fluorescence and see the eggs of fluorescence with your naked eyes, then it's too bright. You should be. It should be dim enough. You can't see it. The camera can see it, but you can't. When ATP is hydrolyzed, uh, magnesium is released. Do you think about the role of other ions like that, like magnesium, for example, or you mentioned zinc earlier as well? Well, just first with magnesium. I mean, some people like to think of magnesium as a, as a way of kind of looking at what's happening to ATP by magnesium changes. And that's certainly true. If you're going to poison the mitochondria, you can see magnesium changes because the ATP suddenly crashes down. But when we've looked at fertilization with magnesium dyes, we can't see any convincing changes in magnesium, for example, people did report changes previously, but I think some of the magnesium dyes that we use, the older generation dyes respond to calcium. The dyes also go into the air. There's all kinds of things they could be picking up. We've used a new range of much more effective magnesium dyes that are much more selective for magnesium over calcium, and we don't see any change. Um, we haven't published that, but it looks like magnesium doesn't really change. But despite the fact the ATP is changing, and I think cells are set up that if they have a physiological increase in ATP, they're able to cope and buffer out with magnesium because magnesium is is dynamically buffered in cells like most ions. So I think magnesium doesn't change at fertilization. We haven't looked at zinc, and I don't think there's any good evidence that zinc changes in the cytosolic compartment at fertilization. Um, the zinc is is being released from the cell through exocytosis. And that's clearly occurs. But and zinc clearly has to be homeostatically controlled at a certain level. But I don't think there's any evidence it plays a signaling role. And what is not available for zinc is a dextran linked dye. What you'd really want to do with zinc at some stage is to have a fluorescent dye that is dextran linked, so you can put it in the cytosolic compartment and know it's there. And then you have to also make it very sensitive to zinc, because zinc is is much lower than calcium. And that that dye hasn't been designed yet. And you would have more of a buffering problem there because the zinc is is even lower than the calcium. Do you think glycolysis and mitochondrial oxidative phosphorylation contribute energetically to distinct processes during egg activation. In simple terms, eggs um don't have any glycolysis. Mammalian eggs are talking very much stuff based on mouse eggs and to some extent, hamster eggs. Eggs and cleavage stage embryos don't seem to have any, um, glycolysis, or at least not enough to contribute to ATP production. I mean, we've shown this again using the luciferase probe that if you if you just take away what you take away all the substrates, um, the ATP goes down and you add back pyruvate, it goes back up again. But if you add back glucose, it doesn't do anything or in fact lactate or glutamine. So the eggs, um, primarily metabolize pyruvate to generate their ATP. And that's consistent with many studies showing that basically the the mitochondria are active, but the glycolysis is not. That's in an unfertilized egg in an early stage embryos. So glycolysis is switched off. However there are studies showing that at fertilization, glycolysis seems to or at least glucose utilization switches on. And there's certainly some of that is glycolysis. So there's there's almost like a little burst of glycolysis at fertilization that is unexplained and and forgotten by most of the field. But it was shown in about twenty odd years ago that glucose seems to be needed for the sperm to fuse and be incorporated. It's not sure what step is it's needed for, but there's kind of some requirement for glucose during fertilization. And if you're doing IVF, you always put a bit of glucose in there in the medium to get the sperm to fuse and to form pronucleus. We don't know why. And let's say there's a little kick of glycolysis and that's unexplained. And I would like to understand that because it's a sort of forgotten problem. We don't even know quite why it's important. And possibly the pentose phosphate pathway as well. Glucose. But it's quite hard to measure glucose uptake dynamically. The probes that measure it, you can measure it if you're doing a static sort of incubator and then wash out and look type analysis. But you know, there aren't many good probes for glucose uptake that are dynamic, that you could do at the same time as calcium. So it's kind of difficult to address that problem. Do you know if that changes uh, at later stages? I mean, it must change at later stages of embryonic development. But maybe the question is when. Well, it kicks in around sort of as you're going into sort of moral stage and certainly by blastocyst stage, you've got a lot a large amount of glucose uptake and glycolysis. And so all that kicks in later. But um, yeah, it's one of the mysteries I think, of, of early development is why eggs have once they're ovulated and they've no longer got cumulus cell support. They're on their own to make their own ATP. And they're just using their mitochondria, which are not even terribly active for mitochondria. You know, they're not thought to be particularly active mitochondria. There's a lot of them I guess, in the cytosol there. But they've decided to just let their mitochondria generate all their ATP for fertilization, although possibly a little bit of glycolysis. But after that, then you've got those first three cell divisions at least where it's it's it's all mitochondrial ATP. Why would they do that. Because it's a bit of a risk if you've got a got to have the ATP for cell cycle progression. And they've decided to rely on mitochondria, which are otherwise thought to be fairly low activity. There's this idea of mitochondrial quiescence. But in mammalian eggs at least, they're not that quiescent. They're certainly supplying all the ATP. There's some really great work in tumor derived cells. Uh, a couple of years ago that was showing that glycolytic enzymes are enriched near the plasma membrane, and that they sort of dynamically support transients in plasma membrane potential, and that mitochondrial oxidative phosphorylation, though it's more efficient on a molar basis in terms of like the fuel that gets oxidized turns into more ATP. It's actually slower rate wise by comparison to glycolysis. So the idea was that glycolysis at the plasma membrane can buffer very rapid demand that that just kind of comes and spikes, whereas the mitochondria can buffer kind of long slow demand of growth potential. I wonder if changes in plasma membrane potential are less of a concern for some of those early cleavage divisions, because they're sort of inside of a protective housing. Yeah, and certainly fertilization. Um, the plasma membrane potential mammalian eggs is a is a bit odd because it's around minus thirty, minus forty. And, uh, in the mouse egg, there's no particular change. Um, there's no evidence for action potentials that you see in other species, in hamster eggs and in fact, in human eggs. There's this odd thing where you get a hyperpolarization. You know, there's the plasma membrane actually goes more negative because you have a calcium activated potassium conductance. Um, and so and in fact, it's one of the ways in which calcium oscillations were first described in mammalian eggs, effectively, was that, um, a guy did a postdoc with Shunichi Miyazaki in Japan found that there was this membrane potential hyperpolarization at fertilization, and they were repetitive. And then he figured out it was a calcium activated potassium conductance. And therefore there were calcium oscillations. So the membrane potential goes negative in hamster eggs, but not really in mouse eggs. It's a few millivolts. It's hardly noticeable. Whereas in hamster eggs it's quite clear. Um, we show that that occurs in the human egg actually during calcium oscillations as well. So human eggs have the same hyperpolarization, but we don't know what the function of that is and we don't know why they would, you know why it would happen in one species and not the other. So that's a bit of. So even the membrane potential changes themselves are a bit of a mystery and tend to get just kind of ignored because I think the mouse egg is particularly uninteresting with regards to membrane potential changes. It also turns out that mammalian eggs are generally pretty hard. Uh, for patch-clamp, it's very hard to get a giga seal, so there's not a lot of people patch clamping mammalian eggs. Do you think Superovulation affects the quality of egg activation? That's a good question. Um, actually, Carmen Williams's lab looked at this recently in a paper where they they show that the calcium oscillations are pretty much the same, whether you do natural ovulation or superovulation. So that was reassuring because kind of no one had checked. And it's kind of somebody should have checked at some stage, but that kind of has been done now. Um, but as for quality of egg in terms of things like how well they develop subsequently, um, it's been suggested. I mean, there are studies in humans suggesting this might be the case because sometimes you can get twenty Oocytes from humans ovulated, and there's a feeling the quality isn't going to be the same as when you have the sort of five or ten, but we're not sure how that would relate to calcium. It might relate to mitochondria. The general consensus is that the paternal line mitochondria do not ultimately reside in the early embryo. But but they're also degraded, I think I think in the mouse, the sperm mitochondria do end up in the egg and then are later degraded. Do you know if sperm mitochondria are functional or is everything I just said wrong? No, no, it's it's pretty much right. The sperm mitochondria don't contribute to development and they appear to be degraded. There's lots of questions about you know, because it's the DNA from the mitochondria. That's probably the critical thing. And there's some suggestion that the DNA is kind of mostly lost from the sperm's mitochondria before fertilization. That's another idea. But, you know, they're outnumbered massively is one thing because I've been interested in ATP changes at fertilization. There's always a question of, well, maybe the sperm's mitochondria goes into the egg and makes more ATP. Is that a possibility? But and the egg's mitochondria are generally thought to be quiescent, relatively inactive. The sperm's mitochondria are sort of super active. They're generating as much ATP as possible. However, they're outnumbered about a thousand to one, you know, so it seems pretty implausible that the sperm's mitochondria could, could actually make any ATP that was significant at all. Can egg activation be improved for therapeutic benefit? Yeah. This is something I'm I'm sort of developed an interest for over a number of years because as I say in mammalian eggs, it's generally considered. And there's evidence for the idea that multiple calcium pulses activates the egg better than a single calcium pulse. And this has clinical relevance because, um, a lot of clinics are using Icsi. This process where you intracytoplasmic sperm injection, you're injecting the sperm to activate the egg. In fact, I think across the world it's either two thirds or three quarters of all ivfs are actually XY. And sometimes that fails completely. So all ten of the eggs injected with sperm fail to activate or they fail to fertilize. And that's mostly due to activation. So they fail to fertilize, mostly because they fail to activate. And it appears that that's because they fail to have calcium oscillations. And there's some evidence that a number of those cases are due to problems with PLC zeta, lack of PLC zeta deficiencies, mutations in PLC zeta. So that could explain some of those cases. I'm interested in the fact that actually Icsi itself has a problem in the sense that about one in four times even sperm that appears to be normal will fail to cause fertilization or egg activation. There's a sort of recurrent problem, and there's about a third of cases where you get low fertilization. So there's not only just failed fertilization, there's only one or two percent of cases total failed fertilization. But you might get about a third of patients in IVF clinics who are given Icsi. You get low fertilization fifty percent or lower. I think it's a significant potential clinical problem because it looks like it might be due to calcium. There's a group in Belgium who have led by Bjorn Hendriks, who've done some really nice experiments where they they did Icsi in like a clinical scenario. They took human sperm and injected it into human eggs, and they measured the calcium and they did it with different groups of, of men. And what was interesting that even with the the controlled donor sperm, they found that about a about thirty percent of the time, the sperm either doesn't trigger calcium pulses or just triggers one or something. So even normal sperm is often poor at triggering calcium pulses, which suggests that that could explain why about a third to a quarter of the time, you you don't get fertilisation, even when you've injected what appears to be a normal looking sperm from a ostensibly fertile man. So that's I think there's quite a lot of IVF and Icsi going on around the world where you're not getting enough calcium pulses, and so if you can get more calcium pulses, that would probably improve things. Um, now the only treatment that's available at the moment, and it's used in many countries, although I believe not in the US, where it's not allowed is to use calcium ionophore. Um, a two three one eight seven as ionophore that's first shown to cause calcium changes in eggs in the nineteen seventies and even in human eggs in the nineteen eighties. So it's been around a long time. It causes one big calcium rise, and it's really not clear how well that works, because even in the early studies, some people said it works well and then others studies on human eggs, for example, said it wouldn't work at all in mouse. Some labs find it works okay. We generally get about twenty percent egg activation. Another clinic. Other labs have found that. So you know, ionophores are it seems to be very variable. And often less than fifty percent of the eggs are activated. So this is a worry. But you know, the the treatment for patients is, you know, plausibly not very active. And I think when they've done clinical trials, um, randomized controlled trials, the efficiency is not very clear at all when they do sort of parallel sibling control studies. So, you know, I think we need a better way of activating eggs. And I think that will be something that causes multiple calcium transients. Well, that pretty much wraps up the questions that I had. And I've already kind of gone over time. So I apologize for that. That's okay. Is there anything that I didn't ask about or any additional thoughts that you have that you'd like to share before we wrap up? I mean, we're working on trying to get a better way of activating eggs. That is one of the projects in my lab, and it's something that we're trying to put together and hopefully get something out on soon, because we're trying to find a way of of causing more than one large calcium transient in a mammalian. It shouldn't be that difficult. Doesn't mean it's easy. Um, so that's one thing that I, I want to try and fix that problem. Um, so that clinics have a better option than calcium ionophore. Because I feel like that's nineteen seventies technology, and we should have moved on and, you know, needs to be simple and safe. I guess that's the, you know, the minor criteria that we want to make sure it's not going to do any damage. By the way, calcium ionophore does appear to be safe. It's people were worried about that. But you know, one big calcium transient doesn't seem to perturb the egg too much. And it's been used clinically. Hundreds babies at least have been born with calcium ionophore activation. But its clinical effectiveness is is unclear at the moment. Thank you so much for your time. This has been absolutely fascinating. That's a pleasure to be on this SSR program. Well, listeners, that does it for another episode of the Future Conceived. This podcast was sponsored by SSRs Virtual Education Committee, whose mission is to develop virtual programs that aid in education, highlight the lives and careers of society members, and bring updates on the latest scientific advancements in reproductive biology. If you're not a member of SSR, now is the perfect time to join this incredible network of researchers and professionals in shaping the future of reproductive science. For more information, please check out our website at SSR. If you enjoyed this discussion, please like and subscribe wherever you get your podcasts and join us for our next episode in this series, when we learn about the systems biology of cell signaling from the legendary Jim Farrell of Stanford University. Until next time.