EP 48: The Calcium Code—SSR Research Award Interview with Dr. Carmen Williams

In this highly insightful episode of The Future Conceived, host Cam Schmidt talks with Dr. Carmen Williams, Senior Investigator at the National Institute of Environmental Health Sciences and the 2025 winner of the SSR Research Award.

Dr. Williams, whose lab investigates the critical events of fertilization, shares groundbreaking findings on calcium signaling during egg activation. You will learn:

• Why the simple components of in vitro culture media, specifically calcium and magnesium ratios, dramatically control the frequency of calcium oscillations and influence the quality and development of the resulting embryo.
• New, compelling evidence linking these early calcium signals to metabolism (TCA cycle) and ribosomal RNA synthesis in the early embryo.
• The continued importance of research into endocrine disruptors, particularly plant estrogens, and their impact on reproductive tract differentiation.

Dr. Williams also discusses her journey from electrical engineering to clinical medicine to basic science, and closes with a powerful message acknowledging the essential role of trainees in driving scientific success.

Transcript

And so I really want to say, you know, if you think a pie is very successful, just look next to that pie and see who's standing there. And that's really where a lot of the energy and the success comes from. 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. Today, I have the pleasure of chatting with Doctor Carmen Williams, senior investigator of the Reproductive and Developmental Biology Laboratory and deputy chief of the Reproductive Medicine Group at the National Institute of Environmental Health Sciences. Doctor Williams is the twenty twenty five winner of Sers Research Award in recognition of her work on how interactions between male and female gametes during fertilization influence how the newly formed embryo develops during the first few days of life. I immensely enjoyed learning about the cutting edge research that she and her team have been doing in the last few years, and I hope that you will too. Thank you so much for joining us on the show today. Thanks for having me, Kim. I'm looking forward to it. How long have you been a member of SSR? I joined the SSR right after I finished my PhD around nineteen ninety eight. Although I might have missed paying my dues a year or two here and there. But it's been a very long time. I've been associated with the SSR. Do you have any standout memories from SSRs annual meetings that you would like to share? The the main thing I really like about SSR meetings is the the sense of community. So having been to not all but multiple SSR meetings, it's really where I go not only to learn science, but to network and see and meet new friends. Um, in in particular, I was very struck when I went to the first meeting after Covid. My first meeting was the meeting in Spokane, and just being able to talk to people was so powerful at that time. Um, because everybody had been behind a screen for so long, and it was really uplifting to be able to go there. The, the most recent meeting I just got back from last week in DC was very similar, where it was really a very cohesive group and, and a lot of fun to do science in that environment. Were there any standout presentations that you really enjoyed last week? I have to say, um, one of my favorite presentations was by Amy Gladfelter. She was one of the plenaries. She's not really a reproductive biologist, as she stated, but a cell biologist. But she gave a talk that was just very intriguing, beautifully illustrated, with simple models that were very compelling. As far as what they were showing. And I learned a ton about the placenta, which I've always struggled with. So that was one of my favorites. I didn't realize that it was sensitive, and I worked on skeletal muscle in my PhD, so I was a little bit familiar with sensitive cells, but I didn't know that about the placenta. Well, I mean, I've known that there were sensitive trophoblasts for forever because, I mean, I've been in reproduction and human reproduction for a long time, and that that was one layer, but something that Amy said that I, I'm still going to check up on is that it's a single cell, like the whole sensitive trophoblast is one cell. And my view of it was always that it was many cells, all of which were multinucleated. And so I'm actually very curious if I'm just wrong about that or if Amy was just sort of more focused on one region of the placenta, would have one large cell that did something. So that's something I need to figure out, because if it's one incredibly convoluted, large cell, that's just so cool. So in twenty twenty three, you were the recipient of SSRs Trainee Mentoring Award. I'd like to start by encouraging our listeners to go check out that episode, where they can learn more about Carmen's journey from electrical engineering to clinical practice, and ultimately to senior investigator of the Reproductive Medicine group at NIH. And the episode number for that episode is eighteen. For the listeners who are looking at up wherever they get their podcasts. Um, so you've had a somewhat atypical career trajectory. Do you feel that your interdisciplinary perspective has shaped the way that you do research? Yes, absolutely. I mean, I, I started life in being a bio major and really liking biology, but then moving into engineering because I didn't want to be a pre-med, and engineering was a lot of fun too. And I eventually went back to the biomedical type physician career, which I really enjoyed, but kind of around, you know, at the end of the day, what really wanted, you know, what I wanted to do that really, I thought I could make a difference and that a lot of people didn't like, but I actually liked better than patient care was being in the lab and figuring out how things work. And so even though my training it it was, I enjoyed it. I learned a ton. I, I learned a lot about what people suffer from with infertility. And most importantly, I, I learned about how important it is for the research to really improve what we're doing and what we're able to offer patients in the clinic. So when I started my infertility fellowship, the success rates for IVF were like eight to ten percent. They were terrible and we would have patients coming back for like ten or twelve cycles and never succeeding. And it was just very, very draining on everyone. And you wanted to do better, but we couldn't. And so the concept of doing research in the area of assisted reproduction always was incredibly important to me, because I knew it would eventually get translated. And in fact, we found out so much, mostly in the in the years after, I was no longer really involved in infertility clinics, but just about how you treat the eggs and the embryos in terms of keeping them warm. Like, we didn't really keep them warm in the beginning. We just put out a room temperature. So of course there's spindles. Were not doing really well. And so you learn so much just by focusing on how these things work and how you get eventually healthy embryos at the end of the day. And my more recent work is thinking about culture, media and how the culture media can mess with the ability of embryos to develop. So really, those early experiences had a lot to do with what I do now and how I've selected the research area that I have. Can we jump into the work that you're doing on culture media? Yeah. So one of the major things that drives embryo development is calcium. And calcium in the cytoplasm of the egg goes up and down for a set of hours. These are called calcium oscillations. And the calcium oscillatory pattern is actually critically important for not only activating the egg and and helping it develop into an embryo, but it can also influence offspring characteristics, as has been shown mainly in mouse models. So if you have too much calcium, it's bad. If you have not enough calcium, it's bad. And each of these things can differentially impact how the offspring grow or what their metabolic characteristics are and what's their fat content, for example. So all this work mainly was done by Jean-Pierre Oziel in the early two thousand, where he actually manipulated the calcium levels using an electrophysiological setup and a perfusion chamber that was fairly artificial. And so we decided to see if it was also true when the calcium signals were changed without having to take the embryos out and put them in culture. And so what we did was to do that was we had to figure out, how do you genetically control calcium oscillatory patterns? And so what we decided to do was to prevent calcium from going back into the cell after the calcium was triggered. So when you trigger a calcium spike, the cell responds to it. But then it's like, wait a minute, I need to get rid of this calcium and put it back into the endoplasmic reticulum where it has come from. But the cell also pushes the calcium outside of the cell to bring the cytoplasmic levels back down to normal. But because it's pushed out of the cell, there's an overall loss of calcium inside the egg. And so it has to let calcium back in through plasma membrane calcium channels. So what we decided to do was to mess with the ability of the egg to bring calcium back in as a way to simply reduce the calcium signal overall during these oscillatory changes. So basically, if you reduce the amount going back into the cell, it means that every calcium spike, um, is slower. So there's a slower frequency of calcium oscillations during that process. And so if you want to do all this in vivo instead of in a culture dish, what you can do is you can knock out one of the channels that lets calcium back into the egg. And if you do that, it turns out that you dramatically can reduce the amount of calcium going into the egg and change that calcium pattern. And what we found was if you do that, you end up disrupting the growth of the offspring without ever having the embryos be in culture. What that gave us an opportunity to do, though, was also to think about how is that calcium channel normally regulated. And so the calcium there's actually two calcium channels that do this. One is a voltage activated channel which is not in this case activated by voltage. It's just open some of the time and allows calcium in through what's called window current. Um, but there's another channel called Trpm7. It's a transient receptor potential channel. And that one is kind of interesting because it's just constitutively open. It lets in a lot of different ions. Um, for example, it lets magnesium in. It's one of the major channels to let magnesium go back into a cell, and it's actually inhibited by magnesium. And so what's kind of interesting about this channel is that if there's magnesium in the medium, it can inhibit the channels function. And if there's a lot of magnesium in the medium, it can inhibit it even more. We were using very low magnesium levels in our medium because we were using a very standard one called um csom csom, which is a standard culture medium for mouse embryos. And it just so happens to have only zero point two millimolar magnesium. And when there's that little magnesium, the calcium signals are very, very rapid and you get a high calcium exposure. And so all you have to do if you want to block that channel or slow it down, is add more calcium to the medium. And when you do that, the frequency decreases and there's less of a calcium signal. And what we ended up showing was that if you have mice that don't have that channel, once again you have differences in offspring growth and development. Well, what's also interesting is that embryo culture media that is used for humans is is in most ways uncharacterized because it's made by private companies, and they don't want to tell you what's in it. And so just having people start thinking about how much magnesium is in the culture medium. And similarly, I haven't talked about this, but the level of calcium itself is also highly relevant because the more calcium you have in there, the faster it goes back in the egg and the more frequent the oscillations will be, giving you a higher calcium overall signal if there's high calcium in the medium. And so how much calcium and how much magnesium can completely drive the the calcium signal at fertilization. And this is all in a mouse. Now, we don't know exactly what happens in a human. However, humans express based on RNA data, the same two channels that I'm talking about in the mouse. So I expect that they would be regulated in the same way. Although, um, as a government employee, I'm restricted from doing any work on human eggs, but I think that would be a very important type of study for somebody to do who's able to do that type of work in the clinic and see whether it really matters. What's the calcium and magnesium in the culture medium in terms of embryo development and hopefully eventually offspring health are the the calcium oscillations. Those are, uh, always happening in the egg. Are those triggered at some point? Yeah. So those are triggered by sperm egg fusion when a protein that comes in with the sperm a phospholipase C, that sperm specific called PLC zeta that causes production of a lipid signaling molecule, IP3, that activates the IP3 receptor on the endoplasmic reticulum membrane and causes a release of calcium. And then the egg is special because when it releases calcium, the calcium itself actually then activates additional IP3 receptors, and so it activates this cascade of calcium release that goes in a wave across the cell. Once that wave happens, you have to bring the calcium levels down by the mechanisms I mentioned, where it gets pumped back into the ER and it gets pumped out of the cell. But the key initial trigger is sperm phospholipase C zeta. So the the oscillations would be generated by some time delay between the rate of pumping calcium back out of the cell and then re-uptake in the endoplasmic reticulum. Exactly. And are those oscillations fairly consistent in terms of the shape of the waveform, or the timing from egg to egg? Consistent is a stretch. There's actually even even if you get a bunch of eggs from a single mouse, there are clearly some very different calcium oscillatory patterns. There are features of the first calcium oscillation that differ from the remainder. The first release event is typically longer than the others, so it lasts anywhere. Depending on the strain of mice you have, it'll last from two or three minutes to up to eight minutes even can be perfectly normal. Then it goes down again. The subsequent release events typically are one to one and a half minutes or so. Um, and then the time in between each spike is truly dependent on how fast calcium goes back into the egg. It doesn't seem to be tightly related to how fast the serca pumps put the calcium from the cytoplasm back into the ER, probably because that is such an efficient process that it at least what we've seen that that doesn't have a big influence. The big influence appears to be how fast it goes back in. And that's dependent on the magnesium and calcium ratios in the, in the medium. And then those oscillations continue for a period of time. They all stop by about four or five hours after Fertilization, but sometimes they stop for a while and then restart. It is not clear why that happens, and sometimes they just stop and don't continue for very long at all. We kind of expect that those eggs are ones that aren't going to develop very well, because they have very little calcium exposure, but that's not been that's not been definitively shown because once we image calcium, it's not really great for the eggs over time because of the light exposure. And also and the methodology we use doesn't really allow us to then try to transfer these guys back into a mouse. So it's hard to do a one to one on what the pattern is like with the outcome. Do you use the acetoxymethyl ester calcium binding dyes, things like Indo one or fura or those kinds of things? Or do you use something else to report the calcium. We almost always use Fura two Am, which is the version that will actually get through the membrane, and then the Am gets cleaved so it stays inside. We sometimes will inject um, another type of dye like calcium green dextran or something. If you really want to avoid the having a, having a chemical that can change over time. Um, but more often, if we're trying to do something that that requires more of an in vivo type setting, what we'll use is a genetically encoded calcium indicator. So there are a bunch of those based on a a protein that was designed called Gcamp. And that's um, yeah. Cam the Cam is for Cam kinase. So essentially it takes two portions of the calcium and calmodulin dependent protein kinase. It splits it and then puts a calcium binding site in the middle. And then that ends up having a conformational change when calcium binds. So it brings the two portions of the molecule together. That then causes fluorescence a green fluorescence to occur because it's a conformational change that allows that to happen. And gcamp has been modified. We actually have a version called Gcamp eight. Right now we have gcamp eight, F for fast and S for slow. And there's also a Gcamp eight M, I think. I'm not sure what that's for, but I also believe they're already on a gcamp nine. And what has happened over the years is that the features and characteristics of these have been changed to be more efficient or less efficient. Sometimes you don't want to detect calcium as efficiently if you're trying to measure super high levels, for example, inside the air, you would need something that wasn't as sensitive because otherwise it would just be on all the time. So the genetically encoded indicators are very powerful and we take advantage of that. Um, but our go to is Fura two because you can just take a wild type bag or genetic knockout of some other type and just load them. Takes thirty minutes and you're done. And it's very, very reproducible. I didn't mention this, but the advantage of Fura is that it's a ratiometric calcium indicator. And so it it actually gives you a baseline and it gives you a increase. And so. What you can pick up for each egg doesn't depend on how much is loaded in or anything like that because it gives you this ratio of what's there to what is activated by calcium. And instead something like calcium green, um, is going to differ in intensity based on how well it's loaded or how well it was microinjected or whatever. And so that's the advantage of something like Fura. Do you ever worry that the, the only way that we can measure calcium is by adding molecules that bind to calcium, thereby modifying the free calcium concentration? I don't worry about that at all. And the reason I don't is that there are a gazillion calcium binding proteins in the cytoplasm already. And so what fura is doing to the system is likely incredibly negligible relative to that makes sense. What I do worry about though, is we're making all these calcium measurements in addition to be true. So What's going on in the oviduct in vivo? We don't know. Nobody knows. Um, we have a good sense, based on work from starting back in the seventies and 80s of what is in the oviduct. So a lot of people have tried to, you know, gotten oviduct fluid out from rhesus monkeys or from cows or from other species. You can do it. Mice. I pretty much don't believe anybody who tells me they've measured what's in the oviduct of a mouse, because it doesn't have any fluid at all that you can do anything with until ovulation, and then you can get a little bit, but that's probably mostly what's come in from the follicle. And then it goes away quickly. So it's hard to believe that. However, if you try to mimic what's in the oviduct, you can. But how do you know you're really mimicking it properly? So what we've been doing lately is developing mouse lines that can emit, that can express gcamp in the egg. And then our plan is to actually watch calcium signaling in the oviduct in vivo. And we're we're close. We haven't done it quite yet but we're very close. We have a, um, we have a mouse line that is expressing gcamp sufficient signal to be able to see it in the oviduct. And it's just a matter of getting some some of the experiments done now. So we're hopeful to see, you know, maybe all this calcium oscillatory behavior thing is something we see in vitro and it doesn't happen in vivo. Or maybe the frequency is different. Or maybe having just one spike is what happens. Like we have no idea. So it's going to be pretty fascinating to figure that out. I'm excited about that project. You would image directly through the wall of the oviduct. Yes. And what's cool about the mouse oviduct is it's so thin that you can actually see the eggs right through the wall. Um, and through the Bursa, which is, you know, a very, very thin, thin layer, like just a couple cell thickness. The trick is that you have to do this while the mouse is living, right? I mean, in theory, you could take the oviduct out and then do it in vitro, but that's going to totally mess with your culture. Medium, right? What's in the oviduct? It's going to change. So you have to do what's called intravital imaging. So the mouse would be fully anesthetized of course. And then they have to have made it shortly before so that, you know, you're going to catch the sperm actually successfully entering the egg and having that first oscillatory, that first oscillation that's so different than the others. So you have to kind of catch it at the right time. And, you know, the timing thing is kind of crazy to think about. But then you're imaging in vivo with an anesthetized mouse and we'll have to see what happens. Do you superovulate when you when you do these experiments? I guess the in vitro experiments we routinely superovulate because there are options. And we've actually done some comparisons of superovulation versus spontaneous ovulation in terms of calcium signaling. We, we we published a very simple paper a couple of years ago in, um, I think Frontiers in Cell Biology or something like that, the frontiers Journal essentially showing that there's no difference between calcium signals that you get from superovulated versus spontaneously ovulated mice. But to get the spontaneously ovulating mice, what we did was we used vasectomized males, and we mated them to the mice so that we could actually make sure of the timing of ovulation as close as possible, because if you just sort of hope to catch an ovulatory cycle and use vaginal swabbing and such to see when they might ovulate. It's challenging at best. And so our strategy instead was just to make a bunch of mice to vast males, vasectomised males, and then be able to collect the eggs and do the study. And I really thought there would be a difference. It was very disappointing that there wasn't. I thought we were going to be able to say, yeah, you know, superovulation is great, but you have to be cautious with your interpretation because it may be different than spontaneous. And then at the end of the day, it was actually pretty reassuring that it was no different. That's probably good news, at least for models of IVF, I would guess. Well, right. And also, I mean, I would hate to have to use a gazillion mice spontaneously ovulating because they each only are going to have on the order of ten or maybe fewer if you're using black sex. But we use mice that ovulate better than black six mice in twenty twenty. Your lab published a review article titled Modulators of Calcium Signaling at fertilization. And there's a really wonderful figure in there that contrasts the change in intracellular calcium across species with a host of different dynamics, from oscillations to these kind of switch like kinetics. What do you think about those maybe in a species dependent context? Would we expect that, uh, the effect of calcium will be similar or or different or or what are your thoughts about that. Yeah. So most species really don't do this calcium oscillations thing. I think muscles and mammals are it who really do an oscillation. Most of them, it just goes up and it stays up for a while and then they're done. I don't know how important the patterns are for any other species, with the exception of some like birds with huge eggs when they when the when the sperm comes in it, it gives a calcium spike. But the egg is so big that little bit of calcium doesn't do enough. And it actually needs what's called physiological polyspermy. So multiple sperm actually have to fertilize the egg before there's sufficient calcium to then trigger resumption of the cell cycle and to really begin egg activation, which I think is kind of cool. So then what do you do with all these sperm? It turns out that only one gets chosen to become the male pronucleus, and the other sort of get pushed to the wayside, and I guess they vanish eventually. So it's a very interesting system. I don't know why mammals oscillate or have to oscillate. I thought it was to protect themselves because if calcium levels go super high in a cell, it's very, um, it's very common for that cell then to undergo apoptosis and die. Right? You end up having too much calcium in the mitochondria, and they trigger the cascade and they die. And in fact, I thought that would be true in an egg as well. It's just that they would have a little bit more capacity, because the calcium that does go up normally for a minute or even up to eight minutes and then come down. I mean, that's still a high calcium exposure. But then we made a double knockout for calcium pumps that pump the calcium out of the egg, and the calcium goes up and it stays up for anywhere from three to five hours. And I fully expected them to die. No they don't. So here we are watching them on the screen. Calcium is up. It's staying up for hours. And what are they doing? They're forming pronuclei. It was incredibly disappointing, but it was still kind of cool. It's like they are so good at handling high levels of calcium that it can just be up for hours. So why do they oscillate? I don't know, it's it's kind of Fascinating. I mean, for sure the things that are downstream of calcium end up oscillating as well. So the calcium signal activates. Cam kinase to gamma is one of the major downstream regulators of the subsequent things that have to happen in the egg. And if you just measure Cam kinase to gamma levels, which was done by a set of incredibly challenging techniques by Tom Dulcibella in the I think in the two thousand, he basically watched calcium go up, and then as soon as the calcium started to come down, he would pluck that egg out and then do an assay for Cam kinase two activity. And what he showed is that Camkk2 activity oscillated just past the calcium oscillations. That was kind of cool. But clearly, given what we saw with the Pronuclear development, it doesn't matter. It can stay up and go up for a long time. and so I cannot answer the question about why they need to oscillate. It's just that they do and mammals do it. Everyone else seems to do fine with one long spike, and that's sufficient. It. You know, a high calcium spike triggers cell cycle resumption through its impact on cell cycle activators and inhibitors and and the proteasome in terms of degrading cell cycle proteins. And so that's sufficient for most species. It's just mammals are weird. But then again we don't even know if they do it in vivo. Right. As I as I said, I suspect they will. But you never know. Do you think the oscillations have a cumulative effect then? Like if you, uh, if you were to have a sustained high calcium level, does that speed up the process or does everything still happen on the same at the same rate? We showed a number of years ago when I was working with Richard Schulze. Um, that if you micro inject an active form of phospholipase C beta into an egg, which causes the calcium oscillations to go faster, that the pronuclei develop a little bit faster. So it seems like it does from that experiment. But when we have this knockout model where calcium goes up and stays up for a long time, it really doesn't seem to change the pace of development. So I don't know that it's an interesting question. I would have said a while back that yes, it did, that too much calcium would end up pushing the cell cycle faster, but I'd say it's still an open question. How important are egg intrinsic variations in calcium kinetics in determining fertility and or development? What changes calcium signals in the neck if we're not messing with it, right? If you're not doing us. Genetically encoded molecules or knocking out channels, one of the things that should affect calcium signaling is the health of the mitochondria that are in the that are in the egg, because every time you have a calcium spike, it activates ATP production from the mitochondria. That was shown by Greg Fitzharris a number of years ago. And so if you don't have good mitochondria, in theory, every time calcium goes up, maybe you're not making as much ATP as you should, or maybe you're generating a lot of reactive oxygen species or something like that. However, there doesn't seem to be any good data showing that if you don't have good mitochondria, it really does much to calcium signaling, and I'm not sure why that's the case. I suspect it's because there's a sufficient function. I mean, there are hundreds of thousands of mitochondria in every egg. I think a mouse has like six hundred thousand or something. And so it may be that even if they're not functioning at full capacity, there's plenty of ATP to go around to power the Serca pumps and the PMC pumps on the membrane so that they can do their jobs and regulate. If you block a mitochondrial complex one function using rotenone, which is a pesticide, you can disrupt the calcium signals a little bit, but not that much. So I think mitochondria are an issue. Most likely it makes sense that they would be, but it's not clear how much it changes things. The other place that has been looked at, um, out of Anna Archduke's lab is what is the impact of age, maternal age on calcium signaling. And they showed that some mouse strains had diminished calcium signaling with age, but others didn't. So it's pretty variable. And I mean, that's why I'm so focused on the medium and all this in vitro stuff because it has a huge impact. Whereas the more physiological parameters that you think about obesity is another one, like obesity affects mitochondrial function. That's been shown by probably a bunch of people, but certainly from Rebecca Roethke's work. And does that then have an impact on calcium signaling? I don't I don't think anyone's shown that that's the case either. Does it have an impact on embryo development? I think that's much more likely. Um, because mitochondrial function obviously is very important for development later. And there's certainly a lot of data associating obesity and aging with not as good embryo development, but whether or not that links back to calcium, I would not specifically link it back, even though going in to studies where I was trying to look at calcium signaling and in the setting of disrupted mitochondrial function, I was expecting to find something. And so far we really haven't seen anything dramatic at all. Does glycolysis play a role in energy metabolism? You want the short answer? No, that's the short answer. Although, um, my postdoc, um, Virginia Savvy, said that she had a long conversation at the Gordon conference recently with Pablo Visconti, who's convinced that oocytes and early embryos actually do glycolysis, even though there is a forty to fifty year literature on the idea that they don't that they don't do glycolysis, they don't have the enzymes, they don't need glucose. Up until about the eight cell stage. Then they can start using glucose. But we'll see. Maybe Pablo will turn the field on its head. So egg intrinsic variation seems to not play a very obvious role, at least in variation that you see. Um, do you think that, uh, environmental or kind of microenvironmental variation would have effects? So, for example, you kind of mentioned this, the metabolic state of the mother or, uh, in vitro capacitation media conditions, um, or, uh, toxicological effects. Yeah. So I think all of those things are going to impact the early embryo. I, I don't know how many of those things are going to change the calcium signaling at fertilization in a way that then has the impact. I suspect it's going to be more direct impact on other proteins, enzymes that are regulating what needs to happen in the early embryo. Because if you think about it, at egg activation, you know, you resume the cell cycle and you sort of kick things off. The sperm head condenses, you know, but then you begin this whole process of genome reprogramming. And so, you know, you have to get rid of protamines, bring in histone proteins on sperm side, the maternal side, you have to complete meiosis and kick out the other polar body. And then you have to start reprogramming the whole genome as the embryo first begins transcribing from its from what we would consider an embryonic genome rather than what was given to it from the mom, which is essentially a lot of stored maternal RNAs. And so that whole reprogramming that in the mouse occurs between the one cell and, you know, blastocyst stage is where it's all happening, but it begins at the one cell stage and critical events all the way up to the time of zygotic genome activation at the two cell stage are going on. One of the things Virginia Savvy has shown now, and has out in a bioRxiv preprint right now, is that the calcium signals actually drive the TCA cycle in the mitochondria. And on top of that, having too much calcium signal completely reduces the amount of ribosomal RNAs that are made in the early embryo. So one of the things that happens in the early embryos, you click on ribosomal RNA synthesis in a big way as soon as fertilization has happened. Well, in in the presence of too much calcium, that doesn't happen. And so you're not making ribosomes most likely. And therefore your ability to translate RNA into protein is diminished. And you've also she's also shown that the calcium signals, when there's too much mess with epigenetic marks that are important for reprogramming. And so even if you're even if your calcium signaling alone isn't doing a lot at the time of egg activation, the implications for what's happening to the later embryo, I think, are very nicely tied back to what's going on with the calcium in ways that you may never know what the impact was, because these embryos go on and make blastocysts. Was there anything that I didn't ask about calcium and egg activation or metabolism that you'd like to talk about? I would make a plug for Virginia's work and the bioRxiv preprint she's got out there, because it really shows a nice connection between the calcium signaling and metabolism that I think is going to be super relevant for embryo development later. Are there any other ongoing or recently completed projects that you'd like to highlight that we haven't already discussed. I haven't mentioned at all the work that Wendy Jefferson has been doing on the impact of estrogenic chemicals on development, and Rachel Bainbridge, as well as a postdoc who's working on that project. And half of my half of my lab actually focuses on endocrine disruptors. And why, what is it about a developing organism that makes them particularly sensitive to what's out there in the environment? And I guess that aspect of the work that we've done is really coming to a head now in terms of thinking about what is it that these steroid hormones do during development that changes differentiation trajectory. That's important for fertility. And so I just want to mention that as a as a topic that I think is super important and something that could get attention from a lot of people as a Relatively open area. We've been looking at the estrogenic chemical aspect of it, but thinking about other exposures and how they impact differentially during development versus in adults, I think is a very important area of study because it is so important for kids developing in the world today. For example, what stages of development are you looking at? We're using a mouse model to look at the differentiation of the female reproductive tract. So we use neonatal exposures because that's when the mouse reproductive tract has formed. Like you've got the full sort of tubal structure that's formed. But it really hasn't differentiated yet. And so that's what we're looking at that's relevant to what goes on in a human starting in the third trimester, late second or early third trimester. But in humans, that differentiation process is still ongoing all the way through puberty. So I'd say that this model is particularly relevant to female human development. And if you think about it, prenatal female development in neonatal intensive care nursery, where there's still really doing differentiation of the uterus and all the way up until they begin having cycles. So that's the relevant timing. Depending on what you're looking at, different timing might be more important. For example, for male reproductive tract development or testis sort of questions. That's going to be more maybe peripubertal, although you'd probably know that more than I would being a sperm guy, but it depends on the thing. You also have to think about brain development, right? So brain development is going on in humans, certainly after birth, all the way up until puberty and probably beyond puberty. And so somebody's thinking about sort of neuroendocrine impact of exposures, would want to use models that actually reflect postnatal development to our estrogenic chemicals. Chemicals that are are disrupting normal estrogen production, or are they environmental analogues to estrogen that are acting as estrogens in the organism? So I wouldn't call it an estrogenic endocrine disruptor unless it was behaving as or interfering with the interaction of estrogen with its receptor. In theory, for example, an aromatase inhibitor could be considered a endocrine disruptor that's affecting estrogen, but in itself is not estrogenic. And so when I say something is an estrogenic chemical, I'm really thinking about the acting like an estrogen binding the estrogen receptor or interfering or binding the receptor in a different domain like tamoxifen can do. For example, are estrogenic chemicals common in the environment? Yes, in a relative sense. For example, bisphenol A that everybody worries about being in that plastic water bottle I just saw that you drink from, um, is an estrogenic chemical. It is a very weak estrogen. Um, but that doesn't mean it can't have effects. The bisphenol substitutes, like BPA and BPA and all those are probably the same. So that's one that's pretty common. What I think about more based on amount of exposure is plant estrogen. So phytoestrogens because those are made in soy we eat a lot of soy products, um, particularly babies who eat soy based infant formulas are exposed to a lot of plant phytoestrogens that have estrogenic activity. And that's been shown in studies based out of actually nees. Done by Walter Rogan where they looked at babies who drank breast milk, cow milk formula or soy based infant formula and saw that the girls basically had long term effects at like nine, ten, eleven months of age on their vaginal cytology, indicating that there was an estrogen effect. Now, it's not a super strong estrogen effect. Then again, that's a time they're developing. So because that exposure is so significant, that's the type of exposure I worry about more. Or for example, people who are going to GNC and pulling out a bottle of, you know, whatever it is and using it as a nutritional supplement when it's really not characterized. That type of exposure is what I worry about more. The ones that are just sort of in the environment, you know, if you're unlucky enough to be downstream of a facility that's dumping chemicals into a river like the Cape Fear River in North Carolina, for example, there can be large enough exposures to have impacts. Where do you think your research is headed in the next few years? I would say thinking about metabolism has now entered my vocabulary, whereas it happened a few years ago. Virginia is going to take most of that project with her, but we may continue to at least think about how metabolism is impacting early embryo development in whatever she's not doing. So that's kind of that's that's a fork in the road that we're thinking about. I've also started a project in the last few years looking at how organelle interactions might be changing calcium signaling. So one thing that's pretty well established in somatic cells is that mitochondrial ER contact are very important for adjusting the amount of calcium that goes into and out of the mitochondria, basically because it fits released locally when they're very close together, then the mitochondria are going to take up a lot more. So we've actually just been trying to characterize what kind of membrane contact sites are there between ER and mitochondria in oocytes. And do they change during maturation in an early embryo development. So that sort of organellar interaction thing. I've gotten very interested in lately. I think it's a it's kind of cool and maybe important. It's a bit challenging to look at because the techniques are are tricky. We're using a lot of em and, and doing fib-sem just focused ion beam scanning em to do volumetric reconstruction of oocytes, eggs and early embryos to be able to figure out what's going on with these contacts, as well as some live imaging methods. But it is tricky. What do you think is the most important open question that scientists coming into the field should focus on. Well, when thinking about egg activation in early embryo development, all the reprogramming. I think the most important question is what can we learn that can then improve what you do in an assisted reproduction clinic? And so finding ways to sort of translate this, you know, almost a wealth of basic science information from both mouse and bovine and other models to what goes on in the clinic, I think is it's a big challenge, but it's super important. And some of the clinics appear to be very interested, at least in trying to do some substantial research. That means that they're doing a better job and making the embryos healthier at least. Although of course, they have to worry about take home baby rate, and they're not so worried about whether the babies then have high blood pressure later or something else. It's it's tricky, but I think that's a huge area where the basic science could be translated in a way that's important. Is there anything that I didn't ask that you would like to discuss? Yeah. I mean, the main thing I would like to say, and I kind of highlighted this when I received the award and, and talked about it at the meeting itself is all the work that we've been doing. And the basis for this award is on the backs of so many really, really outstanding fellows and students and so on. And it's a lot based on their ideas and their energy that somebody like me can succeed in thinking about. Um, a lot of sort of disparate topics. I mean, all the metabolism work that we've done is because Virginia got interested in it and is really excited about it, and she actually taught me a whole lot of this stuff. And so I really want to say, you know, if, if, if you think a Pi is very successful, just look next to that Pi and see who's standing there. And that's really where a lot of the energy and the success comes from. Thank you again so much for taking the time to do this. Sure, I appreciate it. It was a lot of fun to talk about the research and also about the people who've done it. Well, listeners, that does it for today's episode. 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. 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