E42: Sperm Capacitation: Molecular Mechanisms with Dr. Pablo Visconti

Welcome to Future Conceived, the official podcast of the Society for the Study of Reproduction.  In this episode, host Cam Schmidt chats with Dr. Pablo Visconti, Professor of Animal and Veterinary Science at the University of Massachusetts Amherst. Sperm maturation doesn't end with ejaculation, but instead continues in the female reproductive tract through a series of complex biochemical changes. This maturation process, known as capacitation, can make or break sperm success and its independent discovery in the 1950's by Austin and Chang helped pave the way for the first successful in vitro fertilization. Dr. Visconti has been studying the molecular mechanisms that control mammalian sperm capacitation for more than 40 years. This discussion offers a fascinating window into Dr. Visconti's approach to this problem and how his mechanistic insights have evolved as knowledge about sperm capacitation has accumulated over the course of his career.  

Transcript

This is going to seem a little backward, but I have to welcome you to the show so I can get that audio. And then. And then I'll thank you. Great. We're putting the sperm in the testes. Hello and welcome to the future. Conceived the official podcast of the society for the Study of Reproduction. My name is Cam Schmidt, assistant professor in the Department of Biology at East Carolina University. As you probably know, reproductive biology is complicated. Molecules organize into cells, cells organize into tissues, and reproductive tissues form and degrade over time under the control of complex hormone cycles. Studying these processes in a systematic way is no easy task. How should we think about how all this complexity fits together? Perhaps more importantly, how do the models we use to describe these systems influence the way we conduct our research? The philosopher William Barrett once said, we have come to understand the phenomena of life only as an assemblage of the lifeless. We take these mechanistic insights to be ultimately concrete and fundamentally real. This concept of mechanistic insight defines what we do as researchers. Mechanistic insights are codified in the scope of peer reviewed journals, and often demanded by peer reviewers of our manuscripts and funding proposals. But what exactly is a mechanism? Does mechanism mean the same thing at the scale of molecules that it does at the scale of cells? Does mechanism mean the same thing to a physiologist as it does to a physicist? This season on the Future Conceived will be conducting long form interviews with researchers across disciplines who are trying to understand how biology works. We'll learn from geneticists, biochemists, systems biologists and physicists about sperm, eggs, and embryos. Our goal is to understand how the pursuit of mechanistic insight Influences their approach to research and what they see as a comprehensive explanation for the phenomena they study. Today, we'll begin by exploring the cell signaling mechanisms that control sperm capacitation. In nineteen fifty one, two researchers, Minghui Chang and Colin Russell Austin, independently reported rodent sperm are not competent to fertilize an egg immediately following ejaculation, but instead must gain this ability during residence in the female reproductive tract. This discovery had enormous impact on the ability to perform successful in vitro fertilization in rodents, which ultimately paved the way for human IVF. Doctor Pablo Visconti has been studying the biochemical mechanisms that control sperm capacitation for more than forty years. He's a professor in the Department of Veterinary and Animal Sciences at the University of Massachusetts Amherst. I had a lot of fun recording this conversation with Pablo, and I hope you will enjoy learning from him as much as I did. Pablo, welcome to the show and thanks so much for joining us today. Thank you. Thank you very much for inviting me, Cameron. It's it's a pleasure. Maybe we can start by introducing Min-hsiu Chang and just understanding. What role did he play in this question to begin with, and. Yeah. I don't want to forget Austin, but the reality is, like, I don't remember Austin's first name, and I always like to remember that, but I don't remember the that at this moment I have a blank. But obviously I remember and yeah, I can I can talk about him. He, he also had done the first successful in vitro fertilization in rabbits. Is that correct? Yes. That's correct. The whole story about Ming Cho Chang, because a Doctor Chang is my idol. I have many idols, including him. Doctor And my former mentor Greg Kopf. A but but is the one that preceded all that. Uh, he, uh, he came to the to the US to work in the Worcester Foundation. That is also a very interesting story, because Gregory Pincus was the director of that foundation, and he was the first one to claim to have done in vitro fertilization in rabbits. However, nobody could repeat those data. And people don't believe that to to much because of that paper. He or despite that paper, the it was extremely controversial. It was early twenty thirty something like that. I don't remember exactly. Uh, he did not get tenure in Harvard and with friends that believed him. He was a very brilliant scientist. They got money to get the Worcester Foundation. Now, the Worcester Foundation was not what it would be later. And it was not what we, we, we think about the modern lab. It was mostly in, in I have never been there, but it's it was not like high level laboratory there a now Mincho Chang he wanted to do it forever. It's like he has that in mind and he has some projects and all that. And so the person to do that with was Gregory Pincus. So he wrote him to come to, to the US and he finished working in the Worcester Foundation. Now he, I although I met him, it's not that I know him, but my impression is like he was a storm of nature. It's like he has so many ideas, so many things. So I'm in the Worcester Foundation, did three amazing things, and I will describe them more or less in, in, in the, the the range. The first one is a was part of Gregory Pincus a team. And he did the first paper I believe it's the first one showing that an injection of progesterone block ovulation in rabbits. So he's the father together with Gregory Pincus of the contraceptive pill. So that paper was published in the Acta Latin America now or something like that. But it's one of the Most highest impact, a discovery because it gave the possibility of doing the contraceptive revolutions in the seventies, sixties and 70s. Okay, in nineteen fifty one, a completely independent, the one who was working in Australia didn't know the work of Ming Cho Chang Ming. Cho Chang was working on the same foundation, but Austin and Chang published a two amazing papers and high impact papers. A both of them, probably. They were working in a well, I know Chang, but Austin I believe is similar. There was a big problem despite Gregory Pincus paper in some time before that nobody could repeat IVF. In mammals, in vitro fertilization was common ground in Grounding amphibians in other in other species, but in mammals was impossible. So they have this a paper in which they showed that the sperm needs to be in the female track. In order to gain fertilizing capacity. These type of experiments were done first with a like kind of in vivo is like they they I it's it's I have read these papers before but I don't claim that I remember exactly the all the experiments that they did. But both they came with the same conclusion that the sperm after ejaculation is not able to fertilize. I always say this, that if you put an egg and sperm, the sperm will be very happy around the egg, swimming and all that, but will not fertilize the egg. A so they discover that they need to have like a this exposure to to female track conditions to gain that fertilizing capacity. And they call that capacitation and believe that Austin was the person that coined the term capacitation. Both papers publish, as I said, nineteen fifty one, very important papers. But the most important part of those papers is what came later. That is a once a doctor, Chang realized that Capacitation was needed. He used capacitation to get in vitro fertilization. And that's. This is how in vitro fertilization started. So when I think about the physiology of sperm. I like to say that they have two fundamental tasks that they have to find the egg, and they have to recognize that they've bumped into the egg and not something else. Do you think that's a comprehensive summary, or is there there more that you would add to that? Those are obviously two very important things to find the egg and and to recognize the egg. That's the basic of species specific recognition. Now the sperm is bringing chromatin to to the egg is is a transport. And that that chromatin is very modified during spermatogenesis. And it is also at least this is current research by many people, including my colleague Oliver Randall, that the sperm is bringing information that is beyond the. The DNA sequence. Okay. Now our research, although obviously research I cannot tell you that this is correct or not, is like a. We have results kind of indicating or kind of suggesting that you can also during Capacitation, you can modify certain aspects that will be important in post fertilization events like chromatin condensation and maybe some epigenetic changes. So I, I tend to think that that the sperm function is a what you said is recognizing the egg and all that, but it's a lot about information. The obvious part is that the spam brings all the mail, a genetic information to to the embryo. And the second part is like what happened to the sperm during the epididymis, during maturation, during capacitation in the female track. It might also be relevant for future events after the sperm fertilize. So in the context of what we know, maybe focusing on the in vitro condition, what changes do sperm have to undergo during capacitation with regard to those kind of fundamental physiological tasks? The part of the chromatin condensation we don't know yet. In theory, what is textbook is that the sperm is condensing the testes. I'm starting from the last part to take that away. A, A and a. Once it is inside the egg, the the protamines are released. And that's how chromatin decondensation starts. Now, if there is something going on during fertilization and during capacitation that helps that process. It's only hypothesis that we will need to test very carefully. So taking that away then we have other aspects of capacitation and and signaling. So what's going on. Is that what you can observe in most mammalian species? I'm talking mostly about mammals. In in this talk you have changes in the motility pattern. The in some species is kind of fantastic. I, I, I hate that hamsters are so expensive because they they are such beautiful, beautiful species to work with the sperm. Um, I work with them during my, my PhD, and, uh, it's kind of a rocket in comparison with the mouse sperm or human sperm. They move really fast. They are beautiful. You can see the acrosome in phase microscopy. You don't need to stain. It's a very interesting species. And the motility is fantastic. And you can distinguish is textbook. It's like you look at the hamster and it's textbook. You the hamster moves like completely progressive, very fast, progressive motility. And suddenly when they become hyperactive, all the sperm start doing eights. Like, like if you do a textbook and I want to explain hyperactivation, I would explain it like this in reality. In other species is not as fun as in the hamster. The change in the symmetry of the flagellum movement is not as obvious as in the in the in the hamster sperm, but you can see it with your own eye and you can quantify it using computer assisted sperm analysis. Casa that many, many people use, at least in basic science, to measure motility and to quantify the number of sperm, the velocity and that type of thing. So during capacitation, these changes are important. And hyperactivation has been shown with genetic models that those sperm that cannot hyper activate, they cannot fertilize, they are sterile. And the type of movement is very important. So that's one of the things. So a big part of research, uh, on, on these aspects has to do with the signaling pathways that promote a sperm hyperactivation and sperm fertility in general, as an example. That is a very common example thanks to the work from David Clapham, follow now by Jin Ju Chung. Uh, I, I have big admiration for all this work. Uh, the catsper, uh, knockouts of catsper are not able to to fertilize. They are sterile. The sperm moves, but it has a very strange movement. And when you put it close to the egg, you can put tons of sperm there. None of them will fertilize. Uh, and One important aspect there at the physiological sperm physiology level is the movement of of the sperm and the lack of hyperactivation at at the same time. Obviously, catsper is a calcium channel and it has problems with calcium. And calcium is very relevant for for this this movement. So there are other models, for example, has done the first knockout of a potassium channel that is specific potassium channel that is called slow three. And this potassium channel also is essential for motility. But also it has a role in the acrosome reaction in the preparation for the Acrosome reaction that we have not talked too much. Up to now. And I was going to it. It's a good transition to talk about the second physiological change that you can observe in in sperm. So this depends a lot of in different species is different and also depends on the capacitation conditions and all that. But. Chang doctor Chang when he described capacitation he described described it and define it as everything that happened to the sperm that prepared sperm for fertilization. And an essential part is the Acrosome reaction. So Doctor Chang considered the Acrosome reaction part of Capacitation. Now, for many purposes of study, the process we tend and this come from from Harvey Florman man. Also, a very important review with Donald Babcock. Many years ago that he started to define capacitation more as the preparation for the action reaction. So you the capacitation the sperm is prepared for the acrosome reaction, but is not acrosome reactive. And then when a. At the time the hypothesis was the sperm arriving to the zona pellucida, zona pellucida induced the Acrosome reaction regardless if it is the zona pellucida, because it has been kind of demonstrated that in the mouses that's not the case, but still in other species is an open question. The. But regardless what it is, we for research reasons and to organize and classify our our mind a Most researchers in the field. We use that definition, that capacitation induced hyperactivation on one side and prepare the sperm for the acrosome reaction on the other one. And then it's more philosophical. If then after the Acrosome reaction is part of capacitation or not, but but for instrumental reason for designing experiments. We said that. Now, Cameron, I can tell you one part that is for me a completely amazing and that is the part that okay, even if I have never seen anything else for me to see, that is something that. Changed my mind about all this. Okay, the Capacitation media is a very well defined media. It's a very simple media. You can do in vitro fertilization practically with some salts, some energy substrate. You need BSA that nobody understands exactly what it does. A different forms of BSA. Obviously bovine serum albumin is not like sometimes I need to discuss with reviewers because they said, well, bovine serum albumin is from bovine. It's not physiological. Yeah, we know that. So yeah. But okay. It's a trick to mimic albumins in in the, in in the female track. But but you need albumin and there are differences with albumin and all that. But that's it. Nothing else. It's not like you need serum. You need unknown things. You need amino acids. You need you can do everything with a very, very simple media. Now, every part of that media if you remove it. The sperm cannot get capacitated. So. Maybe there are some things that are less important, but let's say bicarbonate and BSA. When I see sperm that is incubated in the absence of bicarbonate and BSA, and you add, for example, solubilized zona pellucida or progesterone, nothing happened. And we can follow iron changes like calcium changes and acrosome reaction and that kind of things. Nothing happened. You incubate them with bicarbonate and BSA. You put progesterone, calcium increase and many sperm acrosome reacted. So sometimes I see the traces and I think it's completely incredible because the there is kind of a clear thing that the bicarbonate and the BSA are doing there. Okay. So. For me, that has always been the the the big mystery is like what these compounds not only BSA and bicarbonate because we also work with sodium and chloride. Uh, obviously with calcium. So all these components, what they do in order to capacitate the sperm and change so completely the response to, to agonists or to many other things. So the questions about a non cap and cap are those many times it's like how how you do that. So as a trick for many years. I started these type of experiments when I was a PhD, but I continued this one. It was with Greg in Philadelphia. The approach has always been, okay, we have this sperm. We incubated in the media that they are very happy, but it has no bicarbonate, no BSA. And we call that non cap media. And then we start replacing the. We add bicarbonate. What happened. We add back BSA. What happened. And that sperm suddenly become fertile. We did the same with calcium. We did the same with sodium with chloride with all these components changing these type of things. And this was a way of starting dislocating the the system and trying to understand what each of these compounds that the most interesting for, from my point of view that most of of our research went on that were bicarbonate, BSA and calcium. So. That generates a lot of puzzles. A several signaling pathways are cross talking and have a positive and negative feedback, one on the other one, making the problem much more difficult of what we usually think about it, because it should be a very simple linear signaling, and it's not. There are many parts involved. Now, what is clear is that the main action of bicarbonate is to activate the cyclic AMP levels and that initiate all the processes in. Mammalian sperm. So at least the ones that I have studied. So without bicarbonate nothing happened. And you need bicarbonate cyclic AMP is induced. And then it becomes the problem with cyclic AMP is doing okay. The obvious part is that it activates protein kinase A. That is the main what I would not say the main the most the the best studied a cyclic AMP effector is protein kinase A, and that for sure is happening. We have shown that many people are investigating on that. And if you do a knockout of the protein kinase, say That is a sperm specific splicing variant in in the sperm. The sperm are infertile. So there is one of the parts. But cyclic AMP is doing other things also. And you have a sodium proton exchangers that appear to be regulated by cyclic AMP because they have a very clear cyclic AMP binding domain. The system becomes more complex. I'm not going to talk about all these little peculiarities, but what I want to to to tell you about this little story of cyclic AMP, that everything that the chemists like that are linear pathways. I love chemistry because of that. All the linear pathways. Well when you have a cell they are not like that. You have system biology and you increase one. You put bicarbonate. And yeah the first part is Parties likely to be only synthesis of cyclic AMP, maybe increasing a little the pH. But after that many, many things happen and there are many puzzles there that I'm most interested in in solving like temporal and spatial. A is like the sperm is a very peculiar cell. It has a, morphologically speaking, it has a head that is compartmentalized in many domains. It has a tail that is compartmentalized in many domains. You have a midpiece with the mitochondria, you have a principal piece. You study metabolism. And although kind of controversial, the dogma today, and I believe that dogma is that all the glycolytic enzymes are in the principal piece and oxidative phosphorylation occur in the midpiece. So they are very compartmentalized because obviously there is a crosstalk between them, but it's not obvious. It's not like in other cell types where things happen very close. And hexokinase in many cases are are attached through porins to the to the mitochondria. And everything is happening in that place. In the case of the sperm is different. We don't know how difference, but we know that what we try to dissect in a way that making make this comprehensible, to write a review a it's in reality, it's much more complex, and we are all aware of that. Nobody can say that the whole story is a bi carbonate bicarbonate activity. Activate. Suck suck suck. Activate cyclic AMP, then pKa activates calcium entrance or something like that. By the way, that's controversial. Also, some people say one thing and some people other, but doesn't matter at this moment. What I'm trying to to to the take home message is that there is a complexity in pathways that we try to simplify in order to study a particular parts. My questions here is how those are translated to prepare the sperm for the acrosome reaction. How if this is diffusion, if there is some automatic change when you modify the tail, that goes to the head. And one of my favorite hypothesis is that membrane potential plays a big role on that. Because once you activate a slow three, potassium goes out, the sperm gets hyperactive. A not so hyperpolarize. The membrane potential is hyperpolarized. Not to confuse with motility. And once the sperm is hyperpolarized, this is transmitted almost immediately to the sperm. Because the membrane potential is is something that that it will be the same in the head and and in the tail. So those changes in, in in the membrane potential has the potential to be signaling to very different places in in the cell. So that's one of my favorite hypothesis. But I cannot discard also that some of the ions like calcium for example, can be going from one place to the other in in the cell. That's another possibility. Same with pH And that type of things. There could be diffusion that also is part of the signaling. Many interesting puzzles in in the spawn world. So you mentioned that there's a kind of minimal media composition of, uh, bicarbonate and some sort of serum albumin. Um, and in the absence of those exogenous factors, then you don't get capacitation and you don't get fertilization. So, so I think it's interesting that like, I would imagine, um, you know, sea urchin sperm probably don't depend on bicarbonate and serum albumin. I think the connection there is that this environmental chemistry is probably indicative of the ovulation state in some way, or correlated with the ovulation state in some way, or maybe correlated with the process of ejaculation, like you mentioned that, uh, bicarbonate is in seminal plasma as well. Do you think that those chemical conditions reflect the timing of fertilization in some way? Like, is this a way that sperm anticipate that timing through this biochemical mechanism? Well, first the question is very good. And I will divide it in two, because you mentioned the sea urchin sperm and you say bicarbonate will not do anything there. First of all, I'm not completely sure because the soluble is in sea urchin sperm. Obviously BSA will not do anything there, so the pathways are different. Before answering the second part of the question that is extremely interesting, I want to to to tell you a little about sperm species, is that I believe that the at least my hypothesis is that evolutionary. What has been changing is the regulation of very basic components that are important in every species. And they changed the way that these work. And in some species they might use more one type of uh, of mechanisms, in others more one another type. But in most species you will have all these basic molecules present. And what will change is the type of way that you regulate the, the, the intracellular changes in concentrations. As an example, fish, one of my favorite, uh, maybe because I have a colleague that works with cichlids at the University of Massachusetts and the cichlids are amazing. I always wanted to work with with that, but not not a lot of money to to do this type of research. But but these evolutionary are great because secrets are like thousands of species that live in completely different environment, in many different lakes in Africa. The most common one that is delicious is tilapia. But that's a secret. But the interesting thing is that they they live in places that has such a high salinity and a species that lives in freshwater. Okay, fish sperm are activated after releasing water. And when they are in freshwater, it's easy. We have done these experiments and they are great because you release the sperm in a media, physiological media or a little high sodium media. The sperm are completely in model. You dilute that and the sperm start I'm moving. So this is an osmotic a mechanisms. I will not talk about the mechanism because I don't know enough about the signaling aspects and all that. But obviously it is doing something with the with the ATP indices and all that. But so the other thing is like the same, a very related species because it's also cichlid living selling water. And the sperm there are not activated in that way. So what I always wanted to in my mind I would love to investigate is like you can transport the slowly selling living fish to a fresh water. You cannot do it immediately because they will die. But if you do it, you start really reducing the salinity. You will. You will be able to, to to to convert one. And my question is how the sperm behave is like they become sterile because sperm is not able to fertilize anymore. Or sperm proteins adapt completely to the new media. And now they become expressing receptors that are regulated by osmolarity. Many of the things inside sperm, in sea urchin sperm and all that. Obviously they are regulated in a completely different way from the outside, but the molecules inside, they are very conserved. You always have calcium, you always have cyclic AMP. In some cases, like the sea urchin, you have cyclic GMP. That is essential. They are all those signaling mechanisms has a lot of things in common. Changes in membrane potential So one very interesting collaboration that I have is probably the closest because we have been collaborating for many, many years is with Doctor Alberto Garzon, and he's an expert in both. He has published many, many papers in sea urchin sperm together with Benjamin Cao, probably are the two big people in sea urchin sperm. Well, obviously there are many others like Viktor Bakir and there are other people. But in terms of the signaling aspects. Benjamin and Alberto has done amazing work. Both of them are very interested in how these type of things are correlated with with mammalian sperm. So that's a part of the the the answer that evolutionary in many of these systems, from my point of view are A conserved and what it changes the way that you regulate them. Not the signaling aspects per se. That is a really interesting way to see that. Yeah. So just so I'm understanding the the internal signaling process, which in this case would be using cyclic AMP as a second messenger system for kinase activation that remains evolutionarily conserved. And it's probably controlling the same kinds of behaviors like a transition from progressive to hyperactive motility and initiation of an acrosome reaction. I would say I would not go back that far. That cyclic AMP will always do a very similar thing and all that, but cyclic AMP will be involved in many species. Calcium will be involved in many species. Obviously ATP without ATP the the sperm will not move. Dyneins of course are involved in all the species and what it change is like the type of regulation. In some cases you will need a receptor mediated signal. Like in sea urchins you have the peptides and all those that that will initiate certain parts. Sorry if I I'm not an expert in sea urchin sperm, but but there are a many things that are different than to mammalian sperm. I'm not claiming that they're the same. Even for metabolism in the sea urchin, sperm is completely mitochondria a release a production of of ATP uh and in mammals glycolysis is extremely important. And so you have these type of differences. But the fundamental molecules are always regulated to to get Fertilization happening in nineteen ninety five. And I think you were in Greg Coffey's lab. You wrote a two part report that's collectively been cited more than two thousand times. It demonstrated a correlation between capacitation state and protein tyrosine phosphorylation. And then the second report detailed the cyclic AMP dependent pathway that regulated that effect. Can you talk a little bit more about the significance of that finding? Okay. Yeah. Well, the significance of those findings is that they made my career. So thanks to those paper, I got positions. I got my green card also, and many things, because many people wrote me support letter for my my green card in the in the United States. But let's talk about the science. Okay. I, I have different views of that. A I. I think those papers were so highly cited. Not so much for the discovery of tyrosine phosphorylation, although we will talk about that also, but because I maybe I'm bragging about this, but I, I believe that that gave a methodology of how how to study capacitation process because it was a very a textbook way of saying loss of function. Gain of function is like, okay, this is the media. And the first observation was that we in this media, over time increased tyrosine phosphorylation. That probably is figure one of of the paper. I don't remember exactly. So then I will I would say, well this is in media that contain this, this and this and this. And I chose three components by carbon and BSA and calcium. And I started very simple loss of function experiments. So I took bicarbonate. Nothing happened. I took a no tyrosine phosphorylation and took BSA. No tyrosine phosphorylation I took calcium. No phosphorylation. And then I showed that the sperm were not able to do this. And that's it. Of course, the paper is a little more complex because we we correlated that with capacitation using some old assays discovered by Byatts story. The it's called it's called Chlortetracycline assay to follow Capacitation. We we we measure also the the zona pellucida induced I would call it now Solubilized zona pellucida because. yeah, as I said before, it's kind of controversial. How physiological is that? But it is very clear that the solubilized zona pellucida induce the Acrosome reaction. And I show that all these systems correlate with that. Now, because of my background during my PhD, working with with what we know now is suck. But at the at the time we did not know that was a a a sperm site that was dependent on bicarbonate. When I saw that bicarbonate was so important, I decided to bypass bicarbonate with cyclic AMP. And again this was gain of function. So I do the loss of function and the rescue experiment with the gain of function. So now cyclic AMP induced tyrosine phosphorylation. It Brightest. Burn like a lamp. Now what? Make those papers? So cited is. Well, first of all, as I said, the methodology was very simple. So it was extremely reproducible. I'm not saying that other papers are not reproducible. Probably most papers are reproducible. What I'm saying is that there are some techniques that are complex. And to repeat that in every lab in the world, it's not so easy. The techniques that I put there can be done by an undergraduate student, learn that in no time, and repeat my papers in in a couple of months and will have exactly the same results. I believe that that also make the the the relevance, not relevance, relevant, but the fact that it was so reproducible and so easy to do that everybody repeat it, and when they repeat it, they cited the original paper. So I think that that methodological part, not methodology because of the method that is Western blot, the the theoretical method, like the logical approach to Capacitation was what has been a good contribution of those papers. Now, going about the discovery per se, it's still passing, but the tyrosine phosphorylation is and. I obviously all these I learned from Gregg and I was always in agreement with that. It's like I'm going to say something very important for research. At least for me. Eliminate the work. The the word demonstrate from research vocabulary. It should be eliminated. It's not in the dictionary. We don't demonstrate absolutely anything. We only have experiments that are consistent with the particular hypothesis. So if you read all these papers, you will see something that I still use a lot when I write. That is the word associated. So I put capacitation does associated increase in tyrosine phosphorylation. I've never said in any of my papers and I publish a lot with tyrosine phosphorylation. Tyrosine phosphorylation was the the most relevant thing of capacitation. What I said is like in all the conditions that I try that the sperm is capacitated you get tyrosine phosphorylation. The thing is, like many people repeated this in different species, in almost all species. You see the increase in tyrosine phosphorylation. But now I come to the a very important paper. As always, I, I put Doctor Jin Joo Joong in one of my high, high levels of of research. Sperm researchers I present at this moment and she published a for many reasons. A very important paper in life, but for particular reason. One of the most important parts there. Is that her hypothesis and her results suggest, and in a way indicate for not using demonstrate. Because I hate that word is that the fertilizing sperm has perfectly aligned catsper channels and no tyrosine phosphorylation. So he he came with an hypothesis that I always I always thought about that. And that's one of the reasons that call it associated and not a proof of capacitation. A the hypothesis is that A. Phosphorylation is is marking those sperm that are not going to fertilize, not those that will fertilize. A still more work needs to be done about that, but it perfectly well. Might. Be the the reality is like I I well everything that Jinju writes I take it seriously But in this particular case, I take it very seriously, because the reality is that we found out the tyrosine kinase that phosphorylate tyrosine phosphorylation pathways because of tyrosine kinases. That is called for T. It's another long story. We found it. Finally, the knockout of thirty has no tyrosine phosphorylation. And in vitro we can see some things. But in vivo is completely fertile. So in theory you don't have tyrosine phosphorylation and the sperm fertilize completely normal. So tyrosine phosphorylation at this moment fascinating topic because something is doing there. But maybe it's negatively correlated with capacitation and not not with the capacitating sperm. What is a Capacitated sperm, the one that fertilize and chinchou. Experiments suggest that those that fertilize are exactly the ones that has no tyrosine phosphorylation. So all these stories are. Very interesting. And in a way, as always, we're always starting. Because if it's not there, why it is all correlated with capacitation. Is there kind of a signal for protein degradation? I don't know, we we don't know about that. It might be that tyrosine phosphorylation is important in the beginning of Capacitation but then needs to be shut. That's another possibility. But we don't know about that. So we know this change in biochemical conditions activate sperm. And this specific biochemistry is responsible for that. So bicarbonate and serum albumins. So I imagine there's sort of two possibilities there that the the sperm are now on this kind of temporal trajectory toward a maturation state that can fertilize. And either they get to that state and they remain in that state for a while, or let's say, indefinitely. Or do you think this is maybe more parabolic, like they mature and then die or something like that? Are they necessarily working within a finite time window to fertilize, and how would that affect the way that we interpret seeing these changes in the sperm? Okay. That's a very good question. That is more a is extremely difficult to answer, at least with my tools or even with present tools. The people I mentioned before, the people that have been more productive on these were people like Susan Suarez, Mariano Masahito, Ikawa, and there are others also, of course, that has been trying to answer some questions of what's going on in Xvivo situation. It's very difficult to follow that. And in reality we can say if we follow Chunk's definition of capacitation, that only one sperm is capacitated because it's the only one that fertilize. Now, how many are capable of fertilized fertilization? That's a different story. So I believe that it's more than one. Now, what happened in the female track is extremely interesting because my view of this is by only by biochemistry. I, I cannot. I cannot see how such a accident is not immediately activated as soon as the sperm is ejaculated. It's like that's happening automatically. So I believe that PCA will be active from the the starting point of of the female track transit. Now, what happened with the other systems? I don't know, eh? Now, I can tell you a little more about kinetics in vitro. And those are very interesting. I will put only one example here. PCA is PCA. Phosphorylation is activated in minutes. I would say maximum in one minute. Maybe I'm wrong plus minus because Western blots are not the best real time type of experiments, but one minute you get practical, Max. Practically maximum a phosphorylation. Now we know that pKa is upstream of. The increase in tyrosine phosphorylation by fur. So if pKa is activated in one minute tyrosine phosphorylation. So it should be activated in one minute also or two minutes at the most. It's like this signaling works like that. It's very fast. However it can be fifteen minutes, twenty minutes depending on the experiments to start seeing a little tyrosine phosphorylation. So in the world of a cell, fifteen minutes is an eternity. So what's going on there? I have no idea. Other very interesting things are the changes in the membrane potential is not automatic. You put membrane potential and it goes down. More than that, if you put ionomycin or or eight two three one eighty seven calcium ionophores, they induce acrosome reaction. No doubt about that. But it's not instantaneous. In most cases. It takes time to to do it. So what usually when you read a paper and I do exactly the same. We try to simplify the story. Calcium ionophore induced acrosome reaction. But there are many things in the middle there that are are not known of what is going on. And for me, what troubled me in many times make me think a lot is like, why this time dependence on, on on all these? What what is regulating that that kinetic. For that reason Real time experiments are so interesting because you can follow certain signals that you with fluorophores or or genetic in a proteins or genetic probes that that can give you a time frames that are not possible to do it in, in other ways. Of course, those experiments are complex and in vitro, but but they are very informative. I guess I hadn't really thought about this before that the kinetics of the Acrosome reaction, the kinetics of the motility pattern changes have been of interest for some time, and there are studies that look specifically at that. Do you know if anybody has studied the kinetics of the lifespan of the sperm over time? Like, I would think that, you know, the thing that ultimately determines the fertilization window is how many live sperm are there that are capable of fertilizing. Yeah. You're right. And in I think it's a very important question. Now, we know that human sperm can live for seven days. And in in semen is like, if I never done the genetic. But a two day, three days. No problem. It's like the anecdote is that in Argentina, there was a course done by my good friend Patricia, who has done an IVF course for many, many years. And when I was a student, that course was there and she was showing hamster sperm fertilization, probably right. Sperm because our models now she works more with mice. But at that time I don't remember. But it was in different species and and suddenly she said, oh, it's a pity that I don't have human sperm to to show you. And because that course was taken by, by clinical people, also a woman said, well, it's a little embarrassing, but I have some here. So she takes a tube from the handbag. And they were human sperm and people were able to see it so and they were motile and very alive. And we have done work with human sperm also. And twenty four hours is a logical time frame to, to capacitate them and and work. Although now I think processes are faster in many clinic clinics, I know they have been capacitating for twenty four hours. I don't know what is the procedure today, but but uh, but it can be done Unbeaten in that in that time frame. So humans are very resistant. On the other hand, mouse sperm tend to die. We we are doing some experiments now with four hour incubation. And although they fertilize well a the amount of motile sperm is, is reduced after a certain time. So I don't think at this moment there is something very solid about how much sperm can live. And that will depend always on the media and the conditions. It's like a I'm pretty sure oxygen stress in the media that we use that is exposed to oxygen and all that plays a role. A there are many things we are doing some experiments with sperm Um and a and they they can last longer also with so a the kinetics of the lifespan of the sperm a my believing system at this moment because I don't have evidence is that is a extremely variable within the species and will depend also in the incubation conditions. It is I believe, well known. But again, it's not my expertise that bovine sperm can be frozen. Human sperm can be frozen. However, in the pig industry, it's very rare that people freeze sperm. And at least when I talk with experts, they told me that they are not good after freezing. So they usually what they use is, uh, something that is called extenders. And extenders are different type of extenders where you put the big sperm and you leave it there for transport for days or weeks. I don't know exactly, but of course in those extenders sperm leave more than in in the usual capacitation media. So some people are investigating in translation. And investigation is a lot about how to make the best extender and that type of things. We've been using a metaphor of signaling where there's a signal and a receiver. What an alternative metaphor. Be that the sperm can sense bicarbonate in the same way that, for example, an eye can sense light. And would, using a different metaphor, add something to the way that we think about sperm? Capacitation I will answer first the first question, and then I can go a little longer with the the the second one is this. The first one is about sensing receptor. There is a whole body of literature talking about chemotaxis. This work was pioneered by Michael Eisenberg in Israel, followed in Argentina with his mentor Laura Tsoucalas. That has also done a lot of work on that. And then in in Germany, I hate not to to remember the, the name because he's great. He's a great scientist. He has done very interesting work with Pergonal a in a in human sperm. And one experiment that he did in public, like not everybody can smell pergonal. And he show also the pipette with pergonal and this from going to the pipette. So chemotaxis is a possibility. I don't. I don't know too much about that. There are people that believe that this is correct. People that don't believe that this is correct is like many things in in science, like we have different hypotheses there. But. Those experiments are still very, very interesting. And a and I hope somebody take them on and continue working on that. The other question about or I think I am adapting your question to something that I want to say is that and maybe I'm doing some advertisement here about what we are doing now is like, because I talk a lot about my past. Uh. Okay, so the present hypothesis in, in my lab is that manipulating sperm capacitation you can increase fertilization rates. That's one. I would say that that's part of my job description, after all. Uh, but but the the the hypothesis that is more dramatic or interesting, of course, is an hypothesis. It could be right or wrong, but we have some evidence for that is the. Is that manipulating sperm capacitation. You can manipulate also post-fertilization events and you can manipulate embryo development rate and the quality of the embryo, and that's part of what we have been doing in the last ten years. And everything started like everything in, in at least in the present for most researchers with the great doctor Ryuzo Yanagimachi. And one day he emailed me that he wanted to collaborate. He wanted me to do some tyrosine phosphorylation. And my my take on that is like, if if Gianna tells you to do something, leave everything you're doing. I'm sure this will be great. And in fact, it was so. What he was doing was an experiment that I did also in when I was a graduate student. You put calcium ionophore to the sperm. The sperm get completely dead. It's like I can take pertussis. And he's like, I did it for other reasons when I was a student. But that was done by many, many people in, in. In my lifetime. I saw that experiment many times and always with the same result. Sperm gets completely dead. The only one again, that has some hint of what I'm going to tell you is Susan Suarez. That is also another person to follow the literature, because she did similar experiments of what I will tell you in the bovine sperm without going as far. But but but she showed that also. So the thing is, you know, for it's a poison. It kills the sperm. You can get acrosome reaction and all that. But sperm is dying because you see this under the microscope is completely dead or very like quiescent movement of the day. So, you know, sending us samples. And suddenly I didn't understand exactly what it was. Or I wrote him an email and he explained, well, we are trying to recover the sperm after a uniform. So the system was we put the ionophore. There for ten minutes, and then we washed the ionophore and the sperm recovered. But when we did the experiment in our lab later on, because in the beginning was mostly samples from from Hawaii, that or from Japan, because he was collaborating with Doctor in Japan. And what we did was the, the, the Western blot. But slowly we started to do all the, the, the whole thing. And the sperm is not only that it becomes a, it recovered motility, it becomes super sperm. So the hyper activate and all that. And the interesting part of our first paper with, with Doctor Yanagimachi was that a Yana was saying, well, tyrosine phosphorylation is not important because in reality we got we after treating with Ionophore, we got fertilization in minutes and their phosphorylation was taking longer to happen. And that was a very fair. Consequence or reasoning from from Yana. But they said, well this is tyrosine phosphorylation. Phosphorylation has other thing and we have and you can touch me tyrosine phosphorylation, but please don't touch me PCA because PCA is the most important protein ever. So everybody is in love with the particular molecule. But anyway, PCA for me was always so important. So we did the experiments with the ionophore and we, uh, we I, I put the conditions a little rough to say this will never work. So the rough conditions were the following. We put H eighty nine. That is not even a specific for pKa good inhibitor, but it has several off target events. But it blocks completely pKa. And we did the Ionophore experiment okay. With the Ionophore. We bypassed completely the the pKa inhibition. So. What this shows is that calcium is down. Well again my conclusion and what what the experiments is supported and consistent with this hypothesis is that calcium is downstream the cyclic AMP pathway. And when you put ionophore the sperm is completely overwhelmed with uh with uh information there probably ATP is going down. We measure that and it's a paper from a Japanese group also. It is going down because everything is is terrible because calcium is really high. We are using ten to twenty Micromolar Ionophore. When you wash that calcium start reducing. We show that in a paper with with Claudio Sanchez and Alberto Claudio. Calcium comes to a certain threshold level and the sperm start to move like crazy. So we call that super sperm. Okay, so that was the initial experiment that we started doing this with Yana and Doctor Patino in Japan with a craft that was my postdoc at the time we did those experiments. I was completely in the sky. I've never seen something like that. It's like you bypass the cyclic AMP pathway. You don't need cyclic AMP to initiate everything and to get everything. So obviously this is pharmacological disturbance of the system. But anyway, so the idea that when I was writing the paper, the idea that I have at the moment is like, well, if this is true, we can use it to, uh, to induce fertility in sterile sperm. And the obvious case was cats. And that was done by Philippe Navarrete in my lab that was working with calcium and all that. We got the cuts from David Clapham and a and a. And we did the critical experiment. The critical experiment was put Caspar put ionophore a the sperm. That was not very good movement because it was a it was a knockout. And then we washed the ionophore and the sperm become hyperactive and fertilize what opened a lot of see a series of very interesting. Questions about the physiology and all that because, as you know, Caspar is a localized in very specific places. So Gatsby is not only calcium channel, it's a structural protein in the sense that you have a very particular place where catsper is. And in this case, this is a knockout. So it has no catsper at all. So in conditions of in vitro capacitation, where you have like a lot of sperm, the only the calcium can make the trick. I would not claim that the, the, the ionophore can make the trick also in vivo, because I believe that the the type of movement that is regulated by by the very exquisite structure of catsper is also very important. But anyway, in vitro we got thirty percent of a of fertilization. It's not great, but from zero to thirty it's an infinite more so. And then we because we learn all these with with Diana. Up to that moment, my lab I It was always one person completely sperm centric, and for me, IVF was okay because it was the end point. But once you fertilize, you have a zygote. That's not sperm. Who cares? But we we. Diana I learned a that a that the zygote is the beginning, not the end. And so we started to learn a lot. Also thanks to Jesse Major in, in in our department. He's a great embryologist. And he taught us a lot of things. And we learned how to to continue to get blastocyst. So what we got there is that those sperm also make blastocyst. And then we learn how to transfer those blastocyst to pseudopregnant female using nonsurgical approaches. and we got beautiful pups that were catsper cycles. So we have the fold in the paper and all that. For me, that was a beginning of another era in in our lab because I started to to see other things. A very interesting experiment there that we we have not I want to continue pursuing now is that it's not only that we got better fertilization. It is about post-fertilization events. Because when we did this with the controls, with the block six, we went from forty percent, fifty percent fertilization to eighty percent. But then when you take the two cells, those two cells went to eighty percent development when you use the ionophore and to forty percent development when you don't use the ionophore. So then we started It's a very interesting interested in the lab on those effects that you can render. You can give to the sperm during capacitation. Let's call pharmacological capacitation whatever. But capacitation is the process that prepared sperm to fertilize. And we are preparing sperm to fertilize a that can improve embryo development. So that is one of the questions that I started with that that work the rest of the story. You you know it better because we collaborate on that. And and we are interested in the same in similar aspect that is sperm metabolism. So I'm very interested is like I always when I present this data, I said, well, all this is great for signaling, but we don't have an elephant in the room. Probably the most important molecule. Even with a lot of pain in my heart. Even more important than cyclic AMP. That and calcium, that is ATP. ATP is central to everything because you need energy for many, many things a motility, regulation of channels, regulation of ion homeostasis and all that. So I became interested in in the study of metabolism. Now, what I did not know at the time is that metabolism is so complex. Things. We have people like Melanie that I knew that can understand other things there. So I started learning a lot. Of course, I'm a biochemist is not like foreign to me at all. I have even written a small review on on metabolic metabolism. But for me it was a starting point. All this work was pioneered by Eddie and Debbie O'Brien that you know very well because they are there in North Carolina. And I have always been fascinated with their papers, and also by Herb Goldberg with LDH and all that. So I have the board, those papers, and some of them are my Bible. It's like I have it there because I in particular one from Debbie, that is again a very simple minded paper like my tyrosine phosphorylation. It is fantastic paper. It should be part of the collection of everybody that is good in bio. Debbie O'Brien is the last author. I, I love that paper. And it's a very simple one because they have published Higher impact paper. This is a beer that is. But it's amazing. It's so good and you can learn a lot about that. So when we started doing that, I wanted to do metabolomics and many things, and suddenly we do our first experiment. And results are so complex, so terribly complex because I'm used to, to to have very simple mind cyclic and being is PCA. PCA will be upstream of tyrosine phosphorylation and do a western blot. And I produce many papers on that and some of them good. However, when you talk about metabolism you talk about. A and I would not say infinite, but a large number of metabolites. Some of them you can measure, some of them not, but and nothing makes sense. Luckily ATP makes some sense and cyclic AMP also, but in general it was very difficult. So I was working on this with my student Felipe Navarrete, the one that I mentioned before, and I told him, look, this is impossible. I cannot, I cannot follow this. Let's start from the beginning. Let's do an experiment that was done also by by Davey O'Brien. Like remove everything. I don't want any glucose there. I don't want pyruvate. I don't want lactate. I want no energy there. Let the sperm lose motility completely. And then we can add whatever you want. We can add glucose, we can add pyruvate, we can add lactate, we can add fructose. We can add everything. And that's when the experiments A start to make sense from my point of view at least. And that opened a Pandora's box, because suddenly Philippe called me when we are not used to, to talk at ten pm about experiments. But that day in particular, I remember because he called me pretty late, say, Pablo, I'm doing the experiment and the sperm becomes super sperm. They are crazy. They are completely crazy. So removing the the energy substrate and then putting back the substrate improve sperm motility and sperm activation a lot. So then we did IVF and improve IVF in this block six Subfertile model. They are not as good as CD one. But the most amazing part of this was that the also the post-fertilization events because we got more embryos, and when the embryos were transferred to pseudopregnant females, we got more pups. So that opened another very interesting story in the lab that is is at this moment lasting. So in a way. Our overarching hypothesis for my lab is that a capacitation is. A series of sequential and concomitant events that. Induce fertility in sperm otherwise infertile. The second part of this is that by understanding the signaling pathways that are happening in this firm. We can hijack them using tricks like metabolic tricks or pharmacological tricks, and improve the possibility that the sperm fertilize a the third part of this overarching hypothesis, and this is the most controversial one, even for me, is like, okay, I see it. Seeing is believing, but still is is a very ambitious in that way is like sperm capacitation all these artificial methods of sperm capacitation are not only important for sperm fertilization or the acrosome reaction or hyperactivation, they're also important for post fertilization events And a we are actively working on understanding what we are doing to the sperm in order to improve their embryo development, a potential. That's one of the the parts that we are interested in. So thinking about the model of cell signaling, regulation of sperm capacitation. Um, the way that the model is typically represented is, is sort of like a kind of causal diagram. Like, you know, bicarbonate comes into the cell and stimulates adenylyl cyclase activity. Dot dot dot. And in reality, every single sperm is a different representation of that same mechanism potentially playing out over time. So I know you've mentioned Mariano Buffon's research and Alberto Garzon's research. I'm sure I'm leaving some people out of this, but I know that they've also done some great work showing how heterogeneous sperm populations can be. And you also mentioned, I guess, in your ionophore experiments and the starvation refeeding experiments, not all of the sperm recover. So what do you think the role of heterogeneity is in capacitation? Are there a subset of sperm that are always going to be fertilization competent and they're the only ones, or do you think there's some like rotating maturation waves or something? Cameron. This is an excellent question. And it has been in my mind since mostly since I started doing my first experiments as a PhD student is like, why not every sperm brain behaves the same. Now, having said that, hamsters are more synchronized than than mouse sperm or human sperm. But. But you're completely right for mouse. And human is like. That is terrible. It's like a it's a it breaks your head thinking about why. So I believe that every lab will have their own explanation. I tell you mine. That is not mine is other people also. But it's so from my point of view. There are so many. Of the the sperm population comes from different stages of epididymal maturation. So in some species this will be. Like most biological processes, They are stochastics. They are at random. So I don't believe that there is like a particular linear pathway that the sperm needs to be here. And something happened exactly here in the epididymis, and something happened exactly here in the corpus. I believe that they are involved in exchange of information with the PDMS a all the time. And some of them are right to the cow. Without receive any information. So it's like I don't want information. So again this is a very simplistic view. But the thing is like when we get epididymal sperm or ejaculated sperm, some of them have different stages of maturation. And. That's why we It's like. Like things that are from an observer. Completely crazy. If you put twenty micromolar or ten micromolar ionomycin to a sperm, and you're putting a bomb of calcium like you're bombarding the sperm with calcium, it's like calcium is everywhere. Ionomycin even more than eight to three. One eighty seven. That has to do with the pKa, not pKa. The protein kinase, the the the acid constant of the of the Ionophores. But you're putting a lot of calcium there, and usually you get between sixty and eighty percent of acrosome reaction depending on the experiment. We will not always get even eighty percent. Why that's crazy This is the the prototype of synchronization. You're putting a bomb of calcium there. You increase, you leave it for thirty minutes, and you have twenty percent that has perfect accuracy there without any problem. So for me, that's those type of observations are the ones that that are essential. A maybe because I'm old. There are others that are more interesting of course, and all that. But but that thing of the of the population has a lot to do with that. And when you use ionomycin as a pharmacological agent, you know that this is not receptor, that is not capacitation, that is not something like that. Because, you know, myosin is bypassing everything and we are not putting any inhibitor or nothing. It's like you put there eighty percent at maximum of that, but twenty percent. That's not react Hyperactivation. In the best case, depending on the case analysis, the type of media, how good you are and all that. In general, with CD1 you get twenty percent hyperactivation. Some people report over that, but I believe that that's a mathematical a part. But you don't get all the population hyperactive at all. A. Witch with black seeks even less. A ten percent is what you get of hyper activation. Twelve percent. So what happened with the others? Why the other? Don't don't don't respond. So my main hypothesis here is that this has to do with epididymal maturation and different stages of maturation. And the observation that I can And that is part of my, our research. Now, if if you had ionomycin to a caput sperm, they will not react. What for me is incredible, but that would be a well it's not incredible. It's it's an interesting observation because why they will not react with this amount of calcium there. And this is an exocytotic event. Obviously there are thousands of explanation. I, I still like to think about the magic of that and not. But there are a lot of explanation that has to do with the maturation of the Snare system, the maturation of the exocytotic machinery and all that. So they are reasonable things. But I like to to say, wow, you're putting ionomycin and they are not reacting. That's amazing. And then you get it from the cow and eighty percent acrosome reacts, but twenty percent don't. And that explanation of the normal stage of maturation is a logical one. Now, the other part is that the sperm has a long pre-history before becoming sperm. Sperm in the testes during spermatogenesis. And you know that every sperm should be equivalent because a during spermatogenesis and spermatogenesis, you have seen systems that British that make sperm functionally diploid because it receives a genetic material RNA and proteins from their their cohorts, so they are all distributed. However, there are some very good papers claiming that this is not exactly like that, but there is some random distribution also, and that some RNAs has a more or less probabilities to to to go into the the different upload a sperm. So that's another alternative hypothesis that you have during this distribution of RNA. You can say it's equal. Well that's not an explanation of heterogeneity. But it could be also that that there you have like some sperm that don't receive all the material that they need and they are less functional than others. So that's another possibility. You have the mixture of both. I don't have many more explanations. I'm sure that people have have thought about other things once you are in vitro. All the sperm are receiving exactly the same condition. So what happened? Deterioration of the sperm? From my point of view, should come in vivo in either the testes epididymal maturation. And. Those type of processes. There's not I don't have many more explanations because yeah we know that the sperm are heterogeneous population. And I can say that my non capacitated conditions are great because they don't capacitate the sperm, but they will never say that the capacitation conditions are great because I know that I'm only capacitating a small portion of the well, a limited portion, I don't know. I will not say the percentage, but it's not all the sperm that are capacitated because not all the sperm will be able to fertilize. So in a way, very simple and silly observations are the ones that get my attention. Big experiments are great for seeing a seminar for journal clubs, and I'm completely amazed because the great techniques are are completely amazing at this moment. And they moved me a lot. But intellectually, those little questions are the ones that are always in my mind. Why sperm can improve embryo development. That's not very logical. Why sperm will not react when you put Import. There. This these type of things. And I would like to have more. We have a lot on temporal and spatial spatial. We are we are working a lot on on that that part to to to really maybe with single molecules like calcium or pH that we have probes even with magnesium a that is a correlate of ATP in a way a when we have a fluorophores that can, can, can do that, we can do real time experiments in single cells. And those are very informative of how some of these pathways work. And, and we are very interested in that. Cameron I don't want to to to stop completely this since talking about without talking about my other hat because I have two hats depending on what I'm talking about. The other the other part that I'm very, very interested is in contraception. In contraception. Uh, and um, I wanted to mention two things. One, that I started being interested in on this topic, uh, by John her in the University of Virginia, where I worked there for a while, not as a postdoc. I was more a research assistant professor, but I learned many things about his interest on male contraception. So I want to to pay honor to do that. And also at present we doctor that I, I have a very active collaboration trying to find a inhibitors of testes specific kinases. And. In that part also, I want to acknowledge the amazing help on all this project for the whole community of male contraceptive initiatives, uh, that has supported not only my research but a lot of research in, in contraception and. Yeah, of course, NIH always support my research and many other people, but NIH has been more involved in supporting my capacitation research, not the contraception is more this collaboration, although Brenda and I, because I collaborate with Linda, also has a lot of support from NIH. But MCI has been extremely good at this. When will we know enough about this mechanism that controls capacitation? So for example, like you mentioned, if if we can use this knowledge of the biochemical signaling pathways that regulate this process to design effective male contraception, that we must have some pretty thorough understanding of how the mechanism works. Is that how you would define it? Um, maybe my my actual question is what what counts as a satisfying explanation to you? My initial answer to that is that there is no time frame, and we will never understand completely anything, so we can advance in our knowledge and we will start putting little stones in the way to find a lot of answers. And what we understand now is it's amazing. It's like a again, I would mention Jin Soo Chung and David Clapham, a work on on Catsper and and to see the the lines of catsper distribution in the sperm are it opened my mind. It's like it's something that that I I consider completely amazing that you can distribute. I always saw this porn like a bug and the membrane, all the molecules distributed at random there to see that they have this strict localization. Uh, talking about the calcium channel change completely my mind. However, this is the beginning. It's always the beginning. It's like finding a cat. By by David Crawford and David Harbour's independently was an amazing discovery. Like you have the calcium one finding sack for me was by Lonnie and Johan. That was one of the most amazing discoveries because they they discovered the the protein that was involved in, from my point of view, very biased point of view. The most important molecule in sperm that is cyclic AMP. I'm talking about that. Obviously all the molecules are important. So but that's the beginning. Uh, all these these these papers are fantastic. But, uh, they are the starting point from what what it is the end of one project is the preliminary results for the next one. So and that's what I for me distinguish certain researchers from others. Uh, I'm talking specifically about the sperm research because we have people that are fantastic in many in other systems that they have a. High impact discoveries in sperm. And they did not continue. And that doesn't talk about the researcher. But what for me makes a party researcher is the continuation of story. So and a yeah. You discovered that catsper uh is the calcium channel and it publishes I believe nature science paper high impact in a about that the sperm is infertile. Okay. If you have that. Yeah, it's still really great literature. But what what makes David Clapham apart. On that although he's he has never been sperm expert is that he continued. He published several papers and he formed a whole cohort of people that continue. Working on this, including a. Shinshu A ran a descent ran a and many others. Betsy Navarro is like and and and his work has opened the mind of many other researchers also a. The same with I would say with other of the big Discoveries like slow three of molecules or lonely and yoking with suck is like they are not publishing one paper. They continue trying to get the maximum of that. But at the end, whatever they publish is the end of that story. But it's the beginning of the next. It's not the end of science like I have in my in in my bookshelves at home, a book that is called The End of Science. That was very interesting. But, um, that's not true. Uh, it was inspired by Fukuyama end of of history that it was if something was proven incorrect, was that thesis from Fukuyama about the end of history? Uh, and in the case of the end of science, it's exactly the same. Science is never ending, and our knowledge about fertilization and reproduction Action will give enough material to investigate in the years to come, and I cannot see a limit on that now. We will improve and we are improving all the time. But that's that's my take on your question. Every conversation I think we've ever had, I look down at, at my watch and it's been several hours and I've learned an enormous amount. I guess I'm going to kind of wrap things up now. But before I do that, I want to kind of give you an opportunity to say anything else you'd like to say. Um, so are there are there any questions that I didn't ask or any, any topics that I didn't bring up that you'd like to talk about before we wrap up? No, Cameron, I think I talked too much and people tend to say that I talk too much. What a learning particular will laugh a lot about if he listened to this podcast, because he knows that I talk a lot, and the only thing that I wanted to say before I said it, that it was all the other part of my research that has to do with contraception and give knowledge. Very important people in in my life. And because you give me the opportunity. Last thing I want to to to say something that is obvious, but it did not come up during the talk that all the work that I did is supported by my students and my postdoc and the my mentors, and I would have not been able to do absolutely anything on, on my own. This is a collaborative work. And collaborators, many collaborators all over the world. I mentioned Yann, I mentioned Lonnie and Johan, Greg. Goff, John. Hurst, son di Alberto da San, Claudio Sanchez Gerardo a all my postdocs Jessica Escoffier, Dario, my students, Eva, Gaia, many, many people. And I'm sorry that I cannot mention everything. And of course, my wife and Maria that has been on my side and helping me to organize my mind all these years in the lab and outside the laboratory. Also, thank you so much for joining us today. Well, as always, it's it's great to talk to you, Cameron. This was more formal than usually, but you know that I always enjoy these conversations. Well, that does it for today. Listeners. Thank you for joining us on this 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. If you enjoyed this discussion, please like and subscribe wherever you get your podcasts, and join us again for our next episode. In this series, where we learn from Doctor Melanie Balbach how the unique adaptations of sperm metabolism support the energetic demands of Capacitation.