EP47: Egg Activation: Molecular Mechanisms with Dr. Mariana Wolfner

Join host Cameron A. Schmidt, PhD, as he kicks off a new series celebrating the vital role of the egg in sexual reproduction. This episode features a conversation with pioneering developmental biologist Dr. Mariana Wolfner of Cornell University.

Dr. Wolfner has spent nearly fifty years utilizing the powerful genetics of the vinegar fly, Drosophila melanogaster, to dissect the fundamental mechanisms guiding sex determination and development, spanning from genetics and molecular biology to evolutionary development.

Key Topics in this Episode:

• The Power of Model Organisms: Discover why Drosophila is an indispensable tool for uncovering conserved biological mechanisms shared across diverse species, including mammals.
• Defining Conservation: Dr. Wolfner explores the difference between biochemical functional conservation and developmental functional conservation in evolutionary biology.
• The Mystery of Egg Activation: Learn about this critical, yet poorly understood, process where a terminally differentiated egg transitions into a totipotent cell capable of creating an entire organism.
  • The Calcium Signal: Discuss the pivotal role of calcium in triggering activation and how the initiating impulse differs between organisms like mammals (sperm-delivered enzyme) and insects (mechanical/environmental cues upon ovulation).
• Sexual Conflict and Cooperation: An updated perspective on the role of male accessory gland secretions (seminal fluid) and how molecules transferred during mating influence female physiology and behavior, challenging older interpretations of sexual conflict in favor of a more nuanced view of cooperation and cross-talk between the sexes.

This discussion offers a fascinating look at how small-scale molecular events ultimately inform large-scale developmental and evolutionary phenomena.

Transcript

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. A letter written in sixteen seventy seven contained the following quote. Man comes not from an egg but from an animal. Cule that is found in male sperm. For my part, I would say that the male sperm and seeds of plants have been penetrated so far that there is nothing further to discover in this great secret. But I could air. In my opinion, the originator of that quote was none other than Antonie van Leeuwenhoek, developer of the earliest microscope and discoverer of spermatozoa. Just over a century later, in seventeen eighty five. Lazzaro Spallanzani conducted what are arguably the funniest experiments ever performed. He put pants on male frogs prior to mating, and deduced that fusion of sperm and egg are required for fertilization. Recently, we spoke with researchers who study the mechanisms that control sperm maturation. However, it seems that eggs two are quite important in sexual reproduction. The next few episodes in this series are a very well deserved homage to eggs. We'll be speaking with scientists who study the mechanism of egg activation, and how key events around the time of fertilization. Set the stage for the greatest show on Earth, the building of an organism from a single totipotent cell. Today, I have the pleasure of chatting with Doctor Mariana Wolfner, professor of developmental biology at Cornell University. Doctor Wolfner is a pioneering scientist and dedicated teacher who has been studying the mechanisms that guide sex determination and development in the vinegar fly Drosophila melanogaster, for nearly fifty years. Her insights span from genetics to molecular biology and evolutionary development. I immensely enjoyed learning from her unique perspective and I hope you will too. A quick technical note for our listeners while recording this episode. We had some occasional audio dropouts. We tried to clean them up as much as possible, and they only last for a fraction of a second, but you may hear them on occasion throughout the episode. Apologies in advance and thanks for your patience. And now on to the show. Mariana, thank you so much for joining us on the show today.
Thank you for having me. I'm happy to be here.
Many of our listeners think a lot about the biology of mammalian reproduction, but maybe a little bit less familiar with arthropods, including Drosophila. So can we start by just giving a little bit of background into Drosophila reproductive physiology?
Sure. Um, so I'll start out by just saying that Drosophila is model organisms that people have been using to understand the physiology and molecular biology and genetics of pretty much everything. And the argument here is that, um, although there are differences between animals, the fundamental molecules and mechanisms are very well conserved and flies are quick to grow, cheap to grow. Um, and because of that, they've been worked on for over one hundred years. And because of that and the many people working on them, there are really wonderful tools that we can use to tease things apart in flies that you can't in other organisms. Or you maybe could, but far less quickly. And because of those tools, um, we a lot of really highly conserved genes and pathways were either discovered in flies or how they work was figured out using fly genetics like the Hox genes, like the sex determination dmrt genes, flies called doublesex, um, and like a number of signaling pathways like Hedgehog and notch and other ones. So flies are, in a way, a much simpler system that does things basically similarly, but with some unique, um, twists. So in terms of fly reproductive biology. Again, there's parallels to mammals. So uh, for example, uh, flies have gonads. And like mammals, the germ cells develop in a different part of the embryo and then migrate into the gonads and are then supported by somatic cells in the gonads to allow them to make gametes. And that's true for eggs and that's true for sperm. And, um, they are uh, a lot of their basic biology is similar. The sperm have heads and tails and swim. They enter the female with seminal proteins which affect the female's physiology. All of this is very, very parallel. So now I'm going to switch just to the female. That's okay. Um, and just say for example, um, the female germ line is established in the embryo as it is in mammals. And, um, primordial germ cells become the stem cells, the female germline stem cells that are at one end of the gonad. Again, uh, stem cells being very analogous to ones in other organisms. And a lot of what we know about germline stem cells include studies from flies. Oogenesis making eggs begins by the germline stem cell dividing into two. One of the daughters stays a stem cell, the other because it does so by dividing by mitosis four times but without complete cytokinesis. So the sixteen products of the four mitosis stay connected in their cytoplasm. They share a cytoplasm together forming what's called a cyst. And mice for example, do the same thing. Um, the cyst is then surrounded by somatic cells analogous to what happens in mammals. And then one cell in the cyst in Drosophila, um begins meiosis and it will become the oocyte. The other cells, which are its sisters, start to make all the molecules and organelles that will be helpful for the egg when it needs to develop into an embryo. And during oogenesis, they dump all of that into the oocyte by a variety of means, um, that many labs have have figured out. And then the somatic cells on the outside of the oocyte, um, make the membranes and coverings of the egg again, analogous to what happens in mammals. And eventually the follicle cell layer, um, breaks open and then the oocyte leaves the ovary in the process of ovulation. The oocyte then moves down the reproductive tract, um, until it reaches what is called either the uterus or the Bursa, if you want insect terminology, but it's basically the uterus. And they're a sperm which was stored in the female will fertilize the egg and then the egg will get laid. So now we have a couple of differences between us and flies in that fertilization occurs after the egg has begun to activate, which we'll talk about later. Um, and of course, the embryo doesn't implant in the female. It is laid onto the substratum by the female. So the eggs are much bigger than they are in mammals, because they're full of all the molecules that need to sustain the embryo in its early development. But these molecules, again, are very, very, very conserved.
So when we say that something is conserved, the measure, the easy measure for something that's conserved, especially in evolutionary time, is the sequence of the gene. And then we relate the sequence of the gene to a function. But the function has to be kind of defined at some level. So we could say that a gene encodes a protein that has lactate dehydrogenase activity or something like that, right. Even if the protein is not structurally similar, how far do you think conservation reaches from the gene into different processes, especially processes involved in the reproductive physiology, like the physiological process of egg laying?
That is a really good question, and I like your definition. I also tend to think about conservation at the sequence and like biochemical function level. Um, so uh particular kinase or particular protease in a particular family with sequence conservation. Um, having said that, let me, um, give a couple of examples that that fly work has allowed to be done. And I should emphasize that none of this is from my own lab. For example, you can replace a fly Hox gene or a fly major developmental gene like eyeless, which causes which regulates fly development with its ortholog, its sequence counterpart in mice. And it will still some, in some cases work in the same pathway. So work from the Gehring lab long ago, put the, um, mouse, um, small eye gene into fly in place of eyeless and got eyes. But they were fly eyes. They were not mouse eyes. So it was doing its job of turning on the pathway. But then the pathway worked in a fly way. I think that's true for a lot of the conserved Molecules, but molecules also often have additional activities. So a molecule might, for example, be a kinase that ends up functioning in a conserved pathway, but also a completely unique pathway, for example, in eggs. Again, there is a protein that puts poly A onto messenger RNAs in eggs. And again, this is a gold two conserved molecule found in eggs in many species and in flies. It also does this. We have not replaced it with the other species versions. But again when I say conserved, I mostly mean biochemically conserved. But often the developmental function is conserved too.
So when analyzing conservation, I guess I really liked the way that you made a distinction between biochemical functional conservation and developmental functional conservation. So those are two very different scales of thing. And I suppose you can have biochemical functional conservation without developmental functional conservation. And you could also have developmental functional conservation without biochemical functional conservation.
That's correct. And there's two things that I can say about this. One of the arguments for model organisms like flies is that you do have the biochemical conservation often. And then there may be some fly specific processes that one could use to dissect how a conserved molecule works, that then can help us understand the biochemistry of how that molecule does what it does in people or in mammals. Um, so testing inhibitors of certain, uh, disease relevant genes can be quickly done in flies, even if they do different things in flies. Then there is a concept in evolutionary developmental biology called developmental systems drift, where you have an outcome that's conserved, but sometimes you have multiple different ways to get to it. And that's also really interesting because it tells you where the places are that you can wiggle or tweak and what has to be absolutely the same. So it's useful there.
To your first paper explored control over cell cycle progression in yeast by enzymes involved in amino acid biosynthesis. What led you to transition from yeast to Drosophila?
I was always interested in genetics. I was one of those sort of nerdy kids that kind of loved biology and genetics, and even in college, I knew that I was interested in gene regulation, and the Fink lab was studying the regulation of amino acid biosynthesis. And so, um, I was lucky to be able to join it and worked on several genes that are known to be important in the process in yeast. Yeast was at the time, um, the a really good place to study gene regulation. Drosophila was not quite, uh, at the molecular level yet. Um, I worked in Jerry's lab, and then I went off to graduate school. And in graduate school, I first started working in, uh, microbial lab because I was still interested in gene regulation. But Dave Hogness, uh, was in the department, and he had just begun to, um, figure out how to use recombinant DNA to study genes in Drosophila. So this is a long time ago. This was the cloning of the first fly genes was done at that time in his lab. And I realized that, um, it would be a way for me to continue to study gene regulation, but in something that was more complex than a single celled organism. I was really interested in development and in reproduction. And this was going to be the way to do it. So as a grad student, I switched into Dave's lab, and I was part of a group that studied the genes regulated by a steroid hormone. That, um, is what causes metamorphosis from larva to pupa. It's not estrogen or testosterone, but it is a steroid hormone. And we now know that the parallels are many. Then when it was time to, um, do a postdoc, I was still interested in understanding gene regulation and development, but I decided to study something different. I was really curious about how an animal that is genetically specified to develop as a male develops as a male, whereas one that's genetically specified to develop as a female develops as a female with basically the same genome. And unlike all of the other developmental systems that had been studied, like Hox genes, where while where function is normal and failure to function is usually dead or abnormal. Sex determination function. You have two alternative functions, both of which are normal. One takes the male path, one takes the female path. So I went to post-doc on sex determination genes. Um, and then when I set up my own lab, I wanted to understand what those genes regulated. And so I applied the techniques we'd used as a, I'd used at a graduate student to try to find genes that were expressed in either males or females and then study how they were regulated. And that brought us into reproductive biology.
So I think you hit on this a little bit, uh, in the previous response, but I'll ask just in case there's anything else that you wanted to talk about? Um, in nineteen ninety four, you co-wrote a book chapter called Harnessing the Power of Drosophila Genetics. Uh, how do you use genetics to understand the control of cellular processes?
Yeah, that was a really fun paper to write. Um, I wrote it with my friend and colleague Mike Goldberg, who was in the office next door to mine and was also a student in the graduate student in the same lab. Um, as I was long ago. So this relates to the thing I said earlier that, um, with organisms like flies, um, you can quickly assess the function of different molecules. And so the standard genetic approach is to, um, make or obtain, um, mutation in an interesting gene for you and then ask what doesn't work correctly in the mutant relative to a genetically similar or identical organism that has a normal copy of the gene, and there's many ways to do it. A is to do a genetic screen where you don't know anything about the process, and you just screen for mutations that in genes that are needed for it, because the mutants don't do that process correctly. And so this is unbiased. And you will identify many molecules that you never thought were important in the process. But you now know from the organism that they are. So I mean, there are many examples of this. One of the most famous ones is the screen by Nusslein-volhard and Whitehouse that led to the discovery of genes that are necessary for patterning in the embryo, that hedgehog pathway that I mentioned before, or screens for um homeotic mutations by uh Lewis and Kaufman and Goering, which identified the Hox genes because they found mutants whose body parts didn't develop correctly, and in both cases, nobody knew what what molecules were important in this. But by finding mutants that couldn't do the process and then figuring out what they were, what they coded for and how they work, you could figure out the process. So that's one big way to use genetics to understand the control of processes. And that's something that really requires model organisms like flies and nematode worms and mice. You obviously could not do such a screen in people. Um, the second way to to use genetics to understand the control is that once you've identified these genes and figured out what they code for, you can start asking, how do they do this? And that's a different level of mechanism as opposed to say who does this. That level will be how. And so you can make double mutant combinations, which will tell you if two genes work in the same pathway or not. Or nowadays, not in nineteen ninety four. You can make mutations that are in very targeted parts of these genes, or you can turn these genes on or off in the wrong place or time and see when and how they work and what parts of their molecules are important for their work. So all of these are ways to use genetics to understand cellular processes. And then the final thing, which is not exactly genetics, but to look at the conservation of these genes like we talked about before and see who has them, who uses them for the same thing, what parts of the molecule are conserved.
So when you define gene, do you think of gene as a sequence, or do you think of gene as function or do you, I guess, do you have kind of a way of thinking about what you mean when you say, Gene.
That is a really good question. I think I it depends on on what I'm doing and at any particular time. Probably for me, I think of the genus function, um, having come in from the mutant screen. And how do you find the genes that are important for something. But to then understand the function, you have to know the sequence. So I think of a gene as well as its sequence. And then the question is do you think about the whole gene, including its regulatory sequence and the sequence that codes for the protein that it specifies? And again, that depends on what's going on that day and how I'm how we're analyzing it or thinking about it. I think there are many different ways, and you kind of keep them all in your head at the same time because they intersect with each other.
Do you think that we're limited to being able to analyze only a subset of gene controlled interactions by using this mutational analysis to look for scenarios where we cause a mutation and then see a loss of function.
Probably. And sort of yes and no. So your question was are there limitations to the information we can get just by looking at genes.
Yeah, I think so. Or like um, if you're looking for genes that control a process or if you're even just looking for functional relevance of a gene, then the first thing you might do is try to mutate that gene and see if something happens. Yes. Are you kind of fundamentally limited to only certain conditions or genes?
The answer is yes and no. And the answer is that things have changed since nineteen ninety four, so there are more things we can do. So many genes are what we call different functions. And if you make a mutation that knocks out the gene completely, if it has an early function in development, for example, you might never get the animal that you need to look at its function in reproduction because the mutant will be dead. But there are now methods to turn genes on and off with various external factors um, hormones, temperature, uh, little molecules that are added so that you can actually a particular stage, um, so that you could study it later. But still, since genes often have many functions, you're looking at a whole organism where the tissues are interacting with each other, and you may or may not be able always to pinpoint the function of a particular gene in a particular tissue if it's needed, for example, in two tissues to interact with each other. Um. Conversely, development and reproduction are so important that there's a lot of redundancy built in. And so sometimes you'll make a mutant and you won't see a phenotype, or you'll see a very subtle phenotype, and you'll need to figure out who's redundant with it and then remove them all to see a phenotype. But then of course, they may have the other genes may have other functions. So it gets complicated. So the answer is I think so. But it's not always as simple as it seems.
Is that something that that kind of drives us to tend to focus on things like transcription factors? Because that's sort of the only natural way that we can affect many different genes at once, in a way in which their their normal interactions with each other are conserved.
Um, I would again say yes and no, and it depends on who you are. Um, if you're a transcription factor person, you would say that. Exactly. Uh, other people might say that it's a little bit too broad, um, to remove a transcription factor because it affects so many things and at different times in a cell lineage that then has downstream consequences. So I think the answer is yes it is. But there are other valuable ways to another thing, I, I think this is relevant here, but I'm not sure is that we're constantly discovering things in the genome that we had no clue about. When people first did genetic screens, things like microRNAs, which were actually discovered with genetic screens in nematodes, um, but now micropeptides turn out to be important. All these genes that people really didn't pay attention to because they were focused on larger ones in annotations. And so if you have transcription factors, for example, and then microRNAs. There's much more nuance to what a transcription factor does. If a microRNA can then affect the target of that transcription factor to be expressed, even if the transcription factor expressed it. So there's just a whole it's like we were on the tip of the iceberg and there's a whole bunch of stuff below.
Do you think a lot about some of these newer applications, uh, single cell RNA sequencing and things where you can kind of observe some measure of gene functional state over large numbers of genes and also with the resolution of single cells. And maybe my question is, do you think that will lead us toward a more complete picture of how many genes, some to developmental functions?
Yeah, I think so. Um, I think on the one hand, on the genetic side, the fact that we can tweak specific parts of specific genes, and specific timing and place of the gene expression gives us a very granular way to study a gene and what it does. And then on the flip side, we can get a very granular understanding by looking at single cell RNA seq to look at the tissues and cells that carry out that function, discover that there are more cell types than we thought, that they have these differences in gene expression that can be major or subtle, that we didn't know, and that can then give us the basis for understanding the phenotype, because we now have many more probes for what's going on in the tissue. So the combination of those two now gives us, um, a very precise way to understand how genes are influencing function.
I imagine things have changed a bit since nineteen ninety four, as you mentioned. Uh, how has your approach to using the tools of molecular genetics changed since then?
A lot has changed. Um, and, and a lot has stayed the same. So the basic logic of science and genetics, I think has stayed the same, and reproductive biology as well. But what we have now is these tools that let us go deeper. Um, some examples are the single cell RNA seq that you talked about. Crispr is just totally changed everything, including that, um, although the model organisms like flies are still arguably the best way to to get the the deep biochemical understanding of how gene products work together in a process of development. But now you can look in other organisms and see if they're doing the same thing, um, RNA interference and the ability to turn genes on and off at specific times, again, mostly in the model organisms, gives us a whole new set of tools and omics in all forms, um, does as well. It's it almost felt the first time a transcriptome came out. And I looked at it almost felt like this was secret stuff that you weren't supposed to know. And now you could just look and say, oh yes, this is expressed here, this is expressed here. And more so even with single cell transcriptomics, we just have so much more information that we can ask questions at a different level and interpret phenotypes at a different level.
So we mentioned earlier that we would return to egg activation. Uh, can we talk a little bit about what egg activation is and how it fits into the physiological sequence of events that occur during fertilization?
Absolutely. Egg activation is this amazing process where a terminally differentiated cell, which is going to die if it doesn't do any activation, it's done. It's finished. Switches over to a totipotent state that can now support the development of an entire embryo and make everything. And so it's like a reverse stem cell. To determine cell transition. And it's just an amazing process. And it's extremely poorly understood because it occurs internally in many animals. And it uses molecules that are in the egg already. So you can't do transcriptomics to see what was there before and after because it's the same. Um, and yet it's absolutely critical. If it doesn't happen, there's no embryogenesis. So it for example, understanding how it works is important just biologically, but also in, um, fertility medicine, because if eggs don't activate, IVF can be done, but if the egg cannot activate, it will not be successful. So what egg activates the transition from the mature oocyte, which is alive but arrested at certain developmental stages. For example, in most organisms it has begun meiosis but not finished meiosis. It also has cell coverings, membranes and shell that are soft and that are hospitable to fertilization. And it contains RNAs and proteins that were necessary to make an egg that arrested but but should go away to make an embryo. And RNAs and proteins that were put into the egg by the the mother during oogenesis that will need to be turned on in order to allow embryogenesis. And all of these processes have to flip over. Meiosis has to finish. So you get a haploid nucleus to fuse with the sperm nucleus to make the zygote nucleus of the embryo. The egg coverings have to change so that you don't get polyspermy. And that, um, switch over in macromolecules has to happen to get rid of the ones that made oogenesis. Sorry, made get rid of the ones that made an egg and activate or turn on the ones to make an embryo. So it's just this incredible process and we really understand very, very little about it in many animals, but not arthropods. Um, it's triggered by fertilization. The sperm does something that I'll come back to that triggers the process in Drosophila and the several other arthropods have been looked at. You don't need a sperm, for example, in honeybees. Um, males develop from unfertilized eggs. So clearly those eggs had to be able to activate in order to develop without a sperm. So one of the things that we're curious about is if you don't need a sperm to activate, how do you activate? Um, on the other hand, what is common between activation in all organisms, whether or not a sperm is involved? And in fact, almost everything is common except for the use of sperm in non arthropod animals. So egg activation fits into the physiological sequence of events from egg to embryo. Um, it either is triggered by fertilization or occurs immediately before. And in both cases, it changes the egg from a differentiated cell to a cell that once it's been fertilized or in bees, just changing it, um, becomes capable of undergoing all of embryo development.
So I and maybe it's worth mentioning for the listeners that because I don't really think about this very often, but we mammals are diplomatic, meaning we spend most of our life cycles having two sets of chromosomes. And, uh, yeah, some some organisms are haplotypic, so they'll spend a larger portion of their organismal life, uh, only having one set of chromosomes. Uh, plants. I know that kind of. I don't know very much about plants. Not as much as I should, but. Yeah, so that's a good point about the, uh, male bees then, I assume are haploid.
And females are diploid and their siblings, and they live in the same colony.
Part of our life cycles as organisms are spent as two haploid, single celled organisms for a brief period of time. And then we become these diploid, multicellular organisms.
Yeah, exactly. Yeah.
So I know in mammals, the risk of polyspermy is a significant selective factor during fertilization because the sperm outnumber the egg by such a large number. You mentioned that in Drosophila, sperm or fertilization by sperm is not necessary for egg activation. But is polyspermy still a risk for those organisms?
Yeah, that's a that's a good question. So, um, there seems to be a lot less polyspermy in Drosophila than um, is observed, especially in or can be observed, especially in organisms like sea urchins that release their sperm and eggs into like a tide pool where there's really a lot of sperm and a lot of eggs. In mammals, it can it can be a problem. One way to get, uh, decrease the chance of it is that not as many sperm as enter the female mammal make it all the way to the egg, but still. Um, significant number. Do um, and they can potentially fuse with the egg anywhere on the egg, as far as I know. But in flies and fish, sorry. In insects, many insects and fish, the sperm can only enter the egg in one place called the micro pile. It's like a little tunnel that the sperm has to get into and swim in. And fly. Sperm are unusual in that they are really, really long. They're longer than the Drosophila. And as I mentioned, there's not a lot of polyspermy in Drosophila. We don't know why. Um, but there are two, uh, not mutually exclusive suggestions that have been made. One is that fly sperm fertilize eggs as the eggs are moving down the reproductive tract and come to rest in the uterus, and its micro pile is right at the opening of the storage organs for sperm that are in the female reproductive tract. And so sperm, a sperm will then swim out and swim into the egg. And that process is regulated. So you can imagine that not too many sperm get out for each egg that's there. And there has been speculation that given the length of the sperm tail, the entire sperm swims into the egg. It's not Polyspermy is not really my field in in Drosophila. Um, but I think it can happen. But it's very rare.
So what happens? Post activation seems to depend on the maturation steps that precede fertilization. As you mentioned before, that there are many molecules and processes that have to be sort of set up in this differentiated egg. How do you approach identifying these molecular components that control this process, and how do you think about building a mechanistic picture of how they interact?
So you're absolutely right that what happens post activation does depend on what happened during, um, oogenesis to make the egg. Um, so the way to find the molecular components, I think there are several different ways. One is genetic screens looking for mutants that make eggs that look and behave normally but can't start development. And there are mutants in in some genes in Drosophila that do this. Um, for example, uh, the poly polymerase that I mentioned before that's maternally loaded has that phenotype is called Pangu, discovered in the lab again by mutants that make lovely eggs, but the eggs can't develop. Um, another way is that if you have an organism with big eggs like flies or frogs or many eggs like sea urchins or sea stars, you can do biochemistry and see what molecules are there and use that to build a model. But some of the important molecules are not macromolecules. Particularly calcium is critical in activating eggs in pretty much everything that's been looked at. Um, and that was discovered by physiological studies in organisms with lots of eggs that are released to see whether, um, adding, uh, making eggs pick up calcium, changed them. And it did it caused them to activate. And once that was known, different um, researchers on different systems use different ways to test whether it was working in their system. So often using a chemical that will fluoresce when there's more calcium or a protein that will fluoresce when there's more calcium, to see if calcium levels go up in the eggs, which they do, and then working backwards to figure out what makes it go up, and working forwards to figure out what molecules does it affect. So I think it's the combination of all three of those, um, figuring out what's there in the egg and how is it tweaked during activation to change its activity.
So the, the egg in order to activate gets sort of a, some sort of an impulse, like a signal impulse, for example, could be, you know, the impulse could be sperm binding, it could be alternative impulses as well. Maybe there's some alternatives that we could talk about. Do you think there are generally single impulses that start this process, or is it kind of a sequence of different things, or is it a mixture of a more complicated signal process where like maybe multiple things have to happen to initiate activation?
That is a wonderful question. And I'm going to go back to evolution and variation and say that depending on the organism you look at, it could be a single thing or it can be multiple. I'll give you a couple of examples. So in as I mentioned before, in many organisms, um, a rise in calcium triggers the egg to activate. And let me first talk upstream about what triggers the rise in calcium. And then maybe we can talk downstream about what it does. There are organisms like mammals where, um, the sperm will introduce an enzyme into the egg that then releases calcium that was stored in the egg in organelles like the ER or the mitochondria. So that's a single trigger, the entry of the sperm into the egg and the, the, um, donation of this enzyme that triggers the calcium in Drosophila. We also think it's a trigger, but it has nothing to do with sperm. Rather it's when the egg leaves the ovary in the process of ovulation. It comes under pressure from squeezing out of the ovary and it enters a new environment. The oviduct and some combination of those two activates ion channels in the membrane of the egg to take up calcium from the fluid. And so the calcium comes in. But its initial rise is by a different mechanism than what mammals use. After the calcium increase occurs following the opening of the channels and the uptake of calcium, then that triggers subsequent release of calcium from stores, analogous to what happens in mammals. Release of calcium all the way across the egg, so that that's a shared process. So what's not shared is how that initial increase starts. One can imagine if one is a fly person and thinks about evolution, that perhaps the most fundamental process was raising the calcium. But how you raise it can be different in different lineages, but in certain lineages where it's critical for the embryos to be deployed and have both maternal and paternal contributions, you might want to couple the rise in calcium to the fertilization of the egg. Whereas in insect lineages where some insects are fine without being fertilized, you don't have to couple it. So that rise of calcium happens in different ways in different lineages. But then in some lineages like Drosophila, like nematodes, I believe, like sea urchins and sea stars and frogs. So vertebrates included, there's just this one rise in calcium, and that appears to be sufficient to trigger everything else. So the calcium goes up initially at a localized place. And all of these lineages in the ones where fertilization triggers it. It goes up where the sperm enters the egg and then the sperm. The calcium spreads across the egg in a wave, but there's just a single wave, and that's enough. But in mammals, it appears that there need to be multiple calcium oscillations where the calcium first rises when the sperm enters the egg and then spreads across in a wave. But then that calcium is pumped back into stores or pumped out of the egg, and then calcium gets back into the egg or is released from stores, and you get another calcium rise and another calcium. And early studies showed that these different numbers of calcium rises correlated with different events of egg activation. And studies. Um, in, uh, the Williams and Fissore labs have shown that the calcium uptake for these oscillations, uh, involves the use of ion channels in the membrane, at least one of which is the orthologue of the channel in Drosophila that takes up calcium and starts egg activation. So the answer to your first part of what I think your first part of your question is, is that depending on the organism, there can be one impulse sperm binding pressure and swelling of the egg depending on the lineage. But in some lineages, like mammals, um, you then need additional oscillations of calcium. So the second part of your question, I think related to do these, um, do these triggers also cause all the events at once, or are there multiple responses to the calcium that may not all be connected to each other in time? And I think the simplest answer is, except for the mammalian case that I mentioned, we don't know yet. Um, in organisms where there's a real risk to polyspermy the calcium, can, I believe on its own cause release of cortical granules that change the egg coverings and prevent sperm from coming in. And that's just calcium doing what calcium does with vesicle fusion. But in flies and the other organisms I've mentioned, there are also major changes to the proteome of the egg. Specifically, phosphates get put on or taken off specific proteins, phosphates that we know regulate the activity of at least some of those proteins. And that is probably due to calcium activating enzymes that do this. I don't think these enzymes are needed for the block to polyspermy in the organisms that I mentioned. I think that's just calcium doing what calcium does. But then calcium also, um, triggers these major changes in the activity of the stored proteins, which then go on to do things like cause meiosis to finish or cause translation to start.
It's really interesting to think about this process and the control of the process in the context of evo-devo and that, you know, the egg activation process seems to be pretty well conserved, but it's the control mechanism and what it's tied to, or what series of events or the timing or whatever is what's actually changing more quickly, I guess, in evolutionary time.
Yes, I think that's exactly right.
That's really an interesting way to think about. I guess all all processes, there are probably things that happen that are a little bit older than their control mechanisms, and the control mechanisms are the thing that respond most quickly to changes in the environment.
I think that's right. And then with evolutionary time, the processes can also change a little bit. But I think that the I completely agree with what you just said. Yeah. It's the control that's rapidly changing or able to change.
Uh, this this one may be a kind of a hard question, but. So so I guess, uh, protection from polyspermy may be sort of a secondary benefit of having a harder or hardening the, the outer coat of the egg. And even though polyspermy may not be as big of an influence on Drosophila reproductive physiology, I imagine that, um, uh, resistance to desiccation probably is for the egg. So once it's laid, is, is is a similar kind of physical change in the egg coating. Is that something that happens once the egg is laid or is that and is that related to activation?
Yeah, that's a great question. And and you are completely correct. You have to protect those eggs because they're there going out of the female there going into the world? They have to not desiccate. They have to not have microbes get into them and so on. Um, so there are three layers around the egg. There's of course it's plasma membrane, but then there's a vitelline envelope, and around that there's a chorion or egg shell. The vitelline envelope and chorion were deposited by the follicle cells that surrounded the egg during oogenesis. So somatic cells and um they get cross-linked um, during egg activation. So in fact, a simple assay for egg activation that we often use in the lab is whether the fly eggs. Will lice when put into bleach or not. If they, um, if their, uh coverings have. So the bleach takes the chorion off. But if the vitelline envelope has cross-linked, the eggs will not lice in bleach, at least in, you know, twenty minutes or something like this. And that's because it becomes completely impermeable to small molecules or solutes, solutes or anything else. Even gases probably can't get in. So the Drosophila egg, the chorion looks football shaped for most of the egg, but at the front of the egg has two what look like soda straws sticking out called dorsal appendages. And they're little tubes through which the air can get into the egg. Because the rest of the egg shell, I believe, is pretty impermeable to pretty much everything.
So in, uh, in nineteen ninety five, you co-authored a paper that's been cited more than sixteen hundred times that accessory gland secretions from males can shorten the lifespan of female flies, thereby imposing an evolutionary cost on mating for the females. How has your view of the role of male accessory gland secretions in Drosophila reproductive physiology evolved since then.
So that paper was the result of a collaboration with Tracy Chapman and Linda Partridge in England. And it was an example of the genetic, the use of genetics to dissect something that I mentioned before. They had been studying the fact that female flies who mate multiply die younger than female flies that haven't mated, and they had shown that this didn't have to do well, that there was a there was a part of this that was independent of the energetic costs of making lots of eggs, which happens after mating, but they had no way to figure out, um, what components caused it. My lab had been looking genetically at a tissue called the accessory gland, which is basically analogous to the prostate and seminal vesicle of um, of mammals, which produces Duces proteins and other molecules in the seminal fluid that accompany the sperm into the female. And we had generated a genetic strain that whose accessory glands couldn't make protein. But everything else in the male was fine. So what Tracey and Linda and my lab showed, uh, that, um, Jon Kalb was the student in my lab. Well, Tracey and Linda and John showed was that if you mated females to males, that the accessory glands didn't work. The females did not die as young. So that indicated that something from the male accessory gland can somehow shorten the lifespan of the female fly. So I'll just say a couple things about that. This is a Drosophila thing. It happens in Drosophila and in some other insects, but in other insects the lifespan is not shortened by mating. So this is um, but that doesn't mean it's not important and interesting at the time. So this is like thirty years ago. This, um, was looked at in the context of sexual conflict, which is that, um, the interests of males and females are different, and males will want to, in air quotes, manipulate the physiology of females to make the females produce as many progeny from that male as possible, no matter what it does to the female. So if it makes her make lots of eggs, or shift her resources into making lots of eggs as long as she can make more progeny, this is supposedly good for the male. There are other things that were known at that time that the male transferred in seminal fluid, that, for example, made the female less interested in mating with another male that were also interpreted in a conflict sense. Those interpretations are not necessarily wrong, although, as I said, we now know that in or, for example, our studies with Laura Harrington have shown that in mosquitoes, seminal proteins increase the lifespan of the female. So clearly this is not a general thing. Um, but also the whole view of what seminal proteins do to females has become much more nuanced. Now, I think people, although they accept there are some conflicts between what might be, uh, more selected for in the male versus what's selected for in the female. Ultimately, it's actually more of a cooperation, which makes sense because they're having progeny together. So on the one hand, having seminal proteins, for example, increase egg production in the female is great for the male, but it's also good for the female because the seminal proteins can act as a signal that she's now received sperm, and therefore it's worth producing eggs because they can be fertilized, or the seminal protein that makes the female less interested in mating again. Um now has been shown by work from Bellator and others. Doesn't actually make her not want to mate. It makes her choosier so she'll mate with a better male than she'll have his sperm and be able to use his sperm. But if the second male is the same as the first male, genetically or worse, then she's much choosier and not interested in mating. So it's beginning to look like these molecules have evolved by a cross-talk between the sexes to be useful for each of them in their own way. And although there may be conflicts, there's also cooperation and co-option and use of these molecules. Uh, for example, it may be that the shortened life span of the female flies is advantageous in the sense that the female puts so much resources into making eggs quickly that she will have progeny quickly, long before she would die a few days early.
How do you, uh, think about that when, uh, designing experiments, I guess. How does that influence your experimental design?
Well, for example, um, a few years ago, an experiment by my former postdoc, snigdha misra, showed that a male can transfer a lot of a particular seminal protein and that this can actually be useful to other males, like other males around him, which are likely his relatives. Um, in their fertility, is giving more than he needs to give. And work by um former postdoc Laura Surratt and Stu Rigby showed that males can actually tailor how much of the seminal proteins they provide to females, depending on whether there are other males around or whether the female has made it or not. So there's much more flexibility in the system. Um, the other thing is that an impressive fraction, but not all of these seminal proteins evolve very rapidly at the sequence level. And this was interpreted initially as making sense out of a conflict situation, so that a protein from a male would, quote, manipulate, unquote a female. Females evolved resistance and then males made a better version of the protein. But now, again, thinking about why these molecules evolve quickly has to be more nuanced by including, uh, what they do for the female as well as in the female. Can I add some one other?
Oh, absolutely. Go for it. Yeah.
Um, just hearkening back to what I said at the very beginning to the answer to one of your first questions. The fly proteins, although not the same as seminal proteins in mammals, are in the same biochemical groups in many cases. So this is another case where, um, despite a lot of years of evolution, studying how they work in flies can give us insights into how these proteins might be involved in fertility in pretty much everything.
I think that and maybe more familiar cases like mammals, it doesn't seem nearly as surprising. I think, that that certain types of glandular secretions communicate, uh, organismal sexual status to the other sex. Like our, uh, smelly armpits. I guess that's one thing that comes to mind. So I think it's really interesting to imagine that as kind of an extension of the same concept, but in a very, very different scenario and, and also having an effect at a very different scale I guess too, like it's not a an organism level or I guess it is an organism level communication, but it's not happening through nearly as familiar a pathway for us mammals.
You're quite right. Although there are pheromones that are transferred, made in one of them is made in the male one on in glands under his skin that are transferred to the female. Um, so making her smell quote unquote different, um, at least initially, that also makes her more or less attractive. The parallels between our biology and the biology of Drosophila are incredible, even if it's not always, although quite often, the same molecules. But the biological parallels are just incredible.
I read a kind of a popular science article recently that was noting how, uh, how much our social perception of the way that we frame these things influences the stories that we tell about evolution and causes and evolution. So, for example, the article was talking about female choice in the form of egg choice for specific sperm, and then uh, mentioning how, uh, sperm competition was sort of like a masculine ideation. And egg choice is kind of this feminine ideation. My initial instinct is to think that those metaphors are probably not that helpful, that, um, it just kind of adds baggage onto the the more physical explanations of how these things work, that probably doesn't need to be there. Do you read those articles or think about that framing at all?
To some extent I do. And there was a wonderful, um, uh, symposium session at the society. For Integrative and Comparative Biology in early twenty twenty, I think, um, organized by Virginia Hazen at Holyoke and Terry or I'm not sure where she I think she's in Arizona, specifically on this question on whether the way this has been framed has, um. Constrained how people think about it, that sperm competition was exactly what it sounds like was a competition between males. And you just kind of ignored the female. But there's been a lot of literature now. Um, for example, long ago by, um, uh, Bill Eberhard's book Female Choice and Experiments by Andrew Clark, my colleague, um, with Drosophila. Of course, that says that actually it makes a big difference what females those two males mate with conversely egg or female choice, uh, is a newer, somewhat thirty year old or something, but newer concept. We need to be very careful about whether our language is constraining our hypotheses. So one of the things that we often use, um, in my lab now, we're still learning, but we're often using it is things like sperm precedence, removing any words that imply choice or preference, um, or competition, because it's clearly a dance between both the male and the female, just like with the seminal proteins. Yeah. So that I think that what you were talking about is a very important, um, article and method of thinking about the field.
So your research spans multiple scales from cells to whole organisms. And I suppose that's just the spatial scale. So you're also kind of thinking about molecular timescales and even evolutionary timescales to some extent. When you think about biological problems, how do you imagine that small scale events like biochemical reactions, inform large scale events like the behavior of organisms over time?
That is a great question, a fascinating question, and I don't have a great answer for it. First of all. Yeah, I'm interested in all of those levels. I just love each of them and I like seeing them together. But also, um, I have students and postdocs in my lab who may be particularly fascinated with evolution or with biochemistry. And so it's the group together that is fascinated by all of those. And I just love seeing it at all of those levels. Um, how do I imagine that small scale events influence like by like biochemistry influence large scale ones by evolution. I come back to to my identity as a geneticist as well as reproductive biologist, in that the genes function is what ultimately determines behavior, or a physical phenotype or a fertility phenotype. And it's the underlying biochemistry of the gene products and how they interact with other ones in a cell and between cells. That will therefore lead to a particular phenotype. And evolution essentially takes all the potential phenotypes that appear and selects the one that works the best. And so it's a little bit like at some level, making a buffet at a restaurant. And then you go in and you pick the chocolate or whatever it is that you like that works for you. And it's the biochemical reactions and the genetic reactions that set up what's on that buffet. And that's kind of how I think about it.
In the lab, like in practice and doing research, you would compartmentalize those things. Like you study the molecular scale things in a molecular scale context and then study the developmental scale things in a developmental scale context.
Yes. But then we'll usually step back and say, okay, so what does this mean in a bigger context? But as we focus on a particular question, yeah, we'll put on a certain set of glasses to glasses in a certain set of glasses unquote, to focus molecularly or to focus evolutionarily. But then once we have an answer, we're like, well, does that make sense molecularly from the evolution answer, or does that make sense with evolution? Do we know about other flies that might tell us this. So we go back and forth. But I think it would be it would explode my mind to try to do them at the same time. So we kind of go back and forth.
For, for a given phenomenon. I guess just something that you might observe a developmental process or a function or something that's observable. When do you consider that you have a complete enough understanding of the process?
Wow. I have two answers to that. One is never is always when you figure something out. There are new questions that it raises. But and that can lead me in a new direction, which is a new process. But at the same time, when it makes sense together, when it forms a story that makes sense and, and the pieces fit together, like in a puzzle. Um, and I can see where it starts at one level, like where a particular gene coded for a particular molecule, which did a particular function which led to a phenotype which was selectable upon that story would feel very good to me.
Is that what you would consider a mechanism when all those different pieces come together?
Yeah, I would consider it a mechanism, but I would also say that there are some mechanisms inside of it. It's the mechanism for a process, but it has the mechanism for reactions or for evolution inside of it.
That's all the questions that I have. But the kind of final question that I like to ask, uh, is, is there anything that I didn't ask or didn't talk about that you would like to address before we wrap up?
First of all, I really enjoyed your questions. And they were wonderful. And they got it some really deep things. And thank you so much for that. I'll just say one thing which you mentioned to me before you started recording, which is, um, collaboration. We talked about male female collaboration. But, um, one of the things that's also really fun in science is to collaborate with scientists who do different things from you. We like to do a lot of that, um, because sometimes we feel like we don't understand a mechanism or a process from all angles. We understand it from what we do. Someone understands it from a different angle, and it's just fun to put them together and and get new insights from that. I think collaboration is something that happens a lot and that should be fostered, um, working in groups with people who are interested in the same kind of question, but perhaps for a different reason, is just really energizing and fun. The other thing that I wanted to say related to that is that, well, certainly for me and probably for many, many scientists. It's not just the science that drives us, although it certainly is, but it's also the chance to work with other people, including people that we help to train so that they can go off and do their own science, which is as satisfying in its own way as the actual science.
Thank you so much for joining us on the show today. I really appreciate the time that you took to be with us, and I learned a lot.
Oh, this was just so much fun. I really enjoyed chatting with you and your questions were just so great. So thank you so much for doing this and for having this chat together. It was really made me think a lot about things and I really appreciate 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. 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 for our next episode! When we learn from Doctor Carl Swan of Cardiff University about the role of calcium oscillations in mammalian egg activation. Until next time.