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Krista Gert, a current doctoral graduate from Andrea (Andi) Pauli’s lab on the Analysis Institute of Molecular Pathology (IMP) in Vienna, Austria, lately revealed a research on how Bouncer, a small egg membrane protein required for sperm binding, governs compatibility between the sperm and eggs of various fish species. Her research, revealed in Nature Communications in June, juxtaposes two distantly associated, reproductively remoted fish species—zebrafish and medaka. Utilizing a structure-function method guided by evolutionary evaluation, her work uncovers what makes a Bouncer protein appropriate with one species’ sperm however not one other’s, offering an intriguing molecular rationalization for fish hybridization. We caught up with Krista to seek out out extra concerning the story behind the paper. 

What introduced you to Andi’s lab and the way did the mission begin?

I first heard about Andi’s lab by an commercial for the Vienna BioCenter Summer time College program again in 2016 whereas I used to be doing my grasp’s diploma at Linköping College in Sweden. I had beforehand participated in a summer time REU (Analysis Expertise for Undergraduates) program whereas a bachelor’s pupil and was eager on doing a summer time analysis mission once more. Developmental biology fascinated me, so I utilized to Andi’s lab due to a mission on a small protein referred to as Toddler and its position in regulating cell migration throughout gastrulation. I loved being in Andi’s lab and dealing with zebrafish that summer time a lot that I got here again for my grasp’s thesis mission in 2017. For that mission, I modified gears from engaged on Toddler to Bouncer and its position in fertilization with a then Ph.D. pupil, Sarah Herberg. The work I did throughout that point set the stage for my very own Ph.D. mission and led to my discovery that Bouncer retains fertilization species-specific between medaka and zebrafish—a serious discovering in our first publication on Bouncer in Science. These thrilling outcomes offered the momentum for my very own Ph.D. mission. I dove into exploring how Bouncer mediates specificity in sperm-egg interplay between species, the primary purpose of my paper in Nature Communications.   

What was recognized about cross-fertilisation between completely different species earlier than your work? 

On the whole, scientists had noticed a excessive frequency of hybridization amongst fish species in comparison with different animal teams, however nobody had actually sought to know the molecular foundation for it given the truth that we didn’t even know what proteins are typically required for sperm-egg membrane interplay in fish. That’s a part of why it was so thrilling to work on Bouncer—it was the primary egg molecule proven to be required for fertilization in any fish species, opening the door to not solely finding out its perform in sperm binding, but additionally probing the way it may restrict binding to solely sperm from carefully associated species or permit hybridization throughout better phylogenetic distances. 

Are you able to summarise your key findings? 

Once I began engaged on Bouncer, I initially noticed that Bouncer enabled fertilization in a species-specific method: eggs expressing zebrafish Bouncer could possibly be fertilized by zebrafish sperm, however not by medaka sperm. At first of my present research, nonetheless, I quickly discovered that there was extra to it than that—zebrafish sperm turned out to be appropriate with a number of completely different species’ Bouncer proteins, and remarkably, some Bouncer proteins might even work with each medaka and zebrafish sperm. 

Nonetheless, profiting from Bouncer’s medaka/zebrafish specificity, I outlined options inside Bouncer orthologs, each on the amino acid and post-translational modification ranges, which might be required for interplay with zebrafish or medaka sperm. By way of evolution, we discovered that Bouncer stays largely related from fish species to fish species, but we recognized a medaka-specific change within the amino acid sequence (website 63) which contributes to the medaka/zebrafish cross-fertility block. In zebrafish, this website incorporates arginine (R), whereas medaka Bouncer incorporates a leucine (L). We moreover discovered that the N-glycosylation sample of Bouncer actually issues for medaka sperm compatibility however doesn’t affect zebrafish sperm compatibility. On this method, I elucidated a number of the molecular particulars as to why zebrafish and medaka can not cross-fertilize and moreover demonstrated that there are completely different ranges of stringency for sperm-Bouncer interplay relying on the species, possible impacting their means to hybridize with extra distantly associated species vs. solely shut relations. 

Have been you shocked to seek out that seahorse and fugu Bouncer are appropriate with each zebrafish and medaka sperm?

Sure, utterly! This consequence was significantly shocking when contemplating that each seahorse and fugu Bouncer are ~40% an identical to zebrafish Bouncer, whereas medaka Bouncer shares slightly below 39% identification with zebrafish Bouncer. As I discussed earlier than, it was additionally fairly a shock since we initially anticipated that Bouncer would present strict species specificity past simply the medaka/zebrafish mixture. Nonetheless, it was an informative consequence as a result of I might then deal with sequence parts that had been completely different between medaka and zebrafish Bouncer and ignore any adjustments that had been additionally current in fugu and seahorse Bouncer, permitting me to slender the sphere in figuring out what underlies Bouncer’s medaka/zebrafish specificity.  

What was it like working with two completely different species of fish?

For me, working with animals is likely one of the most pleasant components of being a biologist, so I gladly accepted the problem of introducing a brand new mannequin species into the lab. Earlier than my mission, we had solely zebrafish, and I wanted to arrange all of the protocols and instruments for working with medaka. It was not straightforward, however I discovered it very rewarding as soon as I bought all the pieces up and operating! Having the side-by-side comparability of medaka and zebrafish was additionally very informative by way of their intrinsic species variations, significantly within the strategy of fertilization itself. 

Did you’ve any explicit consequence or eureka second that has caught with you?

My largest “eureka second” must be after I noticed zebrafish eggs fertilized by medaka sperm for the primary time. Throughout my grasp’s mission, I had made a number of bouncer mutant zebrafish strains expressing different species’ bouncer genes to check whether or not they might rescue fertilization with zebrafish sperm. I examined the Bouncer proteins from human, mouse, Xenopus, and medaka, however none of them had been appropriate with zebrafish sperm. However what concerning the different method round? Might I fertilize these zebrafish eggs with sperm that was species-matched with the expressed Bouncer protein? The state of affairs most definitely to work was medaka sperm with medaka Bouncer-expressing zebrafish eggs, so I procured a number of medaka males from a neighboring lab and gave IVF a strive. After a couple of makes an attempt and to my nice shock, I caught sight of cleavage-stage embryos within the petri dish as I peered by the dissection scope a couple of hours later—the very first zebrafish-medaka hybrids to ever exist!

And the flipside: had been there any moments of frustration or despair?

Once I began this mission, I believed that I might have the ability to pinpoint the precise adjustments wanted to make zebrafish Bouncer appropriate with medaka sperm. Every time I made a brand new line, I believed for certain that this could be the one that may lastly work. There’s a important time lag between making a brand new line and getting the outcomes since it’s good to wait for 2 generations, and it takes about 3 months for zebrafish to achieve sexual maturity. After making and testing all my amino acid substitution mutants with out a lot luck, I used to be satisfied that combining the R63L change with the medaka N-glycosylation sample in zebrafish Bouncer could be ample for medaka sperm to work. It was disappointing when it didn’t, and much more so after I then tried your complete medaka finger 3 sequence plus the N-glycosylation sample in zebrafish Bouncer, and this nonetheless did not work with medaka sperm. Trying again, although, I’m glad I saved attempting regardless that I didn’t fairly clear up your complete puzzle of species specificity. 

My preliminary discovery of Bouncer as the primary block to cross-fertilization between medaka and zebrafish offered a flexible system for uncovering Bouncer’s interplay associate on sperm. On high of this, my present paper highlights the significance of website 63 in figuring out specificity, and it might subsequently even be crucial for interplay with the sperm binding associate. Present work within the lab by my colleagues Andreas Blaha and Victoria Deneke uncovered that Bouncer might work together with a heterotrimeric advanced on sperm consisting of Izumo1, Spaca6, and a brand new issue, Tmem81, as they report of their current bioRxiv preprint (Deneke, Blaha, et al., 2023). That is groundbreaking for the fertilization subject for the reason that solely sperm-egg protein interplay pair of which we all know at the moment is IZUMO1 and JUNO in mammals. What’s particularly placing to me is that once you have a look at the AlphaFold-predicted mannequin of zebrafish Bouncer and the sperm trimer, the tip of finger 3 the place R63 sits in Bouncer is true on the interface between Spaca6 and Izumo1. It’s thrilling to see how my work was in a position to pinpoint a possible key interplay website regardless that I had solely half of the image. Now that we all know Bouncer’s interplay associate on sperm, we are able to work towards defining the Bouncer-trimer interplay interface and perceive why zebrafish sperm are in a position to bind many Bouncer orthologs, whereas medaka sperm preserve larger specificity.

And personally, what’s subsequent for you after this paper?

Discovering find out how to make hybrids between medaka and zebrafish spawned one other entire mission on which I embarked throughout my Ph.D. I used them to handle one other query of species specificity: how is the timing of zygotic genome activation (ZGA) decided? Medaka and zebrafish have proved to be a really fascinating comparative system for this research as effectively, given their completely different occasions of ZGA onset throughout embryogenesis. At present, I’m ending up work within the lab for a paper on how we used these hybrids to discover this query. In case you might be curious, there’s already a preprint on bioRxiv (Gert et al., 2021).  

I defended my Ph.D. earlier this summer time and am at the moment in search of a post-doc place to proceed engaged on fertilization or reproductive biology. I’m particularly excited to work on understudied species and to determine new animal fashions, as my work with Bouncer in two distantly associated fish species has actually satisfied me of the worth of a comparative system. Sooner or later, I hope to work with endangered species and develop new reproductive applied sciences to help their conservation. 

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