The cellular biology of reproduction is often depicted as an epic journey, in which millions of intrepid sperm fight to be the first to claim the ultimate prize: the chance to fertilize the egg, which has been passively waiting for the sperm to arrive.
But is this an accurate picture? In a 1991 paper, New York University anthropologist Emily Martin argued that this notion was, in fact, “a scientific fairy tale.”1 Instead of representing absolute unbiased truths, she asserted that, “The picture of egg and sperm drawn in popular as well as scientific accounts of reproductive biology relies on stereotypes central to our cultural definitions of male and female.”
While it takes two to tango, research has historically focused on male contributions to the reproductive process, whether in humans or invertebrates. “Fruit flies have been a model system for about a century,” said evolutionary biologist Caitlin McDonough-Goldstein, currently a postdoctoral researcher at the University of Vienna. “But as I was starting to explore the fruit fly reproductive system…when I asked people ‘What does this gland in the female reproductive tract do, the parovaria?’ They would say, ‘Oh, we’re not sure.’”
In fact, a detailed single-cell transcriptomic analysis of the fruit fly female reproductive tract was not published until 2024.2 These findings forced scientists to reconsider earlier findings on fruit fly reproduction: About 40 percent of the genes previously classified as seminal fluid genes were also expressed in various tissues of the female reproductive tract. In the paper, the University of California, Davis research team noted that, “The past 30 years of literature have often explored the rhetorical framing that males use [seminal fluid proteins] to control, manipulate, or harm females.” But if females also produce many of these proteins, these purported functions may need serious re-evaluation.
“So many of the things that we thought were male-specific are actually the results of an interaction,” said McDonough-Goldstein.
There are many reasons why it’s important to understand cryptic female choice—females’ ability to influence which sperm fertilize their eggs—and gain a more complete understanding of the biology of reproduction: These processes underpin mechanisms and potential treatments for infertility in humans and could inform breeding programs for endangered species. They are also an important component of sexual selection, and thus may help us understand the trajectories of how organisms change and branch into new species over time. But without a complete understanding of female biology and behavior, said Jean-Christophe Billeter, a behavioral neurogeneticist at the University of Groningen, “We only know half of the equation.”
Evolutionary biologist Caitlin McDonough-Goldstein explores the variation in processes related to reproduction and reproductive outcomes.
Erin Carter
Cryptic and Not-So-Cryptic Female Choice: Why Darwin Hated Peacocks
Natural selection tends to be a relatively straightforward process, driving adaptation to environmental change. “[For example], if it gets hotter, if it gets colder, if you have food present or not—we know quite well how this type of evolution takes place,” said Billeter. “It explains how different species have evolved to have different functions in their ecological niches.” Natural selection gives us birds with beaks that are the perfect shape to eat particular seeds, polar bears with ultra-insulating fur, and glass frogs which are semi-transparent to hide from predators.
The peacock’s resplendent tail feathers have been shaped by sexual selection.
© iStock.com, eROMAZe
“Now, sexual selection is a bit different because it's not actually based on biotic changes,” said Billeter. “It's based on social interactions, and that's what fascinates me about it. It's not what I want, it's what somebody else wants for me.” In other words, sexual selection produces traits that are attractive to potential mates, regardless of whether they have any inherent benefits for the organism itself. This includes elaborate courtship displays in some spider species, brightly-colored guppies that are actually more likely to be eaten than their duller counterparts, and the peacock’s beautiful but unwieldy tail feathers—which were so upsetting to Charles Darwin because they seemed to fly in the face of natural selection.
Darwin separated sexual selection into male-male competition for access to mates and female choice of the most attractive mate; however, he left out many species, such as jacanas, meerkats, and hyenas, in which the females are the competitors, as well as the prevalence of polyandry throughout the animal kingdom.3 Darwin also failed to note that mating was not the final step: Complex and incompletely understood molecular and cellular processes unfold within the female’s reproductive tract to determine which sperm—and perhaps more importantly whose sperm—will get to fertilize an egg. Historically, this field of research has focused largely on male contributions, seeking to answer questions about how the characteristics of the sperm themselves, or the chemical components of the seminal fluid, could enable one male’s sperm to outcompete another, or manipulate the female’s biology to further his own ends.
So many of the things that we thought were male-specific are actually the results of an interaction.
—Caitlin McDonough-Goldstein, University of Vienna
Answering questions about female-driven processes, as well as male-female interactions in post-copulatory sexual selection—and the underlying mechanisms—has been difficult due to both logistical limitations and lack of funding for female-centered research. In recent years, however, cultural shifts that empowered researchers to ask new kinds of questions as well as technological advancements in microscopy, transcriptomics, and genetic engineering, have allowed scientists to begin lifting the veil on these cryptic processes.
Many processes that were once attributed solely to males are now known to be influenced by male and female components.
Erin Carter and Caitlin McDonough-Goldstein
What do Females Want? Elements of Post-Mating Selection
These female-driven selection processes, called cryptic female choice, enable a female to influence which sperm fertilize the eggs, usually in the context of favoring one male’s sperm over another’s.
In order to formulate hypotheses about how a female biases the paternity of her offspring, researchers first need to figure out what kinds of male traits the female looks for when she tips the scales to favor some sperm over others. “There’s not a huge amount of research on the topic,” said Clelia Gasparini, an evolutionary biologist at the University of Padova. “It's not something that we know everything about.” In general, though, it seems that females are selecting for the same kind of things in post-copulation processes as they might in pre-copulation processes.
Some of these are quite straightforward. First, females generally prefer males from their own species, or conspecifics. To test whether post-mating selection processes also favored conspecific males, researchers observed matings between female fruit flies and males of the same species or a closely related species (heterospecifics).4 Even though the female could produce offspring with the heterospecifics, when she was allowed to mate with both types, the majority of her offspring were sired by the conspecific. As potential mechanisms, researchers observed that females ejected heterospecific sperm after a shorter latency, and changed which sperm storage organ they favored based on which organ contained the conspecific sperm.
While it’s not advantageous to mate with a different species, it’s also detrimental to mate with a male that’s too closely related. Research has demonstrated that post-mating selection plays a role in inbreeding avoidance as well: “Promiscuous” field crickets stored fewer sperm from siblings, and in guppies—a fish species with internal fertilization that give birth to live young—sperm swam faster in female reproductive fluids from unrelated females than in sibling fluids.5,6
Some species have easily observable traits, such as social dominance or coloration, that identify males that females find particularly attractive. These preferences too, extend beyond overt mate choice—they are reflected in post-copulatory processes as well. For example, researchers have observed that female chickens are more likely to eject sperm from non-dominant males.7 In another study, researchers allowed a female guppy to visually assess two males, a test male that was intermediately colored, and an alternative male, which was either very bright or very drab.8 She was then given an opportunity to mate with the test male. Researchers found that females retained more sperm from the test male when he was paired with the drab, or less attractive, male than when he was paired with the brighter, more attractive male, even though the characteristics of the test male himself had not changed. This suggests that female preferences continue to influence the paternity of the offspring even after mating has occurred, and her preferences are governed by the perceived attractiveness of her mate compared to other local males.
Researchers use transgenic flies with green fluorescent sperm to study post-sexual selection processes.
Jean-Christophe Billeter, Groningen Institute for Evolutionary Life Sciences, University of Groningen.
All this begs the question: If these post-copulatory processes select for the same male that the female would choose to mate with anyway, what purpose do they serve? There are several potential answers. In some species, including the chickens mentioned above, forced matings mean that females may not necessarily get to choose every mate, so post-copulatory selection may help bias paternity against unwanted males.
In animals that are external fertilizers, which includes many fish and aquatic invertebrates, eggs and sperm meet outside the body, so traditional mate choice mechanisms may not apply. “In external fertilizers, people believed that because the eggs and the sperm are released into the water, there was no [opportunity] for the female to bias paternity,” said Gasparini. “Researchers thought that there was less cryptic female choice in external fertilizers [than in internal fertilizers] for this reason, but it looks like it's actually the opposite. The less opportunity you have to control which male you mate with…the more you should rely, as a female, on post-copulatory mechanisms.”
Evolutionary biologist Clelia Gasparini studies the interactions between zebrafish female reproductive fluid and sperm, and how this affects paternity.
Clelia Gasparini
Gasparini is using zebrafish to study how the female reproductive fluid (FRF) that surrounds the eggs could influence which sperm are ultimately successful in fertilization. So far, her work has shown that zebrafish FRF generally increases sperm velocity and longevity, and that FRF from different females boosts sperm performance to a greater or lesser extent depending on the identity of the male, which hints at a potential mechanism for selection based on the pair’s compatibility.9 In another fish species, the stickleback, females seem to choose males based on major histocompatibility complex (MHC) genes, selecting for a male whose repertoire of MHC alleles, which are crucial for immunity, are neither too similar nor too different than hers.10 While it has not yet been proven, zebrafish post-copulatory selection processes could also function to produce offspring with optimal MHC diversity.
Gasparini has also demonstrated that zebrafish FRF may not only be able to aid in the selection of compatible males, but may also help attract the highest-quality sperm from an individual male.11 Gasparini’s team separated zebrafish sperm that were attracted to FRF from those that were not, and found that the FRF-seeking sperm had higher DNA integrity and were able to fertilize a higher percentage of the eggs.
Gasparini is currently focused on FRF in external fertilizers, but females whose eggs are fertilized internally also possess reproductive tract fluids with dynamic profiles of hormones, cytokines, and other proteins, which could play important roles in reproduction.12 She hopes to eventually expand the scope of her studies, researching other species to determine how broadly applicable her fish findings are. “Because this fluid is occurring in every single species, I think it’s probably just a question of being able to do the proper experiments, which is not easy…But in almost every species where [researchers] have really looked at this fluid, they’ve found that it does something [related to] selection,” said Gasparini.
Zebrafish are external fertilizers, so molecular processes may be especially important for biasing paternity to favor high quality or more compatible males.
Clelia Gasparini
Flies, Pheromones, and the Myth of Extreme Female Choosiness
In fruit flies, post-mating selection processes may help a female keep her options open, allowing her to be more or less picky about mates depending on the situation. This idea, explained Billeter, is relatively new and contradicts previous beliefs about female choosiness.
“There is an old paradigm in biology called Bateman’s principle, whereby males can make a lot of sperm for very cheap, and females can make only a few eggs that are very expensive to make,” said Billeter. “This creates a situation where males and females don't have the same kind of pressures to reproduce with each other—males basically benefit from being very promiscuous and mating with as many females as they can, and females benefit from mating very carefully with a perfectly chosen male…Now, this paradigm is quickly changing, because it turned out to be a sort of old, male fantasy that was not true at all.”
However, early fruit fly experiments did initially seem to support this choosiness paradigm. Laboratory studies showed that after the first mating, female flies became much less receptive to mating with new males, and that this effect was caused by a particular peptide in the male’s seminal fluid.13 This was interpreted as sexual coercion by the male, said Billeter. “The male basically puts a special kind of pheromone into his ejaculate that a female has to receive if she wants to be fertilized, which then manipulates her by making her not want to re-mate. That way, the male protects his investment, and he becomes the only sire of the female’s offspring,” Billeter said. In this case, it would make sense for a female to be extremely choosy with her first mate, since she might not get chance to mate with another male.
Sexual selection is a bit different because it's not actually based on biotic changes… It's based on social interactions, and that's what fascinates me about it.
—Jean-Christophe Billeter, University of Groningen
Observations of flies in the wild, however, cast doubt on this interpretation. Wild females commonly mated multiple times per day, so clearly females weren’t completely inhibited from re-mating. “This, of course, makes complete sense,” said Billeter, “Because in evolutionary terms, why would anyone allow themselves to be manipulated?”
Billeter’s work has helped demonstrate that instead, females use these signals to modulate their choosiness to an appropriate level. “Drosophila females, in fact, are not choosy at all when they are virgins. Because if you think about it, what is the greatest cost to a female? Is it to mate with a bad male or to never mate?” Thus, it’s likely advantageous to have low standards for a first mate—to ensure that she will not die without reproducing—and thereafter only mate with higher quality males to give her offspring the best genes that are available.
Billeter has also explored the biochemical processes that mediate this increase in choosiness. When females received a particular seminal peptide from males, it triggered the release of juvenile hormone, which was known to promote egg production. Billeter showed that this hormone also acted on specific olfactory neurons in the female fly brain, reducing their sensitivity to palmitoleic acid, a fruit fly aphrodisiac pheromone.14 Thus, after her first mating, these neurons are only responsive to the most attractive males.
The Same Story in Reverse: Changing the Lens Through Which We View Biology
Mariana Wolfner, a molecular biologist at Cornell University, has been studying Drosophila reproduction and development since the 1980s, and has witnessed firsthand how the field has shifted from a focus on male-female conflict to a more nuanced understanding of the interactions between male and female cells and proteins. In the past, not only was the egg seen as a passive participant, but also the prevailing view among insect biologists was that the peptides and other signaling molecules produced by the male manipulated the female’s body and behaviors to maximize the number of offspring he sired.
In other words, researchers largely viewed post-mating changes in the female fly as largely orchestrated by, and beneficial for, the male. The male seminal molecules make the female ramp up egg production, trigger ovulation, and compel her to store hundreds of his sperm cells in specialized storage organs.15 To fuel egg production, she consumes more protein and devotes fewer resources to her immune function, and her lifespan is shortened compared to her unmated sisters.16 One molecule in particular, christened “sex peptide,” seemed to be responsible for many of these effects and became the representative trait for sexual conflict, thought to promote male reproductive success while decreasing female fitness.
There's this idea that both sexes are benefiting from the male proteins that are transferred to the female.
—Mariana Wolfner, Cornell University
But that’s not the only way to look at it. “Now, I can tell you the same story reversed,” said Wolfner. “Making eggs is energetically demanding for females. So, wouldn't it be nice if you [didn’t] make eggs until you received a signal that you have sperm with which to fertilize them? So maybe just as much as the male is using seminal proteins to ‘make’ the female make eggs, the female is using the male proteins to tell her when her physiology should change—at a time that's advantageous for her, when she’s not wasting eggs.”
Instead of conflict between the sexes, Wolfner said that now, “there's this idea that both sexes are benefiting from the male proteins that are transferred to the female.” For example, in addition to processes directly related to fertility, sex peptide from the male also exerts effects on the female fly brain, improving her long-term memory.17
Increasingly, research is revealing potential cooperative interactions between male and female cells and proteins. For example, Wolfner, in collaboration with Syracuse University evolutionary biologists Scott Pitnick and Steve Dorus, demonstrated that four days after insemination, about 20 percent of the fruit fly sperm proteome consisted of proteins derived from the female.18 Many of these proteins were related to energy metabolism or sperm remodeling processes that were important for sperm-egg recognition. Thus, these female proteins could play an essential role in sperm survival during relatively long-term storage in the female reproductive tract and in preparing the sperm for their eventual union with the egg.
Cryptic Female Choice Could Shed New Light on Infertility
Post-mating selection mechanisms seem to be important for successful fertilization in some species of insects, fish, and birds, but is any of this applicable to human reproduction? While the specifics may be different—humans have different mating strategies, different reproductive architecture, and different proteomic signatures associated with sperm and eggs—researchers hope that this foundational work could inspire other scientists to think about human fertility and infertility in new ways.
Studies of post-copulatory selection processes could help guide improvements to assisted reproductive technologies, like in vitro fertilization.
© iStock.com, Peter Hansen
“I work on fruit flies, so I'm not saying that what I work on has any kind of direct implication for humans,” said Billeter, “[but maybe] it leads people to start thinking about other scenarios as to why people are infertile. So, not looking at just sperm or eggs, but maybe looking more at the interaction between the two sexes.”
Indeed, some preliminary work with human samples indicates that there may be important but incompletely understood interaction effects. Similar to Gasparini’s fish studies, other researchers found that in humans, female follicular fluid had differential effects on sperm properties, like motility and viability, depending on the male-female pairing, with certain couples seeming to have higher compatibility than others.19 Researchers hypothesize that such incompatibility could underlie some portion of the 15–30 percent of infertility cases that do not have a readily identifiable cause.20
Gasparini also sees potential for her work to inform sperm selection techniques for use in assisted reproductive technologies. Of the tens to hundreds of millions of sperm in a sample, Gasparini said that there are not necessarily hard-and-fast, objective rules to determine which ones to use during in vitro fertilization, beyond broad tests of motility. “Because every test you [can] run on a single sperm cell will destroy the sperm,” she said.
We are the result of millions of years of evolution…And because reproduction is the one thing that defines us as living beings, whatever increases reproductive success and quality of the offspring has been selected over many, many millions of years.
—Clelia Gasparini, University of Padova
This inability to identify top-notch sperm can be especially problematic for intracytoplasmic sperm injection, in which a single sperm is injected directly into the egg. Gasparini hopes that her work could inspire FRF-mediated pre-selection strategies to help choose not only the best, but most compatible sperm, taking advantage of selection processes that have already been finely honed by evolution. “We are the result of millions of years of evolution,” she said. “And because reproduction is the one thing that defines us as living beings, whatever increases reproductive success and quality of the offspring has been selected over many, many millions of years.” Instead of ignoring theses processes, we could learn more about them and leverage them to help couples experiencing infertility.
While researchers are inching closer to understanding how these cryptic post-mating processes function, there’s still much more to learn. “There’s more to reproduction than meets the eye,” said Billeter.
- Martin E. The egg and the sperm: How science has constructed a romance based on stereotypical male-female roles. Signs. 1991;16(3):485-501.
- Thayer RC, et al. Regional specialization, polyploidy, and seminal fluid transcripts in the Drosophila female reproductive tract. Proc Natl Acad Sci USA. 2024;121(44):e2409850121.
- Fromonteil S, et al. Sexual selection in females and the evolution of polyandry. PLOS Biol. 2023;21(1):e3001916.
- Manier MK, et al. Postcopulatory sexual selection generates speciation phenotypes in Drosophila. Curr Biol. 2013;23(19):1853-1862.
- Bretman A, et al. Promiscuous females avoid inbreeding by controlling sperm storage. Mol Ecol. 2009;18(16):3340-3345.
- Gasparini C, Pilastro A. Cryptic female preference for genetically unrelated males is mediated by ovarian fluid in the guppy. Proc R Soc B. 2011;278(1717):2495-2501.
- Pizzari T, Birkhead TR. Female feral fowl eject sperm of subdominant males. Nature. 2000;405(6788):787-789.
- Pilastro A, et al. Cryptic female preference for colorful males in guppies. Evolution. 2004;58(3):665-669.
- Poli F, et al. Effects of ovarian fluid on sperm traits and its implications for cryptic female choice in zebrafish. Behav Ecol. 2019;30(5):1298-1305.
- Milinski M, et al. Mate choice decisions of stickleback females predictably modified by MHC peptide ligands. Proc Natl Acad Sci USA. 2005;102(12):4414-4418.
- Cattelan S, et al. Female reproductive fluid attracts more and better sperm: Implications for within-ejaculate cryptic female choice. Biol Lett. 2023;19(6):20230063.
- Basuino L, Silveira CF. Human follicular fluid and effects on reproduction. JBRA Assist Reprod. 2016;20(1):38-40.
- Liu H, Kubli E. Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster. Proc Natl Acad Sci USA. 2003;100(17):9929-9933.
- Kohlmeier P, et al. Mating increases Drosophila melanogaster females’ choosiness by reducing olfactory sensitivity to a male pheromone. Nat Ecol Evol. 2021;5(8):1165-1173.
- Wolfner MF. The gifts that keep on giving: Physiological functions and evolutionary dynamics of male seminal proteins in Drosophila. Heredity. 2002;88(2):85-93.
- Hopkins BR, Perry JC. The evolution of sex peptide: Sexual conflict, cooperation, and coevolution. Biol Rev Camb Philos Soc. 2022;97(4):1426-1448.
- Scheunemann L, et al. A sperm peptide enhances long-term memory in female Drosophila. Sci Adv. 2019;5(11):eaax3432.
- McCullough EL, et al. The life history of Drosophila sperm involves molecular continuity between male and female reproductive tracts. Proc Natl Acad Sci USA. 2022;119(11):e2119899119.
- Jokiniemi A, et al. Gamete-level immunogenetic incompatibility in humans-towards deeper understanding of fertilization and infertility? Heredity. 2020;125(5):281-289.
- Quaas A, Dokras A. Diagnosis and treatment of unexplained infertility. Rev Obstet Gynecol. 2008;1(2):69-76.
