Recent work published by Prof. Marianna Wolfner ’74, molecular biology and genetics, and collaborators found that the survival and success of fruit fly sperm after mating depends on proteins in the female reproductive tract. The team found that just four days after mating, 20 percent of the proteins bound to sperm are female-derived.
Scientists have long known that sperm undergo changes in the female after mating, in many insects and even in humans, according to Wolfner. These changes are often fundamental to the sperm’s development. Fruit flies were an optimal organism for further study since their reproductive biology and genome are already well-understood.
Fruit flies have an interesting reproductive feature:Females can retain sperm in their bodies for up to two weeks after mating. This is longer than a generation time, the duration required for an egg to develop into an adult. Wolfner said that this extended duration prior to fertilization may be one reason why female proteins are needed by sperm to survive.
This hypothesis was consistent with the team’s findings: some of the female-derived proteins seem to be replacing those found in the seminal fluid. Wolfner said that a common class of female proteins binding to the sperm were metabolic proteins, which could play an essential role in keeping the sperm alive.
Future work will aim to better understand and confirm what functions these proteins play. Wolfner explained that her team can now use gene-editing tools like CRISPR to “knock out” expression of different female proteins and measure effects on fertility, sperm retention, and survival.
The study’s experimental approach was straightforward. “It’s pretty simple: dissect-out the sperm, break down the protein [into peptides], determine the mass of the peptides, figure out what protein they come from and figure out where the protein comes from,” Wolfner said.
The team used mass spectrometry, which allowed them to calculate the molecular weights of the broken-down proteins with high precision. The weights could then be used to identify the proteins.
Mass spectrometry was also critical for distinguishing male and female proteins – female proteins were labeled with heavy isotopes, which altered their weights. The team could then use these small differences in the expected and actual weights to determine whether the protein came from the male or the female.
The Cornell group also worked in collaboration with scientists at Syracuse University. The two labs repeated the same sperm dissections and procedures and found the same results, increasing the team’s confidence in their findings.
But the role of female proteins on male sperm may go beyond providing metabolic support. It is known that female fruit flies can exert selective control over which sperm fertilizes their eggs. For example, a female will often mate for the first time with any male she can find, Wolfner explained. However, she will select a second mate more choosily, and the second mating tends to produce more progeny, relative to the first one.
The mechanism by which this bias occurs is still unknown, but Wolfner’s team has a theory, based on their findings.
“We had this crazy idea: the sperm from the first male, that have been in the female for longer, will have more female protein on them [relative to the second male],” Wolfner said “So the female could say, ‘Oh, the sperm with less of my protein are the new ones. I’ll use those.’ It fits with our data, but we have no direct experimental proof of this yet.”
Proving this idea is another future direction of this work, according to Wolfner.
The findings are part of a broader theme in reproduction, known as molecular handoff. While Wolfner and her team focused on the role of female molecules on male sperm, this handoff can occur from males to females as well. One example of this is the sex peptide, a molecule transferred during mating via sperm.
“The sex peptide has many different effects on the female – our lab, in collaboration with professor Buchon’s lab in entomology, showed that it causes her gut to grow and to express genes for digestion,” Wolfner said.
She explained that these changes may help females increase food intake in preparation for laying eggs. Such findings add nuance to the traditional views of sexual conflict in biology, according to Wolfner.
“There’s a lot of conflict in reproduction since males and females have different reproductive strategies, leading to different evolutionary pressures on them. But there’s also all this cooperation and handoff,” Wolfner said. “It’s fascinating because it’s both.”