In a new study, researchers led by Serge Y. Fuchs of the School of Veterinary Medicine, have uncovered a detailed mechanism by which tumors can skirt both the immune system and cancer therapies that leverage its power, such as genetically engineered CAR T cells.

Their investigation, published in the journal Cell Metabolism, revealed how tumor-derived factors stimulate trogocytosis — a process derived from the Greek word trogo, which means “to gnaw” or “to chew.” When T cells interact with cancer cells, they can sometimes “nibble” a piece of the cancer cell membrane. When that membrane segment includes an antigen, a molecule specific to the cancer, the T cells may then begin expressing that antigen on their own cell surface, making it appear to other T cells like a cancer cell.

Trogocytosis can affect a patient’s own T cells and those modified to become CAR T cells, an approach in which a patient’s T cells are genetically engineered to specifically target cancer cells, grown in a lab, and then delivered back to the patient.

“Trogocytosis can lead to three different things, and all three are bad for a person with a tumor,” says Fuchs, the senior author on the work. “First of all, the tumor cell did not get killed and has lost an antigen, which may mean that even if another, better equipped T cell comes along, it will not recognize it, giving cancer cells a window of opportunity to grow unchecked. The second problem is, for reasons we still don’t understand, once a T cell takes a piece of the tumor cell membrane, it becomes much less active. And the third problem is very ironic. Because now, a T cell that displays tumor antigen, this ‘sheep in wolf’s clothing,’ may then become victim to ‘fratricide,’ killed by another T cell.” Overall, the result is a decline in killer T cell numbers and activity and a bump in opportunities for the cancer cells to escape detection and grow.

“What we see is that only a small number of cells undergo trogocytosis and then they disappear quickly because they’re killed. So we’re studying a vanishing act. It is hard to do — very expensive and very tedious — but it appears to be very important,” says Fuchs.

With a longstanding interest in how receptors on the surface of immune cells guide anti-cancer immunity, Fuchs and colleagues have made a variety of discoveries showing how tumors can manipulate T cells to escape being targeted and killed.

A particular area of interest is what’s known as tumor-derived factors, or the concoction of proteins, lipids, and other materials that cancer cells secrete into the body. In the current study, the Penn researchers found that collecting these secretions and exposing the resulting solution to T cells hampered the cells’ ability to do their cancer-fighting job.

“Just exposing them to this tumor-conditioned media caused them to kill fewer cancer cells, trogocytose more, and get killed more,” Fuchs says.

Trogocytosis was previously believed to have something to do with cancer’s ability to hinder anti-cancer immunity, but the Penn-led team pinned down the mechanism, showing that T cells exposed to tumor-derived factors experienced a notable decline in levels of the gene CH25H. This gene is known to be involved in altering cell’s lipid membranes and can inhibit two cell membranes from fusing together — an essential process for trogocytosis to occur. When they added back a metabolite produced by CH25H, they could block trogocytosis.

“That was an ‘aha’ moment,” Fuchs says.

Further characterization of the pathway helped the team identify another player, the gene ATF3, which opposes the activity of CH25H. Eliminating AFT3 prevented trogocytosis from occurring and restored the ability of T cells to kill tumor cells.

Not only do the new insights suggest novel targets for anti-cancer therapies, but they may have immediate significance for CAR T therapy. Since trogocytosis could impair the effectiveness of the engineered T cells delivered in CAR T, the researchers surmised that blocking this could improve CAR T performance. “We figured, why don’t we use what is cleverly known as an ‘armored CAR’ approach, and co-express CH25H in the CAR T,” Fuchs says. “This turned out to be more efficient than the old CAR T cells.”

Indeed, delivering the CAR T cells adorned with CH25H improved the survival of mice with cancer compared to the unarmored CAR T cells.

Though only a small percentage of T cells are involved in trogocytosis, Fuchs says it may be an underappreciated process when it comes to cancer immunity and other processes, such as those involved in autoimmunity. With future work, he and colleagues intend to explore the roles of ATF3 and CH25H and other molecules in trogocytosis. And he’s hopeful that other researchers will pick up on these lines of work, bringing the findings closer to clinical application.

“I can see this going into use in CAR T therapy quickly,” Fuchs says. “It’s ready to play.”

Serge Fuchs is the Elizabeth and William Whitney Clark Professor of Oncology at the University of Pennsylvania School of Veterinary Medicine.

Fuchs’ coauthors on the work were Penn Vet’s Zhen Lu, Noreen McBrearty, Jinyun Chen, Vivek S. Tomar, Hongru Zhang, Gianluca De Rosa, and Daniel P. Beiting; the Perelman School of Medicine’s Aiwen Tan, Aalim M. Weljie, Zhen Miao, Subin S. George, and Constantinos Koumensis; Takeda Development Center’s Allison Berger and Gurpanna Saggu; and Case Western Reserve University’s J. Alan Diehl.

The study was supported in part by the National Institutes of Health (grants CA247803, CA240814, CA165997, and CA115299).

The 10^th oocyte collection in northern white rhinos (NWR) was performed by a team of scientists and conservationists from the Leibniz Institute for Zoo and Wildlife Research (Leibniz-IZW), Ol Pejeta Conservancy, Safari Park Dvůr Králové, Kenya Wildlife Service (KWS) & Wildlife Research and Training Institute (WRTI) on July 28, 2022, at Ol Pejeta Conservancy in Kenya. The BioRescue team was able to collect 23 oocytes from Fatu, the younger of the two remaining NWR females. Oocyte collections from Najin, Fatu’s mother, were ceased in 2021 following an in-depth ethical risk assessment. The oocytes were immediately air-lifted to the Avantea laboratory in Cremona, Italy. Following maturation, 7 of the oocytes were fertilized using cryopreserved, thawed semen from the deceased NWR male Angalifu. Eventually, 5 embryos of Fatu were successfully produced and cryopreserved in liquid nitrogen.

This collection followed on from the 9^th oocyte collection held at the same location and by the same team on April 24^th. Out of 16 collected oocytes, 3 embryos were produced in the Avantea laboratory, again using the semen of Angalifu. Successful results of both procedures raised the total number of NWR embryos produced to 22 — all of them from female Fatu, with half of them sired by the deceased male Suni who was born in Safari Park Dvůr Králové, Czech Republic, and the other half sired by Angalifu who lived in San Diego Zoo Safari Park, USA.

Once the protocol to transfer the embryos to surrogate southern white rhino (SWR) female recipients is optimized, the embryos will be the foundation of a new NWR population, eventually destined to step back into their ecological role as keystone grazers in Central Africa.

To set up suitable conditions for a successful embryo transfer, the team has been carefully following the interactions of the sterilized SWR bull Owuan, who serves as the oestrus detector, with the possible surrogate females that share an enclosure with him. Once the conditions allow it, the BioRescue team will attempt to conduct an embryo transfer — first with SWR embryos to demonstrate that the whole procedure works properly before the team uses the extremely valuable NWR embryos. The team is currently considering whether adding more SWR females to the program might increase the chance of achieving the first successful embryo transfer.

Thomas Hildebrandt, BioRescue project leader and head of Department of Reproduction Management at Leibniz-IZW: “In 2019, one day before our worldwide first oocyte collection in NWR I said — tomorrow we will change the world. Today I can say, we did: The 5 new NWR embryos created in one set of procedures are a new record in our mission to save the NWR from the brink of extinction. In total, we managed to produce and cryopreserve 22 pure NWR embryos from 158 oocytes collected during 10 collections: 148 from Fatu and 10 from Nájin. Our next aim is to successfully produce viable offspring by inventing and using new scientific embryo transfer methods and techniques. The ground breaking scientific work we are establishing here will lay the groundwork for future conservation rescue initiatives.”

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The discovery, published in Nature, describes the skeletal remains of a young adult found in a cave in Borneo, who had part of the left lower leg and left foot amputated, probably as a child, at least 31,000 years ago. The person survived the surgical procedure, living for at least another six to nine years.

The find presents a remarkable feat. It is notoriously difficult to prevent infections in surgical amputations, even to this day. Yet 30,000 years ago a community was able to successfully navigate veins, arteries, nerves, and tissue, and keep the wound clean so that it healed successfully. The individual went on the live into adulthood where an unknown cause eventually led to their death.

Bioarchaeologist and an expert in ancient skeletons, Dr Melandri Vlok, at University of Sydney said the find is “incredibly exciting and unexpected.”

“The discovery implies that at least some modern human foraging groups in tropical Asia had developed sophisticated medical knowledge and skills long before the Neolithic farming transition,” said Dr Vlok, who is co-lead author of the paper and a postdoctoral research associate in Sydney Southeast Asia Centre.

Studying bones

The skeleton of the young adult, possibly in their 20s when they died, was carefully buried within LiangTebo cave — located Borneo in East Kalimantan, in a limestone karst area that harbours some of the world’s earliest dated rock art.

The bones were uncovered by archaeologists from Griffith University and University of Western Australia (UWA) just days before borders closed for the COVID-19 pandemic in March 2020. The team was led by Professor Maxime Aubert and Dr Tim Maloney (Griffith University), Dr India Dilkes-Hall (UWA) and Mr Andika Priyatno from the Kalimantan Timur Cultural Heritage Preservation Centre.

The University of Sydney’s Dr Vlok was invited to study the bones when they were brought back to Australia.

“No one told me they had not found the left foot in the grave,” Dr Vlok said. “They kept it hidden from me to see what I would find.”

As Dr Vlok laid the bones out, the left leg looked withered, and was the size of a child’s, but the individual was an adult. She unwrapped the part of the leg that contained the stump and noticed the cut was clean, well healed and had no evidence of any infection. “The chances the amputation was an accident was so infinitely small,” Dr Vlok said. “The only conclusion was this was stone age surgery.”

Dr Vlok ran to the office to tell her research colleagues what she had found. “I told them I thought it looked like a surgical amputation,” she said. “It wasn’t until then that they said they already knew the foot was missing.” Dr Vlok had just confirmed their suspicions. The foot was never placed in the grave to begin with.

An accident

While it is not entirely clear what led to the amputation, the individual also had a very well healed neck fracture and trauma to their collar bone that may have occurred during the same event, said Dr Vlok.

“An accident, such as a rock fall may have caused the injuries, and it was clearly recognised by the community that the foot had to be taken off for the child to survive,” she said.

“It is an extremely rugged environment with steep mountains dotted with caves containing some of the oldest paintings created by our species,” said Professor Aubert.

Archaeologists including excavation lead Dr Tim Maloney had to kayak into the valley and scale the enormous cliff to get into the cave, proving just how remarkable it was for someone with only one leg to have survived in such challenging terrain.

“This unique find challenges assumptions of humanity’s capabilities in the past and is set to significantly advance our understanding of human lifeways in tropical rainforests,” said Dr Dilkes-Hall.

Nature has evolved these structures to harness the sun’s energy through photosynthesis, but these sunlight receivers don’t belong to plants. They’re found in microbes known as cyanobacteria, the evolutionary descendants of the first organisms on Earth capable of taking sunlight, water and carbon dioxide and turning them into sugars and oxygen.

Published Aug. 31 in the journal Nature, the findings immediately shed new light on microbial photosynthesis — specifically, how light energy is captured and sent to where it’s needed to power the conversion of carbon dioxide into sugars. Going forward, the insights could also help researchers remediate harmful bacteria in the environment, develop artificial photosynthetic systems for renewable energy and enlist microbes in sustainable manufacturing that starts with the raw materials of carbon dioxide and sunlight.

“There’s a lot of interest in using cyanobacteria as solar-powered factories that capture sunlight and convert it into a kind of energy that can be used to make important products,” said Cheryl Kerfeld, Hannah Distinguished Professor of structural bioengineering in the College of Natural Science. “With a blueprint like the one we’ve provided in this study, you can start thinking about tuning and optimizing the light-harvesting component of photosynthesis.”

“Once you see how something works, you have a better idea of how you can modify it and manipulate it. That’s a big advantage,” said Markus Sutter, a senior research associate in the Kerfeld Lab, which operates at MSU and Berkeley Lab in California.

The cyanobacterial antenna structures, which are called phycobilisomes, are complex collections of pigments and proteins, which assemble into relatively massive complexes.

For decades, researchers have been working to visualize the different building blocks of phycobilisomes to try to understand how they’re put together. Phycobilisomes are fragile, necessitating this piecemeal approach. Historically, researchers have been unable to get the high-resolution images of intact antennae needed to understand how they capture and conduct light energy.

Now, thanks to an international team of experts and advances in a technique known as cryo-electron microscopy, the structure of a cyanobacterial light harvesting antenna is available with nearly atomic resolution. The team included researchers from MSU, Berkeley Lab, the University of California, Berkeley and the University of South Bohemia in the Czech Republic.

“We were fortunate to be a team made up of people with complementary expertise, people who worked well together,” said Kerfeld, who is also a member of the MSU-DOE Plant Research Laboratory, which is supported by the U.S. Department of Energy. “The group had the right chemistry.”

‘A long journey full of nice surprises’

“This work is a breakthrough in the field of photosynthesis,” said Paul Sauer, a postdoctoral researcher in Professor Eva Nogales’ cryogenic electron microscopy lab at Berkeley Lab and UC Berkeley.

“The complete light-harvesting structure of a cyanobacteria’s antenna has been missing until now,” Sauer said. “Our discovery helps us understand how evolution came up with ways to turn carbon dioxide and light into oxygen and sugar in bacteria, long before any plants existed on our planet.”

Along with Kerfeld, Sauer is a corresponding author of the new article. The team documented several notable results, including finding a new phycobilisome protein and observing two new ways that the phycobilisome orients its light-capturing rods that hadn’t been resolved before.

“It’s 12 pages of discoveries,” said María Agustina Domínguez-Martín of the Nature report. As a postdoctoral researcher in the Kerfeld Lab, Domínguez-Martín initiated the study at MSU and brought it to completion at the Berkeley Lab. She is currently at the University of Cordoba in Spain as part of the Marie Sk?owdoska-Curie Postdoctoral Fellowship. “It’s been a long journey full of nice surprises.”

One surprise, for example, came in how a relatively small protein can act as a surge protector for the massive antenna. Before this work, researchers knew the phycobilisome could corral molecules called orange carotenoid proteins, or OCPs, when the phycobilisome had absorbed too much sunlight. The OCPs release the excess energy as heat, protecting a cyanobacterium’s photosynthetic system from burning up.

Until now, there’s been debate about how many OCPs the phycobilisome could bind and where those binding sites were. The new research answers these fundamental questions and offers potentially practical insights.

This kind of surge-protecting system — which is called photoprotection and has analogs in the plant world — naturally tends to be wasteful. Cyanobacteria are slow to turn their photoprotection off after it has done its job. Now, with the complete picture of how the surge protector works, researchers can design ways to engineer “smart,” less wasteful photoprotection, Kerfeld said.

And, despite helping make the planet habitable for humans and countless other organisms that need oxygen to survive, cyanobacteria have a dark side. Cyanobacteria blooms in lakes, ponds and reservoirs can produce toxins that are deadly to native ecosystems as well as humans and their pets. Having a blueprint of how the bacteria not only collect the sun’s energy, but also protect themselves from too much of it could inspire new ideas to attack harmful blooms.

Beyond the new answers and potential applications this work offers, the researchers are also excited about the new questions it raises and the research it could inspire.

“If you think of this like Legos, you can keep building up, right? The proteins and pigments are like blocks making the phycobilisome, but then that’s part of the photosystem, which is in the cell membrane, which is part of the entire cell,” Sutter said. “We’re climbing up the ladder of scale in a way. We’ve found something new on our rung, but we can’t say we’ve got the system settled.”

“We’ve answered some questions, but we’ve opened the doors on others and, to me, that’s what makes it a breakthrough,” Domínguez-Martín said. “I’m excited to see how the field develops from here.”

This work was supported by the U.S. Department of Energy Office of Science, the National Institutes of Health, the Czech Science Foundation and the European Union’s Horizon 2020 research and innovation program.

A new study by neuroscientists at the University of California, Berkeley, simulated the effects of food insecurity in juvenile mice and found lasting changes later in life.

“We show that irregular access to food in the late juvenile and early adolescent period affects learning, decision-making and dopamine neurons in adulthood,” said Linda Wilbrecht, UC Berkeley professor of psychology and member of the Helen Wills Neuroscience Institute.

One key difference in behavior involved cognitive flexibility: the ability to generate new solutions when the world changes.

“Mice searching for rewards might be inflexible, sticking to only one strategy even when it no longer yields a reward, or they might be flexible and quickly try out new strategies. We found that the stability of the food supply mice had when they were young governed how flexible they were under different conditions when they were grown up,” she said.

Epidemiological studies have linked food insecurity in children and adolescents with weight gain in later life, as well as learning problems and lower scores in mathematics, reading and vocabulary. But these studies are confounded by other poverty-related issues, such as maternal depression and environmental stressors. The new study was designed to look at the developmental and behavioral impacts of food insecurity in a controlled setting not possible using human subjects.

The study has implications for humans. Policymakers acknowledge the importance of good nutrition in early childhood through high school, with federally-funded free or reduced-price breakfast and lunch programs available in schools throughout the United States. The federal Supplemental Nutrition Assistance Program (SNAP) also provides benefits to supplement the food budget of needy families. For families living paycheck to paycheck, these food programs have demonstrated effects — in particular, boosted performance in school and rates of graduation.

But there may be times when kids cannot access food programs, such as during summer vacation. Programs may also inadvertently create a feast and famine cycle when benefits are distributed with weeks between payments, potentially leaving impoverished families unable to afford food at the end of each payment cycle. According to a recent report from the U.S. Department of Agriculture, 6.2% of households with children — 2.3 million households total — were food insecure in 2021.

“I think that we have to understand that even transient food insecurity matters, the brain doesn’t just catch up later. Food insecurity can have long-term impacts on how someone’s brain functions,” Wilbrecht said. “The ability to learn and make decisions is something that’s developing during childhood and adolescence, and we are seeing how these critical skills are impacted by access to food. Access to food is something that we can address in this county. Feeding and benefits programs exist, and we can make them better by making access to benefits or food more reliable and consistent. Supporting brain development is a good reason to support food programs.”

The research, conducted with UC Berkeley faculty members Helen Bateup, Stephan Lammel and their lab colleagues, will appear in an upcoming print edition of the journal Current Biology. It was posted online July 20.

Flexibility under changing rules

Wilbrecht and her colleagues, including Robert Wood Johnson Foundation Health and Society Scholar Ezequiel Galarce, mimicked human food insecurity in mice by delivering food on an irregular schedule while still allowing enough food to maintain safe body weight. This food regimen began a week before puberty onset in mice, equivalent to late childhood in humans, and continued for 20 days through the equivalent of late teen ages in mice. Another group of mice was offered food whenever they wanted it.

They then tested cognition in adulthood using foraging tasks where mice searched a changing environment for rewards. For example, a behavior — in this case, learning which odor led to the Honey Nut Cheerios — might be successful for a short time, but not forever. A second odor now predicted where the reward was hidden.

The well-fed and food-insecure mice were tested as adults in both certain and uncertain settings, with noticeable differences in cognitive flexibility. Food-insecure mice were more flexible in uncertain situations than were well-fed mice, while well-fed mice were more flexible in more stable situations.

“You would have to test in the field to see how these different flexibility profiles affect survival,” she said. “The findings are nuanced, but hopeful, because we identify both gain and loss of function in learning and decision-making that are wrought by experience of scarcity.”

While the effect of food insecurity on cognition in male mice was robust, female mice showed no effect on cognition.

“This is one of the most robust behavioral effects we’ve ever seen when we’ve been modeling adversity,” Wilbrecht said.

Food insecurity had other decidedly negative effects in female mice, however. Those females who were food insecure when growing up tended to become overweight when given unrestricted food in adulthood, something mirrored in humans who’ve grown up with food insecurity. Male mice showed no such effect.

Doctoral student Wan Chen Lin and researchers in the Bateup and Lammel labs also looked at the brain’s reward network, which is governed by the neurotransmitter dopamine, and found changes there, as well, in male mice.

“We found that the neurons in the dopamine system, which is critical for learning, decision- making and reward-related behaviors, like addiction, were significantly altered in both their inputs and their outputs,” Wilbrecht said. “It suggests there are more broadscale changes in the learning and decision-making systems in the brain.”

For example, the researchers saw changes in the synapses of dopamine neurons that project to the nucleus accumbens and also found changes in dopamine release in the dorsal striatum. These dopamine neurons have been shown to play a role in learning and decision-making in numerous other studies.

The researchers are continuing their studies of food-insecure mice to determine if they are more susceptible as adults to addictive behaviors, which are associated with the dopamine network.

Other UC Berkeley authors of the paper are former postdoctoral fellow Polina Kosillo, former doctoral student Christine Liu and senior scientist Lung-Hao Tai. The work was supported the National Institutes of Health (R21 AA025172, U19NS113201) and the Robert Wood Johnson Foundation. Bateup is a Chan Zuckerberg Biohub investigator and a Weill Neurohub investigator.

Their findings are published in Cell Reports Physical Science.

While researchers have been exploring the possibility of converting carbon dioxide to ethylene for more than a decade, the UIC team’s approach is the first to achieve nearly 100% utilization of carbon dioxide to produce hydrocarbons. Their system uses electrolysis to transform captured carbon dioxide gas into high purity ethylene, with other carbon-based fuels and oxygen as byproducts.

The process can convert up to 6 metric tons of carbon dioxide into 1 metric ton of ethylene, recycling almost all carbon dioxide captured. Because the system runs on electricity, the use of renewable energy can make the process carbon negative.

According to Singh, his team’s approach surpasses the net-zero carbon goal of other carbon capture and conversion technologies by actually reducing the total carbon dioxide output from industry. “It’s a net negative,” he said. “For every 1 ton of ethylene produced, you’re taking 6 tons of CO₂ from point sources that otherwise would be released to the atmosphere.”

Previous attempts at converting carbon dioxide into ethylene have relied on reactors that produce ethylene within the source carbon dioxide emission stream. In these cases, as little as 10% of CO₂ emissions typically converts to ethylene. The ethylene must later be separated from the carbon dioxide in an energy-intensive process often involving fossil fuels.

In UIC’s approach, an electric current is passed through a cell, half of which is filled with captured carbon dioxide, the other half with a water-based solution. An electrified catalyst draws charged hydrogen atoms from the water molecules into the other half of the unit separated by a membrane, where they combine with charged carbon atoms from the carbon dioxide molecules to form ethylene.

Among manufactured chemicals worldwide, ethylene ranks third for carbon emissions after ammonia and cement. Ethylene is used not only to create plastic products for the packaging, agricultural and automotive industries, but also to produce chemicals used in antifreeze, medical sterilizers and vinyl siding for houses.

Ethylene is usually made in a process called steam cracking that requires enormous amounts of heat. Cracking generates about 1.5 metric tons of carbon emissions per ton of ethylene created. On average, manufacturers produce around 160 million tons of ethylene each year, which results in more than 260 million tons of carbon dioxide emissions worldwide.

In addition to ethylene, the UIC scientists were able to produce other carbon-rich products useful to industry with their electrolysis approach. They also achieved a very high solar energy conversion efficiency, converting 10% of energy from the solar panels directly to carbon product output. This is well above the state-of-the-art standard of 2%. For all the ethylene they produced, the solar energy conversion efficiency was around 4%, approximately the same rate as photosynthesis.

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To understand the history of the donkey’s domestication, teams at the Centre for Anthropobiology and Genomics of Toulouse (CNRS/ Université Toulouse 3 Paul Sabatier) and scientists¹ from 37 laboratories around the world worked together to build and analyse the most complete panel of genomes ever studied for this animal. It contains the genomes of 207 contemporary donkeys living on all continents, as well as those of 31 early donkeys and 15 wild equids. In an article published on 9 September in Science, the researchers reveal that the donkey was first domesticated in Africa in 5,000 B.C.E, around the time when the Sahara became the desert region we know today.

It was only 2,500 years later that donkeys left their place of origin in Africa and reached Europe and Asia, where this species developed lineages that, in some cases, still exist today.

By analysing archaeological remains, scientists also uncovered evidence of a previously unknown genetic lineage of donkeys that lived in the Levant 2,000 years ago. Furthermore, the influence of this lineage is thought to extend far beyond the region, and still today, fragments of its genetic heritage can be found throughout Europe. These discoveries call for new archaeological digs to find the initial source of domestication in Africa, as well as the sequencing of other early donkey genomes on both shores of the Mediterranean sea, to better understand the role of this animal in the history of trade between Europe and North Africa.

¹ The following French laboratories contributed to this study: Genoscope, François Jacob institute of biology (CNRS/CEA/Univ. D’Every-Val-d’Essonne), Archéorient laboratory (CNRS/Univ. Lumière Lyon 2), l’Institut de Paléontologie Humaine (CNRS/Museum d’histoire naturelle/Univ. De Perpignan via domitia), le laboratoire Archéologie des sociétés méditerranéennes (CNRS/Ministère de la culture/Univ. Paul Valery Montpellier). This work received support from the GeT-PlaGe and Genoscope platforms, members of the France Génomique infrastructure (BUCÉPHALE and MARENGO project), and numerous financial supports, in particular from ), the French National Research Agency (ANR-10-INBS 09), for funding from the CNRS (AnimalFarm IRP), and from the European Union, including from the European Research Council PEGASUS programme.

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The Hiroshima University research team published their findings in the journal Scientific Reports, a Nature publication, on August 17, 2022.

“While previous studies have demonstrated that chicken primordial germ cells possess a characteristic feature in sex determination, its mechanism remains unclear. To solve this challenge, we revealed gene expression profiles of male and female primordial germ cells derived from early chick embryos and then predicted the sex determination mechanism,” said Kennosuke Ichikawa, a postdoctoral researcher at the Genome Editing Innovation Center, Hiroshima University. This research is the first to predict the sex-determination mechanism by comparing the gene expression profiles of avian primordial germ cells at each embryonic stage, as well as by using a stimulation test.

Birds have unique mechanisms of sex determination, that are different from mammals. In mammals, which have an XX (female) — XY (male) sex chromosome system, their sex determination depends on the action of the Y-chromosome. In birds, which have a ZZ (male) — ZW (female) sex chromosome system, their sex depends on the action of the Z chromosome. Yet the molecular mechanism of sex determination remains unclear. The development of the reproductive glands called gonads, into either ovaries or testes, and the development of other sexual characteristics are at least partially cell autonomous. The researchers’ investigation of the sex-determination mechanism in birds provides them insight into the evolution of vertebrate sex-determining mechanisms.

To investigate the mechanisms underlying sex determination in avian germ cells, the team purified male and female primordial germ cells from the blood and the gonads, using fluorescence-activated cell sorting. With this process they achieved a purity of greater than 96 percent. They determined gene expression profiles of the primordial germ cells at each developmental stage for each sex using RNA-sequencing analysis, where next-generation sequencing is used to examine the quantity and sequences of RNA in a sample. Then, the researchers predicted the sex-determination mechanism of the primordial germ cells using bioinformatic analysis, where computer tools are used to understand biological data. To evaluate the prediction, they stimulated male primordial germ cells with retinoic acid in vitro, and examined the changes in gene expression.

Before settling in the gonads, the female circulating primordial germ cells obtained from blood displayed sex-biased expression. The primordial germ cells from the gonads also exhibited sex-biased expression, and the number of female-biased genes detected was higher than that of male-biased genes. The team realized that the female-biased genes in the primordial germ cells were enriched in some metabolic processes. To reveal the mechanisms underlying this process, the researchers performed stimulation tests.

The team used retinoic acid to stimulate the cultured primordial germ cells collected from male embryos. This stimulation resulted in the upregulation — the process where a cell’s components increase — of several female-biased genes. Overall, their results suggest that sex determination in avian primordial germ cells involves aspects of both cell-autonomous and somatic-cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.

“We successfully predicted female-specific potential processes and pathways in chicken primordial germ cells. We believe our data set can significantly contribute to elucidating the avian sex determination mechanism,” said Ichikawa.

Looking ahead to future work, the team plans to use the data set to identify the key molecules directly inducing the feminization of chicken primordial germ cells. “The ultimate goal of this study is to elucidate the sex determination mechanism and then establish a sex selection method in chickens using genome editing targeting the key molecules,” said Ichikawa.

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