"If that goes wrong, then you end up with the wrong number of chromosomes in the eggs or sperm," said Neil Hunter, a professor in the Department of Microbiology and Molecular Genetics at the University of California, Davis. "This can result in infertility, miscarriage or the birth of children with genetic diseases."

In a paper published Sept. 24 in the journal Nature, Hunter's team reports a major new discovery about a process that helps safeguard against these mistakes. He has pieced together the choreography of proteins that connect matching chromosome pairs -- ensuring that they are sorted correctly as egg and sperm cells develop and divide.

Hunter's discoveries required methods to watch the molecular events of chromosome recombination unfold with unprecedented detail. This involved genetic engineering in budding yeast -- a model organism that has been used for decades to discover how fundamental cellular processes work.

"The chromosome structures that we studied have changed very little across evolution," Hunter said. "Every protein that we looked at in yeast has a direct counterpart in humans." His findings could improve our understanding of fertility problems and how they are diagnosed and treated in humans.

Forming chromosome crossovers for strong connections

Humans have 46 chromosomes in each of our cells, made up of 23 pairs of matching, "homologous" chromosomes, with one of each pair inherited from each parent. Early in the process of making sperm or eggs, those chromosome pairs line up, and the parental chromosomes break and rejoin to each other. These chromosome exchanges, called "crossovers," serve two important functions.

First, they help ensure that each chromosome that is passed on to the offspring contains a unique mixture of genes from both parents. Crossovers also keep the chromosomes connected in matching pairs. These connections guide the distribution of chromosomes when cells divide to produce eggs and sperm. Maintaining crossover connections is especially crucial in females, Hunter said.

As chromosomes pair up in developing egg or sperm cells, matching DNA strands are exchanged and twined together over a short distance to form a structure called a "double Holliday junction." DNA strands of this structure are then cut to join the chromosomes forming a crossover.

In males, developing immature sperm cells then immediately divide and distribute chromosomes to the sperm. In contrast, egg cells developing in the fetal ovary arrest their development after crossovers have formed. The immature egg cells can remain in suspended animation for decades after birth, until they are activated to undergo ovulation.

Only then does the process lurch back into motion: The egg cell finally divides, and the chromosome pairs that were connected by crossovers are finally separated to deliver a single set of chromosomes to the mature egg. "Maintaining the crossover connections over many years is a major challenge for immature egg cells," Hunter said.

If chromosome pairs aren't connected by at least one crossover, they can lose contact with each other, like two people separated in a jostling crowd. This causes them to segregate incorrectly when the cell finally divides, producing egg cells with extra or missing chromosomes. This can cause infertility, miscarriage or genetic conditions such as Down syndrome, in which a child is born with an extra copy of chromosome 21, leading to cognitive impairment, heart defects, hearing loss and other problems.

From yeast to humans

Hunter has spent years trying to understand how crossovers form and how this process can fail and cause reproductive problems. By studying this process in yeast, researchers can directly visualize molecular events of double-Holliday junction resolution in synchronized populations of cells.

Researchers have identified dozens of proteins that bind and process these junctions. Hunter and then-postdoctoral fellow Shangming Tang (now an assistant professor of biochemistry and molecular genetics at the University of Virginia) used a technique called "real-time genetics" to investigate the function of those proteins. With this method, they made cells degrade one or more specific proteins within the junction-associated structures. They could then analyze the DNA from these cells, to see whether the junctions were resolved and if they formed crossovers. In this way, they built up a picture in which a network of proteins function together to ensure that crossovers are formed.

"This strategy allowed us to answer a question that previously wasn't possible," Hunter said.

They identified key proteins such as cohesin that prevent an enzyme called the STR complex (or Bloom complex in humans) from inappropriately dismantling the junctions before they can form crossovers.

"They protect the double Holliday junction," Hunter said. "That is a key discovery."

This years-long research project in yeast is broadly relevant for human reproduction because the process has changed very little during evolution. Failure to protect double-Holliday junctions may be linked to fertility problems in humans.

In addition to Tang, the postdoc, seven undergraduates in the UC Davis College of Biological Sciences contributed to this work, including Jennifer Koo, Mohammad Pourhosseinzadeh, Emerald Nguyen, Natalie Liu, Christopher Ma, Hanyu Lu and Monica Lee.

Additional authors on the paper include Sara Hariri, Regina Bohn and John E. McCarthy, all members of the Hunter lab.

Hunter's research is funded by the National Institutes of Health and the Howard Hughes Medical Institute. His work has also received funding from the UC Davis Comprehensive Cancer Center, the American Cancer Society, the Concern Foundation for Cancer Research, and the Damon Runyon Cancer Foundation.

Hunter's research on crossover and homologous recombination uses advanced scientific facilities at the university's Proteomics Core Facility, MCB Light Microscopy Imaging Facility, Genome Center, Mouse Biology Program, and the Comprehensive Cancer Center.

The study analyzed data from almost 500 patients hospitalized with COVID-19 across six countries. Patients who inhaled heparin were half as likely to require ventilation and had a significantly lower risk of dying compared with those receiving standard care.

Heparin, a drug traditionally injected to treat blood clots, was tested in this study in an inhaled form, targeting the lungs directly. As well as acting as an anticoagulant, heparin has anti-inflammatory and pan-antiviral properties. Earlier research results showed breathing and oxygen levels improved in COVID-19 patients after they inhaled a course of heparin.

The researchers believe the drug could also be useful in fighting other serious respiratory infections such as pneumonia.

Professor Clive Page, Emeritus Professor of Pharmacology at King's who co-led the international study with ANU's Professor van Haren, said: "Inhaled heparin is anti-viral, anti-inflammatory and anti-coagulant. There's no other drug that has that unique combination. We know it's only a matter of time until the next pandemic, and there are still COVID-19 patients who get very sick. This is a great weapon to have up our sleeve."

While the findings highlight the potential of inhaled heparin, further development is required before the treatment can be routinely adopted. The researchers believe the drug could also be useful in fighting other serious respiratory infections such as pneumonia, which can be caused by a range of viruses and bacteria.

According to Professor van Haren, the drug would also be helpful for those with a compromised immune system, such as cancer patients, when they experience a respiratory infection.

Professor Frank van Haren, lead author, ANU and Director of the Intensive Care Unit at the St George Hospital in Sydney, said: "It doesn't matter what kind of respiratory infection the patient is dealing with, the drug -- when inhaled -- will stop it from infecting the patient and from damaging the lungs. We're aiming to conduct another trial in Europe to confirm its effectiveness in fighting other common respiratory infections such as influenza and RSV. And because it's inexpensive, it's much more accessible for those from low-income countries"

The researchers are now also developing an improved formulation of heparin, specifically designed to be given by inhalation.

The research is published in eClinicalMedicine and simultaneously presented of at the European Respiratory Society (ERS) International Congress in Amsterdam on Sunday, September 28, the leading annual gathering for respiratory research.