Mouse sperm showing TLR8 expression (red) and the nucleus (blue) 
Umehara et al., Hiroshima University 

The mammalian X chromosome has many genes that the Y does not—a feature that has special implications for sperm, and also for scientists.

The mouse X chromosome carries two protein receptors that when activated by a chemical make X-bearing sperm slower and easy to separate from Y-bearing sperm, a team of Japanese researchers has found. By sorting the gametes using this method and allowing them to fertilize oocytes in vitro, the scientists could selectively generate mouse litters with majority-female or majority-male pups, they report today (August 13) in PLOS Biology.

“It’s definitely an excellent piece of work,” remarks James Knight, a reproductive biologist at Virginia Tech who wasn’t involved in the study. “The whole methodology that they’re describing, given the accuracy of separating X- and Y-bearing sperm, has tremendous applicability to several...

Reproductive biologist Masayuki Shimada of Hiroshima University and his colleagues initially began the research to better understand the genetic differences between X- and Y-bearing sperm and whether they could explain differences in the mobility of X- and Y-bearing sperm, which previous researchers have observed under specific in vitro conditions.

According to the team’s RNA sequencing data, mouse sperm carry 492 genes on the X chromosome, but only 15 genes on the Y. Among those expressed on the X, the team became interested in two that code for cell receptors, the Toll-like receptors (TLR) 7 and 8. The TLR family of proteins plays important roles in recognizing pathogens such as bacteria and viruses. In addition, Shimada’s group has previously found that when stimulated, certain TLR receptors—TLR2 and TLR4, encoded on chromosomes unrelated to sex—interfere with sperm movement.

I don’t think sperm sorting’s going to take over for embryo selection as the way to do it.

—Louise King, Harvard Medical School

By staining mouse testes with antibodies that target TLR7 and TLR8, the researchers confirmed that they were expressed on X- but not Y-bearing sperm. This characteristic was interesting to Shimada, because the products of most X-linked genes that are expressed in sperm are shared with Y-bearing sperm because the gametes are connected through an intracellular bridge as they develop. TLR7 and TLR8 appear to be expressed after the bridge is lost, so they might reveal functional differences between X and Y sperm, Shimada explains.

To investigate this possibility, the team incubated the sperm with resiquimod, an anti-viral drug that activates both receptors. Normally, all sperm normally swim upward when in a tube. But with the treatment, there were significantly fewer X-bearing sperm in the upper portion of the tube, suggesting that these were simply slower. “The linear motility speed [of X-bearing sperm] was decreased to less than half,” Shimada writes to The Scientist.

The team then investigated why resiquimod had this effect. They found that ATP levels drastically fell in treated X-bearing sperm. Further experiments revealed that the drug’s activation of TLR8 suppresses mitochondrial activity in the midpiece of the sperm and its stimulation of TLR7 suppresses enzymes that regulate the energy-producing process of glycolysis in the tail. This leaves X-bearing sperm with less energy, the researchers note.

“This is the first time that the Toll-like receptors 7 and 8 have been identified for this function,” Knight says. “They’re usually just thought of like most of the Toll-like receptors, that is in mediating various immune responses. So this is certainly a novel finding,” he says.

To see if the resiquimod could be used to separate sperm by sex, the team collected the upper and lower layers of sperm from the test tube after treatment, allowed the gametes to fertilize mouse oocytes in vitro, and implanted the resulting embryos into mice. Using sperm from the upper layer, they obtained 77 blastocyst embryos, 83 percent of which resulted in male pups. Sperm from the lower layer produced 83 embryos, 81 percent of which were female.

Sex-sorting in livestock and in the clinic

Shimada says he thinks his method of sperm sexing could be cheaper and faster than methods currently used to sex mammalian sperm. One dominant technique is the Beltsville Sperm Sexing Technology, developed in the 1980s, in which semen is treated with a fluorescent, DNA-binding dye. As the X chromosome is larger than the Y, it absorbs more dye and fluoresces more strongly under UV light than Y-bearing sperm, allowing X-bearing sperm to be isolated through flow cytometry.

Flow cytometry–based technology requires “a pretty expensive piece of equipment, and takes some expertise for operation,” says Knight. He sees several cost and efficiency advantages to Shimada’s method, noting that it has comparable accuracies in sorting sperm as conventional technologies.

Sperm sorting has several applications in the livestock sector, particularly for the dairy cattle industry where it is used to reduce the number of male calves produced. “The males that might be born are going to basically end up as veal calves,” Knight says, which is problematic because they are less valuable for dairy production, and their slaughtering at a young age raises ethical questions.

Shimada and Knight suggest that the research could be relevant for choosing sex prior to IVF in humans. However, Louise King, an OB-GYN and medical bioethicist at Harvard Medical School, cautions that sperm-sorting technology has already been tried in human IVF, for parents at risk of having children with sex-linked disorders as well as for those who wish to choose the sex of their children. “It wasn’t particularly commercially successful because the success rate wasn’t good enough,” she says. One 2014 study, for instance, that tested the efficacy of flow cytometric sorting in influencing a child’s sex found that after sperm sorting, 94 percent were female when selected for that sex, and 85 percent were male when that sex was chosen.

That success rate often isn’t appealing to families who go to the trouble of choosing the sex of their child, given that they have another option that is 100 percent effective, King says. The IVF clinics in the US that do offer sex selection do so by creating a number of embryos and picking the embryo that has the desired sex. “I don’t think sperm sorting’s going to take over for embryo selection as the way to do it,” she says. 

Whether it’s ethical to make that choice to begin with is another question, King says. The American Society for Reproductive Medicine considers sex selection for non-medical purposes “ethically controversial,” and encourages clinics to develop their own policies, while the practice is not permitted in most European countries.

T. Umehara et al., “Activation of Toll-like receptor 7/8 encoded by the X chromosome alters sperm motility and provides a novel simple technology for sexing sperm,” PLOS Biology, 17(8):e3000398, 2019. 

Katarina Zimmer is a New York–based freelance journalist. Find her on Twitter @katarinazimmer.

Correction (August 15): We incorrectly referred to the Beltsville technique as the Beltsfield technique. The Scientist regrets the error.

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