The extraordinary case of ferocious female moles with testicular genitalia

Rafael Jiménez Medina learned to hunt the elusive Iberian moles of the fields of southern Spain, when he was a young PhD student in genetics at the University of Granada, in the eighties. It was a local hunter of Iberian moles (Talpa occidentalis) who taught him how to capture these solitary, aggressive and territorial animals, which excavate underground galleries and labyrinths in the meadows of the Iberian Peninsula, especially in those with soft soils and rich in earthworms, their favorite food. Although their work can be beneficial to the soil, by aerating or mixing it, their presence and constant movement in cultivated lands make them the target of the wrath of farmers, who hire hunters to get rid of them.

But Jiménez Medina's interest in capturing these underground mammals was different. His PhD project involved visualizing and analyzing their chromosomes, a task that involved collecting and preparing samples derived from the testes of males. During this process, however, he faced a curious problem: when he dissected some moles he recognized as males and analyzed them in the laboratory, they turned out to be genetically female; that is, their sex chromosomes were XX (female) and not XY (male).

The confusion was generated, we now know, by the unusual composition of the reproductive sex organs of mole-females. In contrast to most female mammals, which only have ovaries, those of the Iberian mole have, in addition to those same functional ovaries, a testicular tissue that resembles in anatomy the testes of a male, although functionally differs from them: it produces testosterone, but no sperm. These organs, composed of an ovarian part and a testicular part, are known as ovotestes.

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In addition, mole females have a clitoris covered with foreskin and with an elongated appearance that resembles a penis; And it is through this foreskin that they urinate. Another unique anatomical aspect is that during the juvenile stage of these females the vaginal opening is kept closed. The peculiar anatomical characteristics of mole females impacted Jiménez Medina so much that, after his doctorate, he decided to put aside his chromosomal analysis work to immerse himself fully in the study of the enigmatic sexual development of these animals. His work, and that of other colleagues over the past few decades, would turn this tiny species of mammal (which fits in the palm of a hand) into a model for the study of female masculinization and the biological mechanisms underlying it.

Hyenas and fish

These and other females with typically masculine traits (such as the spotted hyena) "have become the paradigms of female masculinization," Jiménez Medina and colleagues write in a paper in the 2023 Annual Review of Animal Biosciences, in which they discuss the fruits and challenges of studying the anatomy and behavior of these creatures. These exceptional cases, moreover, invite us "to reconsider the concepts of femininity and masculinity" in the field of reproductive biology, the authors add in the article.

The presence of both male and female sex characteristics in a single organism has been documented in many species of invertebrates and fish, but in mammals this phenomenon is mainly restricted to isolated cases; that is, reports of individuals, but not generalities of a species. This anatomical amalgam has even been described in humans for centuries: surgical interventions on patients with ambiguous genitalia are already documented in ancient Greece.

Formerly called hermaphrodism (in reference to Hermaphroditus, a character in Greek mythology with voluptuous breasts and male external genitalia), the term now used to describe individuals with these characteristics is intersex, explains molecular geneticist Francisca Martínez Real of the Max Planck Institute for Molecular Genetics in Berlin.

The particularity of moles, however, is that this intersex does not occur in a few individuals: it is the norm in females. And it is not something exclusive to Iberian moles: it has been identified in eight species of moles, including the Iberian, and in a species of desmanes, mole-like mammals that live near aquatic environments, such as rivers and lakes. These male characteristics provide these females with tools to survive in extreme environments.

The Power of Testosterone

A key consequence of the presence of ovotestes in mole females is their high production of testosterone, the main sex hormone of males (essential for the development of growth and male characteristics), which directly influences their anatomy and behavior. But testosterone production is seasonal, varying as the size of testicular tissue in the ovotestes changes throughout the year, explains Martínez Real, who began studying the control of sexual development in mammals in Jiménez Medina's laboratory earlier this century.

In late autumn, winter and early spring, life becomes easier for these moles, as the ground fills with earthworms. At this time, the testicular part of the ovotestes is smaller and, therefore, the females are less aggressive. Between September and May (and particularly intensely between November and March) is when females allow males to enter their territory for copulation, explains Jiménez Medina. Outside the mating period, interactions between the sexes are mainly reduced to territorial confrontations.

All female Iberian moles have ovotestes: gonads containing ovarian and testicular tissue. They do not produce sperm, but the testicular part of these ovotestes secretes testosterone.

In the summer months, things get complicated. In this time of dry, hard terrain, and scarce food, it is more difficult to dig tunnels and galleries, and that is when more strength is needed. The testicular part of the ovotestes enlarges and blood testosterone levels in females reach on average 2.62 nanograms per milliliter in adults and 5.5 in juveniles. These figures are in the same order of magnitude as males at the same time: 4.9 in adults and 3.9 in juveniles. For comparison, an adult woman has testosterone levels of about 0.5, equivalent to one-tenth of those of an adult man on average (about 5 nanograms per milliliter).

How do these seasonal variations in testosterone production occur in these females? It's an unanswered question, which Jimenez Medina and his colleagues are currently investigating. What is clear is that having these levels of testosterone allows mole females to reach the degree of bodybuilding necessary to do their job: build their galleries, make their hunting area to catch worms and defend their territory. "It's impressive the musculature they have. When you dissect a mole (whether female or male) and remove the skin, what is visible is the body of a bodybuilder," says Jiménez Medina.

"Testosterone is linked to aggression in all kinds of mammals and other vertebrates," says Jenny Graves, an evolutionary geneticist at La Trobe University in Australia who has studied sex determination in mammals. Although in most mammal species the male is the largest, the strongest and the most aggressive, says Graves, it is very interesting to see examples such as moles, where natural selection has caused females to protect their territory.

Rearrangements in the genome

In mammals sex is determined genetically, so the intersex of mole females must have a genetic basis. In search of clues, Martínez Real and Jiménez Medina were part of a team that analyzed the genome of the Iberian mole and that of one of its relatives, the star-nosed mole (Condylura cristata), whose females also develop ovotestes. The team found that the genomes of both species have alterations that affect the activity of key genes involved in testosterone production and testes development, modifying when and how much those genes are turned on and off in the females' sex organs, they reported in a 2020 paper in Science.

One of these alterations affects the activity of a gene called CYP17A1, which encodes an enzyme that controls the production of male sex hormones, including testosterone. Iberian moles and starnose moles have three copies of this gene, while most mammals have only one. But the two extra copies of the gene don't work; the important change is in an extra, modified copy of a nearby DNA sequence that regulates the gene's activity. This modification in the regulatory elements increases the activity of CYP17A1 both in the testes of males and in the testicular tissue of the ovotestes of females, which results in increased testosterone production in both sexes.

In addition, in mammals there is another gene, CYP19A1, which is commonly found active only in the ovaries and converts testosterone into female hormones. This gene could reverse the effects of increased testosterone production due to changes in the CYP17A1 gene. However, in moles, CYP19A1 is only active in the ovarian part of the ovotestes, but not in the testicular, so it does not interfere with testosterone production.

A third alteration affects the activity of another gene, known as FGF9, which contributes to the development of the testes in mammals. Both the Iberian mole and the star-nosed mole have an upside-down piece of DNA in a key region for the regulation of this gene. As a result, Martínez Real and his colleagues found that the gene is active in the reproductive sex organs of females during embryonic development, when in other mammals it is only active in males. Its activity delays ovarian development and favors the development of testicular tissue of what will later be ovotestes, explains Martínez Real.

Development geneticist Rafael Jimenez Medina holds a newly captured mole in a forest in Spain.

When these genetic alterations were introduced into mice, different experiments proved that they play a role in the development of females with masculinized traits. Mice (males and females) that were introduced to the extra copy of the sequence that regulates CYP17A1 activity produced higher amounts of testosterone: females generated twice as much as females whose genomes were not altered, and males three times more than males without alteration. On the other hand, female mice that were made a genetic alteration to increase the activity of the FGF9 gene, similar to the increased activity of that gene in moles, developed testes.

Different ways of being female

Do the presence of ovotestes, large muscles and aggressive behavior make these mole females fewer females? For Jimenez Medina, the answer is no. "From a biological point of view, the most feminine thing is to become a mother," she says, since the goal of female and male sexual forms is to carry out sexual reproduction. And female moles fulfill this role without any problem: they have about four offspring a year, which they care for and nurse for about a month. "They build a new nest, specially enabled to give birth and nurse their young," says Jiménez Medina, and they do so using layers of dry leaves that probably serve to guarantee impermeability and thermal insulation.

In addition, mole females are very efficient sucklers. Their young increase their birth weight from around 3 grams to 45 grams after one month of lactation, perhaps reflecting the high quality of breast milk. Females with an average weight of 53 grams can produce on average litters of 144 grams at weaning, suggesting a higher reproductive effort than other mammalian females with which they have been compared; specifically, with various species of shrews.

Thus, mole females make it clear that there is no single way to be female in biological terms and that, in exceptional cases, the development of masculinized traits could be related to adaptation to an unfriendly environment, such as living underground.

Kay Holekamp, a behavioral ecologist at Michigan State University who has studied female spotted hyena for decades, considers both them and mole females to be a strange mix of characteristics. Saying these females are masculinized "is only a fraction of the story," he adds. There are aspects of their nervous system and behavioral repertoires that are not masculinized at all and that "are very feminine." "The interesting thing about these animals is that they are ultimately chimeras," he concludes.

This article originally appeared in Knowable en español, a non-profit publication dedicated to making scientific knowledge available to everyone.

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