by Emily Goering
figures by Andrea Luviano

Did you know that sea lampreys are more likely to grow into males if they aren’t fed enough food? Or that one type of shrimp produces male offspring in the long days of summer, but females in shorter fall days? Sexual determination systems are the methods by which organisms produce offspring with differing biological sexes. Although all vertebrates evolved from the same common ancestor, animals use vastly different sexual determination systems. The evolution of this methodological diversity is remarkable considering that sexual reproduction is required for species’ survival. In stark contrast, development of body parts is relatively similar among all vertebrates. So, how different are the sexual determination systems? And how do they work?   

To understand key concepts involved in sexual determination, we will look into a widely studied species of fish: Danio rerio or, as it is known in the pet store, zebrafish. Zebrafish have two seemingly contradictory sexual determination systems. One sexual determination system relies on genetic material and the other involves input from the environment. In this article, we will dive into both systems to understand the diversity in sex determination in vertebrates.

Genetic versus environmental sex determination  

The most well-known sexual determination system is based on the genes of an animal, meaning that genetic material determines the sex of an offspring after fertilization. Genetic material within each cell is organized in X-shaped structures, called chromosomes, which contain many individual genes. These genes encode for traits of an organism, from eye-color to height to sexual hormones. Humans, like many mammals and some insects, use sex chromosomes known as X and Y to determine sex. Females get two X chromosomes (XX) and males get an X and a smaller Y chromosome (XY). The X and Y chromosomes contain different sets of genes that instruct the developing organism to become female or male, respectively. Since a distinct chromosome is the basis of sex in this system, the XX/XY sexual determination system is called a chromosomal genetic sexual determination system.   

The second main chromosomal sexual determination system is the ZZ/ZW system. This system is used by all birds, some reptiles, some insects, and some fish, including wild zebrafish. The ZZ/ZW system is a reversed version of the chromosomal XX/XY system. In the ZZ/ZW chromosomal system, males are ZZ, which means their DNA contains two Z chromosomes, and females are ZW, which means they contain a W chromosome instead of a second Z.  

From here, the story gets weird: not all animals have discrete sex chromosomes. In some creatures, including laboratory zebrafish and many reptiles, females and males have the same number and types of chromosomes. How, then, do these animals sexually develop? Some, notably reptiles, rely exclusively on environmental cues. Take the alligator, for whom sex is determined by the temperature of the eggs in the nest. Eggs incubated below 86 degrees Fahrenheit become females and eggs incubated at higher temperatures become males (Figure 1). In practice, this translates to warm, sheltered eggs in the middle of the nest becoming male, while more exposed eggs in the periphery of the nest becoming female. This usually results in a male to female ratio of 1:5, meaning 5 females are hatched for every male. Some scientists believe that this has helped the species survive threats such as habitat loss and hunting, since alligator young have a high mortality rate and males can reproduce with several females each breeding season while females can only breed once. Temperature is the most common environmental cue involved in sexual determination, but other factors, including length of daylight, location, dissolved oxygen content, and food availability have all been shown to influence sexual differentiation in other species.

Figure 1. Alligators determine their sex by the temperature of the egg. When alligator eggs are kept at higher temperatures, the offspring are male. When alligator eggs are kept at lower temperatures, however, the offspring are female. This leads to roughly 80% female offspring!

Polygenic sex determination

As we already described, zebrafish in the wild determine their sex through the ZZ/ZW chromosomal genetic system. Why are laboratory zebrafish different? In the 1970s, tropical fish enthusiast George Streisinger bought zebrafish at a pet store to use in the lab. As a scientist, he wanted to find a simple vertebrate model organism to study the development of the nervous system. Zebrafish fit the bill since they develop outside of the organism in eggs that are completely transparent and can be manipulated at all stages. The lack of ZZ/ZW sex chromosomes in laboratory zebrafish was not noticed until years later, after laboratory zebrafish were established as a powerful model organism on which to conduct a host of scientific studies.  

But without the sex chromosomes of their wild peers, how do laboratory zebrafish sexually differentiate? In short: it’s complicated. In the last ten years, significant progress has been made looking into the exact method of sexual determination of laboratory zebrafish. Three different labs looked at the genomes of laboratory zebrafish in detail by performing an experiment called a genome-wide association study (GWAS). In these GWAS, genomes from male and female zebrafish were collected and compared to each other to look at genetic differences between the sexes. Each lab found evidence for multiple distinct genes on different chromosomes that could be contributing to sex determination, but the genes identified by the three groups did not overlap. This work has led to the currently favored hypothesis that laboratory zebrafish sexual differentiation is a polygenic sexual determination system (Figure 2).  

Figure 2. Wild zebrafish (left) have a different sex determination system than laboratory zebrafish (right).

Polygenic sexual determination is a process by which multiple genes on distinct chromosomes interact to cause sexual maturation. In a polygenic sexual determination system, animals of both sexes contain the same chromosomes. In contrast, in chromosomal sexual determination, discrete sex chromosomes in either sex (XY or ZW) contain a “master switch” for sexual determination. Polygenic sexual differentiation doesn’t have one gene that makes the animal male or female. In laboratory zebrafish, sexual differentiation seems to depend on genetic variation in around ten genes as well as environmental conditions. Conditions that elicit stress, including high temperature, low food, low dissolved oxygen, and overcrowding bias the fish towards males, although we do not yet know the mechanism by which environmental conditions interact with genes to influence sexual differentiation.

Don’t judge a zebrafish by its stripes

The small, half-inch fish loved both by pet owners and scientists has many mysteries within its pretty fins. In the wild, zebrafish largely produce expected ratios of males to females due to the presence of a ZW chromosomal system of sexual determination. In the lab, however, there can be tanks of almost all males or females because sexual determination comes from environmental factors (in the case of the author, oftentimes lack of food!), as well as from the influence of several genes located on multiple, non-sex chromosomes. Despite not knowing the exact genes that influence sexual determination in laboratory zebrafish, they remain a powerful representation of the diversity of sexual determination systems among all animals. 

Emily Goering is a fifth-year graduate student in the Immunology Program at Harvard University, and is a one-time fish hobbyist.

Andrea Luviano is a Harvard Ph.D. student in Health Policy in the Decision Science track. 

Cover image by keenyam on pixabay.

For More Information:

  • Check out this review to learn more about vertebrate sex determination systems.
  • To learn more about XY sex determination, read this previous SITN article.

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