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Is it a boy or a girl? Zebrafish have a hard time deciding, say studies

Illustration : Purabi Deshpande / Research Matters

We humans are blessed with a straightforward way of determining whether our babies are boys or girls. We rely on genes – if the fertilized egg carries two ‘X’ chromosomes, we say it is a girl, and if it contains a ‘X’ and a ‘Y’ chromosome, we say it is a boy. There is no meddling from other factors like environment, temperature or others.

However, in the case of organisms like zebrafish -- a widely used ‘model organism’ for many experiments that has zebra-like stripes, the process of determining whether an embryo develops into a male or a female is not quite obvious. It seems that sex in zebrafish is determined by a number of factors, genetic, environmental or both, working together.
There are plenty of examples of other animals such as crocodiles and turtles, where temperature determines the sex of an embryo. Warmer temperatures tend to produce males while cooler temperatures favour females. In addition, there is another way of sex determination where multiple genes in the embryo orchestrate together to decide the sex. In other cases, factors like population density as in mermithid nematodes, social cues or even pH are known to be causal in sex determination. But how exactly do these factors play out in determining the sex of an unborn embryo?

In order to understand this process in zebrafish, Dr. Nagabhushana and Prof. Rakesh K Mishra from the Centre for Cellular and Molecular Biology, Hyderabad, have reviewed several studies related to sex determination and found that factors like nutrition, oxygen deficiency, temperature, density and exposure to hormones are all involved in deciding the sex. For example, high temperatures caused more males to develop from the clutch. Biochemical factors like enzymes also play a role in sex determination, say the researchers. When an enzyme cytochrome p450 aromatase was chemically inhibited, oocytes (immature eggs) in the embryo committed suicide. Oocyte cell deaths invariably lead to the development of the embryo into a male offspring.

Apart from these factors, several genes have been identified as influencing sex determination in zebrafish. This could be an example of sex determination where multiple genes play a role. The researchers have identified two genes dmrt1 and cyp21a2 on chromosomes 5 and 6 that are responsible in sex determination. They predict that the chromosome 4 may be still evolving as a sex chromosome in zebrafish, akin to the X and Y chromosome in humans.

From studies in the past, two genes have been known to be always involved in sex determination in all vertebrates – dmrt1 and sox9. Evolution has not allowed many changes in these genes over time in many animals – perhaps due to the important function they perform in determining sex. These two genes are differently expressed in the gonads of zebrafish, indicating that they may be involved in sex determination in zebra fish too.

In any embryo, a certain type of specialized cells called Primordial Germ Cells (PGCs), are reserved to develop into either sperms or ovum. Studies have shown that loss of PGCs result in oocyte apoptosis – a strange phenomenon where an inbuilt genetic program pushes a cell (oocytes here), to commit suicide. These embryos, which are now devoid of oocytes, develop into males. This observation highlights the important, and yet not clearly understood role, played by PGCs in sex determination. The current understanding points to an unidentified mysterious signal from the oocytes which acts on a bi-potential gonad capable of developing either into a testis or into an ovary, based on the type of the signal.

The gene cyp19a1 is seen in the gonads of all fishes. When the fish sexually mature, the expression of the gene becomes restricted to developing ovaries. This gene in zebrafish is either hyper or hypo ‘methylated’, based on the temperature. When a gene is hyper methylated (more methyl groups are added to the DNA of the gene), it is repressed and cannot be expressed. With this mechanism, temperature can alter the amount of methylation on the gene that could lead to sex determination.

In summary the players in the game of sex determination in zebra fish are many. Multiple genes, primordial germs cells temperature – all of these influence the sex ratio. But which of this is a key player? No one knows yet! All we know so far is that sex, at least in zebrafish, is a plastic and threshold character.