Sexual differentiation in birds is controlled genetically as in mammals, although the sex chromosomes are different.
Males have a ZZ sex chromosome constitution, while females are ZW.
Gene(s) on the sex chromosomes must initiate gonadal sex differentiation during embryonic life, inducing paired testes in ZZ individuals and unilateral ovaries in ZW individuals.
The traditional view of avian sexual differentiation aligns with that expounded for other vertebrates; upon sexual differentiation, the gonads secrete sex steroid hormones that masculinise or feminise the rest of the body.
However, recent studies on naturally occurring or experimentally induced avian sex reversal suggest a significant role for direct genetic factors, in addition to sex hormones, in regulating sexual differentiation of the soma in birds.
This review will provide an overview of sex determination in birds and both naturally and experimentally induced sex reversal, with emphasis on the key role of oestrogen. We then consider how recent studies on sex reversal and gynandromorphic birds (half male:half female) are shaping our understanding of sexual differentiation in avians and in vertebrates more broadly.
Current evidence shows that sexual differentiation in birds is a mix of direct genetic and hormonal mechanisms. Perturbation of either of these components may lead to sex reversal.
The highly unusual “semi-identical” Australian twins reported last week are the result of a rare event.
It’s thought the brother and sister (who have identical genes from their mother but not their father) developed from an egg fertilised by two different sperm at the same moment.
In humans, it’s the sperm that determines whether an embryo is pushed along a male or female development pathway. But in birds, it’s the other way around. Eggs are the deciding factor in bird sex.
There are other fascinating aspects of bird sex that are not shared with humans. Female birds seem to have some capacity to control the sex of their chicks. And occasionally a bird that is female on one side and male on the other is produced – as in recent reports of this cardinal in the United States.
X and Y, Z and W chromosomes
So what is it about bird chromosomes that makes bird sex so different from human sex?
In humans, cells in females have two copies of a large, gene-rich chromosome called X. Male cells have one X, and a tiny Y chromosome.
Birds also have sex chromosomes, but they act in completely the opposite way. Male birds have two copies of a large, gene-rich chromosome called Z, and females have a single Z and a W chromosome. The tiny W chromosome is all that is left of an original Z, which degenerated over time, much like the human Y.
When cells in the bird ovary undergo the special kind of division (called “meiosis”) that produces eggs with just one set of chromosomes, each egg cell receives either a Z or a W.
Fertilisation with a sperm (all of which bear a Z) produces ZZ male or ZW female chicks.
Birds can control the sex of their chicks
We would expect that, during meiosis, random separation of Z and W should result in half the chicks being male and half female, but birds are tricky. Somehow the female is able to manipulate whether the Z or W chromosome gets into an egg.
Most bird species produce more males than females on average. Some birds, such as kestrels, produce different sex ratios at different times of the year and others respond to environmental conditions or the female’s body condition. For example, when times are tough for zebra finches, more females are produced. Some birds, such as the kookaburra, contrive usually to hatch a male chick first, then a female one.
Why would a bird manipulate the sex of her chicks? We think she is optimising the likelihood of her offspring mating and rearing young (so ensuring the continuation of her genes into future generations).
It makes sense for females in poor condition to hatch more female chicks, because weak male chicks are unlikely to surmount the rigours of courtship and reproduction.
How does the female do it?
There is some evidence she can bias the sex ratio by controlling hormones, particularly progesterone.
How male and female birds develop
In humans, we know it’s a gene on the Y chromosome called SRY that kickstarts the development of a testis in the embryo.
The embryonic testis makes testosterone, and testosterone pushes the development of male characteristics like genitals, hair and voice.
But in birds a completely different gene (called DMRT1) on the Z but not the W seems to determine sex of an embryo.
In a ZZ embryo, the two copies of DMRT1 induce a ridge of cells (the gonad precursor) to develop into a testis, which produces testosterone; a male bird develops.
In a ZW female embryo, the single copy of DMRT1 permits the gonad to develop into an ovary, which makes estrogen and other related hormones; a female bird results.
This kind of sex determination is known as “gene dosage”.
It’s the difference in the number of sex genes that determines sex. Surprisingly, this mechanism is more common in vertebrates than the familiar mammalian system (in which the presence or absence of a Y chromosome bearing the SRY gene determines sex).
Unlike mammals, we never see birds with differences in Z and W chromosome number; there seems to be no bird equivalent to XO women with just a single X chromosome, and men with XXY chromosomes. It may be that such changes are lethal in birds.
Birds that are half-male, half-female
Very occasionally a bird is found with one side male, the other female. The recently sighted cardinal has red male plumage on the right, and beige (female) feathers on the left.
One famous chicken is male on the right and female on the left, with spectacular differences in plumage, comb and fatness.
The most likely origin of such rare mixed animals (called “chimaeras”) is from fusion of separate ZZ and ZW embryos, or from double fertilisation of an abnormal ZW egg.
But why is there such clear 50:50 physical demarcation in half-and-half birds? The protein produced by the sex determining gene DMRT1, as well as sex hormones, travels around the body in the blood so should affect both sides.
There must be another biological pathway, something else on sex chromosomes that fixes sex in the two sides of the body and interprets the same genetic and hormone signals differently.
What genes specify sex differences birds?
Birds may show spectacular sex differences in appearance (such as size, plumage, colour) and behaviour (such as singing).
Think of the peacock’s splendid tail, much admired by drab peahens.
You might think the Z chromosome would be a good place for exorbitant male colour genes, and that the W would be a handy place for egg genes.
But the W chromosome seems to have no specifically female genes.
Studies of the whole peacock genome show that the genes responsible for the spectacular tail feathers are scattered all over the genome.
So they are probably regulated by male and female hormones, and only indirectly the result of sex chromosomes.
A Schematic of gonadal sex differentiation in the chicken embryo showing the likely involvement of key sexual differentiation genes, reproduced from Schmid et al.  with permission from S. Karger AG, Basel.
Genes implicated in avian sexual differentiation outcomes are shown with male-expressed genes in blue, female-expressed genes in pink, and circle sizes representative of relative expression levels.
Functional validation has confirmed the involvement of DMRT1, HEMGN, and Aromatase in the chicken sexual differentiation pathways.
DMRT1 is expressed at higher abundance in the male and is central to the initiation of the testis development pathway, including AMH, HEMGN, Prostaglandin D2 (PDG2) and SOX9.
DMRT1 is present at lower levels in the female with MHM (male hyper-methylated) potentially antagonising the effects of DMRT1 in female (ZW) gonads.
R-SPO1 and WNT4 lead to stabilized β-catenin and active Wnt signalling, which are important for female gonad development in mammals.
This pathway is expected to be analogous in chicken sex determination but has yet to be proven experimentally.
GATA4 is present in both sexes and may be activating DMRT1.
The undifferentiated gonad consists of a medulla (blue), which in the male proliferates and differentiates to form the seminiferous cords, and the outer epithelial layer (pink), which in the female proliferates to form the cortex of the ovary. Germ cells are represented by black dots/circles.
BDMRT1 mRNA expression in embryonic chicken gonads, showing the gonads (G), mesonephric kidney (Ms), and Müllerian duct (Md).
Sexual dimorphisms in the adult chicken, reproduced from Mayer et al.  with permission from Elsevier.
Photographs of a male (A) and female (B) adult chickens demonstrating sexual dimorphisms (head ornaments, spurs, breast musculature, and plumage). Distinct differences can be seen in tail feathering (C) between the male (left) and the female (right).
In chicken and in other birds, the sexes also show sexually dimorphic feathering, with males often displaying more colourful or elaborate feather patterning.
Sexually dimorphic feathering is generally attributed to sex steroid hormones.
In the Galliformes (chickens, turkeys, etc.) female feathering is induced by oestrogens, and male plumage is the default pattern in the absence of significant oestrogen (fig. 2).
For example, female peafowls develop the long and colourful tail plumage of males after ovariectomy and hence loss of oestrogen [reviewed in Owens and Short, 1995].
In a line of bantam chicken with a genetic mutation leading to constitutively active aromatase enzyme activity and hence ectopic oestrogen synthesis, both sexes show female-type (‘henny’) feathering [George and Wilson, 1980; Matsumine et al., 1991]. In seasonally breeding passerines (perching birds) testosterone is important for male plumage [Lindsay et al., 2011].
Sex reversal of the plumage can therefore occur when sex steroid levels are disturbed, through gonadal disease, for example. Sex steroid hormones also play well established roles in sexual differentiation of the avian brain, although there is evidence for direct genetic control of the song centre, in at least some birds [Arnold, 1996; Agate et al., 2003].
Photograph of a gynandromorphic chimera chicken, left = male, right = female, with characteristic differences in plumage, wattle, spur, and breast musculature (A). Schematic illustration of the cellular make-up of this gynandromorphic chicken with ZZ and ZW cells distributed predominantly on the respective ‘male’ and ‘female’ sides (B). Reproduced from Clinton et al.  with permission from Springer.
CASI is also supported by our recent studies showing that male (ZZ) chicken embryos, chronically overexpressing aromatase and elevated serum oestrogen levels, nevertheless develop male-type sexual dimorphisms as adults [Lambeth et al., 2016a].
Meanwhile, chronic overexpression of AMH at embryo stages in chicken can block gonadal sex differentiation and sex steroid synthesis, yielding adult birds that have a mixture of male and female traits [Lambeth et al., 2016b].
In these birds, body weight conformed to genetic sex, for example, despite a blockage in gonadal sex differentiation and sex steroid hormone synthesis [Lambeth et al., 2016b].
These most recent studies support the proposal that sex in birds may be at least partially cell autonomous [Clinton et al., 2012]. Hence, DMRT1 dosage may underlie the direction of gonadal sex differentiation in embryos, while differentiation of other sexually dimorphic structures may involve other Z-linked genes as DMRT1 is not expressed outside the gonads.
However, how can this cell autonomy data be reconciled with the fact that a blocking oestrogen action in chicken embryo leads to robust masculinisation of birds?
It has been noted that such hormonally sex-reversed birds are not totally masculine, with comb, wattle, and other features that are not fully male, or that sex reversal is often temporary, thus pointing to a role for direct genetic factors [Clinton et al., 2012]. Our own recent data on experimentally sex-reversed gonads (by blocking AMH or chronically expressing aromatase genetically) support this view. Figure 4summarises the role of direct genetic versus hormonal signalling in avian sex determination and sexual differentiation.
Model for sex determination and sexual differentiation in avians. Avian sex is determined at fertilization by the inheritance of sex chromosomes (male = ZZ; female = ZW), leading to sexually dimorphic gene expression and subsequent sexual differentiation.