How to manage without a Y chromosome

The mammalian Y chromosome is genetically degenerated and small compared with its counterpart, the X chromosome. The X and Y evolved approximately 180 Mya from an autosome pair. While the X has more or less maintained its original structure, the Y stopped recombining, resulting in its degeneration (1, 2); it lost 97% of its gene content and is less than one-third the size of the X. Only the maleness-determining Sry gene and a few other genes, mostly spermatogenesis genes, remain on the Y chromosome. Whether mammalian Y chromosomes, and in particular the human Y, have reached an end point of degradation or whether they will continue to shrink before entirely disappearing (3–6) is controversial. One major argument against this idea is that total loss of the Y would wipe out males. Thus, evolutionary forces would strongly act against such a loss. However, a few rodent species, which have lost the Y chromosome contradict this argument. In the Amami spiny rat, Tokudaia osimensis, it was long known that males have no Y (Fig. 1); however, the mechanism of how sex is determined in this species has remained a mystery ever since. In PNAS, Terao et al. (7) now provide a solution to the loss of Y by showing that a new sex-determining gene was created by a tiny change in the regulatory region upstream of a key pathway gene.

Fig. 1.

Amami spiny rat, T. osimensis (Image courtesy of Asato Kuroiwa).

The Amami spiny rat, T. osimensis, is a small rodent that lives on the Amami-Oshima Island in Japan. It is considered endangered by the deconstruction of its natural environment and an increase in its natural enemies (www.redlist.org/). This species attracted attention about 50 y ago, when it was found to have lost the typical mammalian Y chromosome (8, 9). Both, males and females have a N = 25 X0 chromosome constitution; however, no trace of the Y chromosome that is present in a related species, T. muenninki (10), was detected. A hypothesis to explain the aberrant karyotype of T. osimensis has been that the Sry gene was transferred from the original Y to another chromosome, but the translocated region was too small to be detected on chromosome spreads. This would be similar to the situation in certain human males who have an XX karyotype without a Y chromosome but the SRY gene is translocated to the X (11). However, despite extensive efforts, no trace of the Sry gene was found in the spiny rat (12, 13).

Accumulation of spermatogenesis genes on the mammalian Y is thought to have driven its evolutionary divergence and degeneration (14). Also, these male beneficial genes should guarantee the maintenance of the Y. So, how could the Amami spiny rat lose the Y chromosome that in addition to the Sry gene has harbored genes that are essential for sperm production and thus functional maleness? The euchromatic regions of the former Tokudaia Y chromosome including the genes for spermatogenesis are now found on the single X chromosome shared by males and females (15), suggesting that the transfer happened before the Y was lost. The situation that genes previously placed on the degenerating Y chromosome had moved to other chromosomes—like “rats leaving a sinking ship”—was also observed in two fish species. In the common guppy, which has an only partially degenerated Y, the sexually antagonistic color genes populate the male-specific regions of the Y (MSY). But in a related species, the swamp guppy, which has an extremely degenerated Y, they have left the MSY and are now autosomal or pseudoautosomal (16).

In their manuscript in PNAS, Terao et al. (7) provide now compelling evidence for the mechanism of how sex is determined in the Amami spiny rat. Their genetic approach first required the generation of a high-quality reference genome for this endangered species. It allowed, in a resequencing analysis of several individuals, for the detection of a duplicated fragment which is present only in males. Although, due to the endangered status of the species, only a few males and females could be tested to confirm the male specificity of the duplication. The analyses left only a marginal chance that another male-specific region had been missed. Intriguingly, the duplicated region lies in a transactivation chromatin domain upstream of the Sox9 gene, a well-known component of the sex determination regulatory network. The duplicated fragment contains a sequence, Enh14, that was earlier identified in the mouse as a putative enhancer of Sox9 (17). The authors then went on to functionally test the prediction that also in the Amami spiny rat this same region acts as an enhancer for Sox9 and that a male-specific duplication of Enh14 would give a higher expression of the maleness-promoting gene than in females. Consequently, this next step would have been to knock out the duplicated Enh14 in individuals that would normally develop as males and to add another copy of Enh14 to Sox9 in prospective females by CRISPR genome modification. The expected sex reversals would have provided evidence that the duplication of Enh9 is necessary and sufficient to determine male development. However, because the Amami spiny rat is a red-listed species, such experiments were not feasible. Thus, the authors turned to the laboratory mouse, considering the caveats and limitations of information from a distantly related species that has a Y chromosome with a fully functional Sry gene.

The hypothesis that Enh14 is a Sox9 enhancer was supported by reporter gene expression in transgenic animals and from replacing the mouse enhancer by the duplicated Enh14 sequence from T. osimensis in genome-edited mice with the female XX genotype. The authors found cells expressing Sox9 in XX animals at a stage, where in wild-type male embryos the testis is forming, when this gene is not naturally expressed in non–genome-edited females. Concurrently, the expression of the ovarian developmental gene Foxl2 was weaker compared to normal females. The fact that XX individuals with the added Enh14 from the Amami spiny rat did not fully sex-reverse and develop as males can be explained by species-specific differences in Sox9 expression levels necessary to proceed with testis development. Thus, the claim that Enh14 duplication is the cause for Sox9 becoming the master male-determining gene is very well supported, even when the final proof is not available. Full sex reversal would be expected, however, in an individual of wild Amami spiny rats with a spontaneous mutation in its Enh14 sequence. This would be a rare event, and given the small population of this endangered species, we might have to wait a long time until such an animal is found.

The Amami spiny rat in conclusion shows us that it is surprisingly easy to fashion a new sex-determining gene.

To complete the understanding of how the new SD locus works in initiating testis development, the molecular mechanism of Sox9 activation in the spiny rat, i.e., which transcription factor binds to Enh14 in the embryonal gonad and thus substitutes for the lost Sry, must be elucidated.

The fact that Sox9 became the new master regulator of the SD regulatory network fits well in the general picture with the genes that determine sex outside the eutherian mammals. With only very few exceptions, they all are derived from components of the well-conserved vertebrate downstream network of SD (18). In those species which have Sry, Sox9 holds a key position in the male-determining pathway. It is the direct target of Sry, and once expressed in the somatic cells of the gonad precursor, it activates several testis development genes (19, 20). In the Amami spiny rat, Sox9 has moved up the hierarchy in agreement with the bottom–up hypothesis for the emergence of new SD genes (21, 22) and has replaced Sry. We do not know exactly how this has happened, but it could be that Sry was lost first. Then, in a transition period, sex was determined by chance events, environmental influences, or the action of alleles of many genes in a polyfactorial genetic sex determination system (or a mixture of those). Finally, an enhancer duplication opened the route to a new stable monofactorial SD system. Alternatively, the mutation at the Sox9 locus occurred first and the mutation swept through the population. Thus, a stronger male-determining function of Sox9 rendered Sry as its up-regulator dispensable, resulting in the disappearance of Sry.

The emergence of Sox9 as the new SD factor in the Amami spiny rat makes its home, chromosome 3, function as a Y chromosome. This provides us with an “evolution in action” scenario for early stages of sex chromosome development. Inferring how old the duplication of the Enh14 region is will provide an estimate of the age of the nascent Y. The next step will be a closer look at the DNA sequences of the duplicated region and its surroundings on the homologous chromosomes. Theory predicts that recombination at and around Sox9 should be reduced and lead to divergence of the region between the proto-Y and X. This must be postulated in order to keep the identity of the Y chromosome. If the SD locus would switch its location by meiotic crossovers between the homologous pair, none of the two would qualify to be termed the Y. Enh14 is part of a 17-kb duplication and thus in a region that ab initio does not match the sequence of the partner chromosome. This could impair crossovers at the SD locus and initiate the process of molecular differentiation of a Y-linked region. Expansion of repeats, accumulation of transposons, and indels would be easy to recognize. Results will show whether already enough time has elapsed in the history of the Amami spiny rat Y chromosome for such changes to have occurred.

In conclusion, the Amami spiny rat shows us that it is surprisingly easy to fashion a new sex-determining gene. It should give hope to those that are distressed by a possible shriveling away of the human Y.