What allows TEMPERATURE (of the environment) to influence the developmental pathways that determine sex?

It is well known that the sex (i.e. whether it’s male or female gender) of certain reptiles can be determined by ambient temperatures of the environment, although the precise molecular mechanism has remained elusive. What is it –– that allows TEMPERATURE to influence the developmental pathways that determine sex? The experiments to identify a master sex-determining gene in species (those having the ability of genetic sex determination) are well established: One identifies genes on the sex chromosomes, demonstrates which of these are differentially expressed in male and female embryos early in development, and then manipulates their expression so as to demonstrate reversal of sex.

This approach does not work, however, when identifying mechanisms of temperature-dependent sex determination (TSD). Differences in temperature can exert its effect on any of the many autosomal (i.e. those chromosomes that are not sex chromosomes) genes involved in sexual differentiation –– even those peripherally involved, provided their altered expression is capable of reversing sex. Thus, although TSD was discovered in reptiles in 1966, we have not understood the mechanisms of TSD. However, authors [see attached full article] ound that transcription of the chromatin-modifier gene Kdm6b (lysine-specific demethylase-6B) responds to temperature in the red-eared slider turtle, conferring temperature sensitivity to a sex-determining gene, Dmrt1 (doublesex- and mab-3–related transcription factor-1).

Dmrt1 expression is high at male-producing temperature (MPT) and low at female-producing temperature (FPT). As such, Dmrt1 became a strong candidate for the male sex-determining gene in this TSD species of turtle, consistent with the master sex-determining role of other “DM-domain–containing” genes in certain fish, amphibians, and birds. These “DM-domain” genes initiate and maintain the male sexual trajectory, while suppressing genes important for female development –– during critical stages of embryogenesis.

Authors showed [see attached] that experimental down-regulation of Kdm6b at 26°C (normally the MPT) shifts embryos from a male to a female developmental trajectory –– because the protein KDM6B plays a central role in epigenetic regulation of gene expression. Suppressing Kdm6b expression decreases demethylation of its target, trimethylated lysine 27 on histone 3 (H3K27), a histone modification that would otherwise repress Dmrt1 promoter activity. Thus, high amounts of KDM6B at MPT activate Dmrt1 gene expression and determine male sex, whereas decrreased amounts of KDM6B repress Dmrt1 expression, resulting in female.

These GEITP pages have often described “epigenetic effects” as “non-DNA-sequence effects that contribute to a trait” (phenotype) –– and which include DNA-methylation, RNA-interference, histone modifications, and chromatin remodeling. This [attached] paper shows convincing evidence of a role in TSD for highly conserved epigenetic modifiers such as KDM6B. This exciting study therefore establishes causality, and a direct genetic link between epigenetic mechanisms (histone modifications) and temperature-dependent sex determination (i.e. a phenotype) in a turtle species.

Science 11 May 2o18; 360: 645–648 [main article] & pp 601–602 [editorial]

COMMENT:Very interesting. It would be nice to see if KDM6B plays the same role in certain fish species in which temperature also determines sex…

COMMENT: Excellent comment/question, Linda, i.e. does the ‘same set of master genes and downstream cascade of events’ –– which is seen in this turtle –– also operate in some, or all, of the other poikilotherms (animals that cannot regulate their body temperature –– except by behavioral means such as basking-in-the-sun or burrowing-into-the-ground)?

And, an even “broader, more universal” question: does this same set of master genes and downstream cascade of events also occur in homeotherms (animals that are able to maintain their body temperature at a constant level –– usually above that of the environment –– by their metabolic activity)? And, if not, WHY NOT (i.e. what modifications have evolved in homeotherms –– during the past several hundred million years –– to alter this set of master genes found in this turtle)?

These articles [attached] say that temperature-dependent determination of sex “occurs in certain reptiles, amphibians, fish and birds.” Birds are not poikilotherms, but they do lay eggs that need to be incubated for a period of time before hatching; sex is determined during embryogenesis and fetogenesis, and therefore these processes would take place during the incubation time of the egg. And what about monotremes (egg-laying mammals such as the opossum, duck-billed platypus)? These papers open up far more questions than it answers.

COMMENT: In matters of sex determination, I think this [attached] is a very interesting paper that you might find relevant to the current ongoing discussion.

COMMENT: A question was entertained earlier: Are mammals different from the turtle with regard to sex determination? The attached article describes sex reversal (in the mouse) following deletion of a single distal enhancer of the Sox9 gene. In mammals, the Sry (sex-determining region-Y) gene encodes the protein SRY that is transiently expressed and initiates testis, and subsequent male development, by triggering cells of the supporting cell lineage to differentiate into Sertoli cells (male testis cells), rather than granulosa cells typical of ovaries. Sox9, (SRY-box 9) the main target of SRY, is critical for differentiation of Sertoli cells, subsequently functioning along with other transcription factors –– notably encoded by the genes Sox8 (SRY-box 8) and then Dmrt1 (doublesex- and mab-3–related transcription factor-1, i.e. THE SAME GENE AS WAS DESCRIBED BELOW FOR THE TURTLE..!!).

Both gain-of-function and loss-of-function studies in mouse and human demonstrate that Sox9 plays a key role in testis determination. Notably, in humans, males that are heterozygous (one gene copy of the gene pair) for null mutations in SOX9 develop campomelic dysplasia, a severe syndrome in which 70% of XY patients show female development. Authors [see attached] discovered –– via in vivo high-throughput chromatin-accessibility techniques, transgenic assays, and genome-editing –– several novel sex-determining regulatory elements in the 2-million-base-pair (2 Mb; 2 megabase) “gene desert” upstream of Sox9. Although others are redundant, Enh13, a 557–base-pair element located 565,000 base-pairs (565 kilobases; kb; kbp) 5′, is essential to initiate mouse testis development; deletion of this element gives XY females with Sox9 transcript levels equivalent to XX gonads (organs that produce ova, not sperm). These results are consistent with the time-sensitive activity of SRY, during development, and indicate a strict order of enhancer usage. Clinically, ENH13 is conserved and embedded within a 32.5-kb region whose deletion in patients is associated with XY sex reversal (i.e. changing from male to female) –– suggesting this enhancer is critical not only in mice, but in humans.

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