Transcriptomic analysis of preovipositional embryonic arrest in a nonsquamate reptile (Chelonia mydas)

Abstract

After gastrulation, oviductal hypoxia maintains turtle embryos in an arrested state prior to oviposition. Subsequent exposure to atmospheric oxygen upon oviposition initiates recommencement of embryonic development. Arrest can be artificially extended for several days after oviposition by incubation of the egg under hypoxic conditions, with development recommencing in an apparently normal fashion after subsequent exposure to normoxia. To examine the transcriptomic events associated with embryonic arrest in green sea turtles (Chelonia mydas), RNA-sequencing analysis was performed on embryos from freshly laid eggs and eggs incubated in either normoxia (oxygen tension ~159 mmHg) or hypoxia (<8 mmHg) for 36 h after oviposition (n = 5 per group). The patterns of gene expression differed markedly among the three experimental groups. Normal embryonic development in normoxia was associated with upregulation of genes involved in DNA replication, the cell cycle, and mitosis, but these genes were commonly downregulated after incubation in hypoxia. Many target genes of hypoxia inducible factors, including the gene encoding insulin-like growth factor binding protein 1 (igfbp1), were downregulated by normoxic incubation but upregulated by incubation in hypoxia. Notably, some of the transcriptomic effects of hypoxia in green turtle embryos resembled those reported to be associated with hypoxia-induced embryonic arrest in diverse taxa, including the nematode Caenorhabditis elegans and zebrafish (Danio rerio). Hypoxia-induced preovipositional embryonic arrest appears to be a unique adaptation of turtles. However, our findings accord with the proposition that the mechanisms underlying hypoxia-induced embryonic arrest per se are highly conserved across diverse taxa.

Increasing hypoxia progressively slows early embryonic development in an oviparous reptile, the green turtle, Chelonia mydas

“…Rather, they appear to be progressively modulated by oxygen availability, so that embryonic development is directly dependent on the ambient oxygen concentration. This conclusion agrees with Gárriz et al's [27] report of differences in transcriptional activity between fresh-laid embryos, embryos incubated in normoxia and embryos in hypoxia-extended arrest. They found that embryos in all three groups were transcriptionally distinct.…”

“…Oxygen-sensing pathways dependent on hypoxia-inducible factors have been identified as potentially important mediators of hypoxia-induced embryonic arrest in zebra fish [ 53 ] and green sea turtles [ 27 ]. Our current findings indicate that these mechanisms do not operate as a simple ‘ switch’, either permitting or blocking embryonic development.…”

“…This conclusion agrees with Gárriz et al . 's [ 27 ] report of differences in transcriptional activity between fresh-laid embryos, embryos incubated in normoxia and embryos in hypoxia-extended arrest. They found that embryos in all three groups were transcriptionally distinct.…”

“…If the latter could be demonstrated, it would suggest that embryonic development in turtles is a more complex physiological process than previously understood, dependent upon not only temperature and moisture but also variably affected by ambient oxygen concentration. Transcriptional activity of green turtle embryos incubated in hypoxia is substantially different to embryos immediately post-oviposition and when incubated in ambient oxygen [ 27 ], although the functional significance of the affected genes remains unknown. Knowledge of whether this is a graduated or an ‘all-or-nothing’ response to oxygen concentration would provide helpful information to better understand the molecular mechanisms controlling arrest and the resumption of development.…”

Effects of postovipositional hypoxia and hyperoxia on leatherback turtle reproductive success and hatchling performance

“…Studies of other turtle species have shown that embryos pause development at the gastrula stage and the arrest is maintained by hypoxia (~1% O 2 in the oviducts) (Kennett et al, 1993; Rafferty et al, 2013; Rings et al, 2015; Williamson et al, 2017). Embryos regulate gene expression in response to oxygen availability (Gárriz et al, 2022) and increases in oxygen availability when the eggs are oviposited break the embryonic arrest, even with small increments in %O 2 (Adams et al, 2022; Rafferty et al, 2013). Conversely, artificially maintaining eggs in extreme hypoxia after oviposition maintains the arrest and protects embryos from movement‐induced mortality (Williamson, Evans, Robinson, et al, 2017).…”