TRPV4 associates environmental temperature and sex determination in the American alligator

Highly diverse modes of sex determination have been observed among vertebrates. In most cases, sex is determined genetically (genotypic sex determination; GSD), which has been characterized by the presence of a key sex-determining gene. In contrast, in many reptiles including crocodilians, select chelonians and squamates, differential sexual outcomes have been identified under varying incubation temperatures during a critical temperature sensitive period (TSP) in the developing embryo (temperature-dependent sex determination; TSD)1,2,3. As an alternative to the GSD system, in which the heritable traits dictate subsequent gonadal differentiation, all species studied to date that exhibit TSD appear to lack sexually heteromorphic chromosomes4,5, and sexual development is assumed to be initiated mostly by environmental cues, independent of the individual’s genetic background. However, genetic researches in reptiles are particularly hindered by lack of available genetic manipulation techniques, compared to other vertebrate species, and much of the sex determination mechanism in this pivotal vertebrate clade is yet to be understood. American alligators, Alligator mississippiensis, display TSD, and the developing embryo detects a thermal stimulus that apparently directs the sexual fate of the bipotential gonad; this critical range of incubation temperatures is shared among all other crocodilians studied to date6,7,8. Eggs incubated at a constant temperature of 33 °C yields 100% male offspring, whereas incubation temperatures below 30 °C lead to female biased offspring sex ratio during TSP (Ferguson stages 21 to 24)9. Downstream sexual differentiation processes in alligators follows a fairly similar pattern often shared among vertebrates. Male producing temperatures (MPT) facilitates the medullary supporting cells to enlarge and proliferate, which in turn initiates their arrangement into distinct seminiferous cords by hatching (stage 27), and eventual testicular morphogenesis10. Dynamic anti-Mullerian hormone (AMH) upregulation and ensuing SRY-box 9 (SOX9) upregulation are observed during the sex determination phase11. At female producing temperatures (FPT), however, primordial germ cell proliferation occurs in the thickened cortex while the medulla undergoes extensive fragmentation10,12. By developmental stage 27, irreversible female commitment occurs with the eventual onset of estradiol-17β synthesis by aromatase (CYP19A1), thus completing ovarian morphogenesis12. However, it remains elusive how incubation temperature during the TSP triggers TSD and the subsequent differentiation cascade. Transformation of the initial environmental temperature signal to a biochemical signaling in TSD is not understood in any species, including species with temperature-dependent sex reversal13. Isolated gonads from a TSD reptile, Trachemys scripta, have been demonstrated to be directly receptive to thermal stimuli, suggesting that the initial reception of environmental cues can be triggered through an endogenous sensory mechanism14. This mechanism hypothesized to be shared among TSD reptiles, including in the alligator as well. Various mechanisms of thermal detection have been reported in the past, these include protein conformational changes, structural shifts in nucleic acid, and membrane property changes. In eukaryotes especially, several multi-pass transmembrane ion channels have been the focus for thermal sensing15,16, most prominent of which is the transient receptor potential (TRP) cation channel superfamily. The channels in this superfamily have been the most extensively characterized among vertebrates. The TRP channels in this superfamily mostly function as environmental sensors primarily through Ca2+ signaling, uniquely activated by various internal or external cues including osmolarity, pH, pressure, and temperature17. At present, 10 thermosensitive TRP channels have been identified in humans and rodents: TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM3, TRPM4, TRPM5, TRPM8, and TRPA1. Many of the thermosensitive TRP channels possess well-defined ranges of activation as well as wide diversification in physiological and functional properties18, and are an ideal candidate as potential thermosensor within TSD mechanism, particularly the TRPV4 since the mammalian TRPV4 channel is known to be activated by moderate heat (27–35 °C)19,20. Here, we report the involvement of A. mississippiensis TRPV4 (AmTRPV4) ortholog in temperature-dependent sex determination. Electrophysiological analysis reveals AmTRPV4 functions as a molecular thermal sensor at a threshold near the range observed in TSD for this species, and pharmacological manipulation of channel activity affects the sexual differentiation processes in spite of incubation temperature during development. This is the first demonstration of a link between a well-described thermo-sensory mechanism, TRP channel, and regulation of TSD, shedding light on the elusive TSD molecular mechanism.