TRPV4 associates environmental temperature and sex determination in the American alligator

Abstract

Temperature-dependent sex determination (TSD), commonly found among reptiles, is a sex determination mode in which the incubation temperature during a critical temperature sensitive period (TSP) determines sexual fate of the individual rather than the individual's genotypic background. In the American alligator (Alligator mississippiensis), eggs incubated during the TSP at 33 °C (male producing temperature: MPT) yields male offspring, whereas incubation temperatures below 30 °C (female producing temperature: FPT) lead to female offspring. However, many of the details of the underlying molecular mechanism remains elusive, and the molecular link between environmental temperature and sex determination pathway is yet to be elucidated. Here we show the alligator TRPV4 ortholog (AmTRPV4) to be activated at temperatures proximate to the TSD-related temperature in alligators, and using pharmacological exposure, we show that AmTRPV4 channel activity affects gene expression patterns associated with male differentiation. This is the first experimental demonstration of a link between a well-described thermo-sensory mechanism, TRPV4 channel, and its potential role in regulation of TSD in vertebrates, shedding unique new light on the elusive TSD molecular mechanism.

Figure 1. Developmental expression profile of American alligator TRP channels in gonad during sexual development.

(A) The mRNA levels of various thermosensitive TRP channels were assessed in gonads at the onset of TSP (stage 21) incubated under MPT and FPT conditions. Gene expressions of 5 AmTRP ion channels (AmTRPV2, AmTRPV4, AmTRPA1, AmTRPM3, AmTRPM8) were observed in varying expression levels. (B) Quantitative RT-PCR analysis was performed for AmTRPV4 at various key sexual developmental stages including bipotential (stage 19; n = 13), sex determination (stage 21; n = 14, 14), sex differentiation (stage 24; n = 14, 15), and pre-hatching (stage 27; n = 14, 15) stages at both FPT and MPT temperature conditions respectively; ± SEM. Temperature sensitive period is indicated in gray.

Figure 2. AmTRPV4 is a thermosensitive TRP channel that activates near alligator TSD temperature range.

(A) A representative trace of the current (upper) activated in response to corresponding changes in bath solution temperature (lower) in the Xenopus oocytes expressing AmTRPV4 using a two-electrode voltage-clamp method. (B) A representative temperature-response profile for AmTRPV4 activation by heat. (C) A representative Arrhenius plot for heat-induced AmTRPV4 activation. The average threshold for activation was 37.30 ± 0.54 °C; n = 17. (D) A representative trace of the AmTRPV4 current in the oocyte activated by a TRPV4 agonist (GSK1016790A indicated in a black bar); n = 4. (E) A representative trace of AmTRPV4 current in the oocyte activated by administration of a specific TRPV4 agonist (GSK1016790A indicated by a black bar) and subsequently inhibited by TRPV4 specific antagonist (RN1734 indicated by a gray bar); n = 4. (F) A representative current trace of AmTRPV4 expressing oocyte activated by heat stimulus and subsequently inhibited by a specific TRPV4 antagonist (RN1734 indicated by a gray bar); n = 4. (G) A representative averaged changes of [Ca²⁺]_i in AmTRPV4-expressing HEK293 cells (n = 75) under both heat and chemical stimulation. [Ca²⁺]_i changes in AmTRPV4-expressing cells (indicated as an average trace ± SE; left y-axis) were observed along with temperatures (indicated by open circle trace; right y-axis). Applications of a TRPV4 agonist (GSK1016790A) and ionomycin are shown with a black and gray bars, respectively. (H) Representative [Ca²⁺]_i and temperature changes in mock transfected HEK293 cells (n = 36).

Figure 3. Pharmaceutical activation and inhibition of AmTRPV4 during sex determination alters male differentiation.

Stage 19 embryos were administered AmTRPV4 antagonist RN1734 (0.5, 0.005 μg/g egg) or agonist GSK1016790A (0.5, 0.005 μg/g egg) in ovo and incubated under MPT and FPT conditions, respectively, until stage 27. (A–E) The mRNA levels of major sex differentiation genes, (A) AMH, (B) SOX9, and (C) CYP19A1 in the gonad at stage 27 were examined using quantitative RT-PCR analysis for each treatment: MPT control (n = 12), 0.005 RN (n = 13), 0.5 RN (n = 12), FPT control (n = 13), 0.005 GSK (n = 15). Asterisks indicate statistically significant change in expression; ± SEM; *P ≤ 0.05; **P ≤ 0.01. Markedly lower mRNA expression was observed for AMH and SOX9, both involved with male differentiation cascade. (D) In situ hybridization was performed on gonadal cross sections using AMH antisense riboprobe. White bar indicates 100 μm. (E) Immunohistochemistry for SOX9 and Hoechst was performed on gonad cross sections. White bar indicates 100 μm.

Figure 4. AmTRPV4 inhibition causes rise in Müllerian duct development in MPT.

(A) Histological analysis of sexual development was performed. Cross-sections of HE stained gonad and Müllerian duct at stage 27 for FPT control (n = 11), 0.005 GSK (0.005 µg/g egg; n = 15), MPT control (n = 13), 0.005 RN (0.005 µg/g egg; n = 11), and 0.5 RN (0.5 µg/g egg; n = 11). Instances of ovarian development were observed in RN1734-treated groups. (B) Graph showing number of individuals with ovarian, testicular, or ambiguous morphology in each treatment groups. (C) Graph showing number of individuals with prominent Müllerian duct in each treatment groups. White bar indicates 100 μm.