Expression and cloning of AmTRPV4

To examine the presence of thermosensitive TRP channels in the gonad during the sex determination period in the American alligator, expression for orthologs of the mammalian thermosensitive TRP channels was screened at the onset of TSP and sex determination (Ferguson stage 21) using RT-PCR. Gene expression was limited to 5 TRP ion channels: TRPV2, TRPV4, TRPM3 and faintly from TRPA1 and TRPM8 (Fig. 1A). Of the 5 confirmed TRP channels expressed in the gonad, TRPV4 expression was of particular interest due to ideal predicted activation temperature. Thus, AmTRPV4 was deemed noteworthy for further investigation. Quantitative RT-PCR analysis for TRPV4 was conducted during various developmental stages; at the bipotential stage (stage 19), onset of TSP and sex determination (stage 21), end of TSP and onset of sexual differentiation (stage 24) and latent stages of sex differentiation (stage 27) at both MPT and FPT. The time series revealed a sexually dimorphic expression patterns in the gonad, with suppression at the FPT (Fig. 1B). AmTRPV4 expression was also examined in the chorioallantoic membrane, as well as in the epidermal tissues and its expression was confirmed, although a sexually dimorphic pattern was not observed (Fig. S1A,B).

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. Full size image

AmTRPV4 clone (857 aa) was amplified, slightly shorter in comparison to other reported reptilian TRPV4 orthologs and phylogenetic analysis showed the AmTRPV4 to be more closely related to birds and other reptilian TRPV4 orthologs, when compared to mammalian TRPV4s (Fig. S2). An evolutionary comparison of various TRPV4 orthologs seemingly points toward overall sequence conservation among the higher vertebrates (Fig. S3). Overall similarity in amino acid sequences were observed between alligator TRPV4 and mouse (Mus musculus; 86%), human (Homo sapiens; 85%), chicken (Gallus gallus; 87%), lizard (Takydromus tachydromoides; 88%) and snake (Elaphe quadrivirgata; 88%)21,22,23. Aside from the proline rich domain (PRD), major domains in the TRPV4 channel structure shared high amino acid sequence identity among higher vertebrate orthologs and hence, we expected a similar temperature-induced channel activation pattern in AmTRPV4 as observed from mammalian TRPV4 channel activation.

AmTRPV4 is a potential candidate in TSD initiation

At present, very little of the TRPV4 channel thermosensitivity properties have been well characterized in non-mammalian vertebrate species and hence, characterization of AmTRPV4 was essential before further investigation. Following AmTRPV4 isolation, ion channel functional properties and activation threshold against thermal stimulus were assessed using the Xenopus laevis oocyte expression system24. Administration of mammalian TRPV4-specific agonist elicited a clear response in cRNA-injected oocytes and not in negative control (water injected oocyte), indicating successful expression in the oocyte. Furthermore, thermal sensitivity was also confirmed and heat stimulation successfully elicited clear inward current (Fig. 2A,B). Xenopus oocytes subjected to water (mock) injection showed no heat-induced current, suggesting specific heat activity by AmTRPV4. An Arrhenius plot analysis indicated an average temperature threshold as 37.30 ± 0.54 °C (n = 17), revealing a warm temperature threshold (Fig. 2C).

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). Full size image

Chemical responsiveness to mammalian TRPV4-specific agonists and antagonist administration was examined. AmTRPV4-expressing oocytes displayed current flow with perfusion of GSK1016790A, a potent TRPV4 specific agonist25,26, at a dose of 50 nM (Fig. 2D). In addition, RN1734, a known TRPV4-specific antagonist26, was able to partially and reversibly inhibit AmTRPV4 activated by GSK1016790A (Fig. 2E). Furthermore, RN1734 also was able to inhibit temperature-induced currents in AmTRPV4-expressing oocytes in a reversible manner (Fig. 2F).

AmTRPV4 was next expressed in HEK293 cells and Ca2+ imaging experiments were performed to examine whether activation of AmTRPV4 is capable of increasing intracellular Ca2+ concentration ([Ca2+] i ). [Ca2+] i increased during a heat stimulation above room temperature, as well as after administration of GSK1016790A (Fig. 2G). In contrast, mock-transfected HEK293 cells showed only faint responses to both stimuli (Fig. 2H), suggesting that the Ca2+ influx was specifically mediated by the AmTRPV4 channel activation. These results confirmed the sensitivity of AmTRPV4 to warm temperatures and its responses to chemicals (an agonist and antagonist) were found to be similar to that described for mammalian TRPV4.

Inhibition of AmTRPV4 during sex determination alters male determination and differentiation-related gene expression

Marked expression of AmTRPV4 in the gonad during TSP, as well as heat-dependent channel activation at a temperature proximate to temperature range involved with alligator TSD provide strong evidence for a possible role of AmTRPV4 in TSD. In order to assess the role of AmTRPV4 during TSD, the channel was evaluated via pharmacological manipulation. Alligator eggs were given a single administration of the chemical agonist GSK1016790A or antagonist RN1734 in ovo at stage 19 (bipotential gonad stage), using two different concentrations (0.005 μg/g/egg and 0.5 μg/g/egg). These doses should be considered as nominal, as we lack information concerning the chemicals’ permeation of the eggshell and half-life in vivo. Also, by dosing AmTRPV4 agonist and antagonist to the whole embryo via in ovo exposure, we were able to observe potential full body effects following activation or inhibition of AmTRPV4, replicating an elevated or low thermal effect. The eggs were incubated under MPT (33.5 °C) or FPT (30.0 °C) conditions and subsequent effects were examined at stage 27 (stage prior to hatching), focusing on various sex differentiation related genes and specifically on AMH, SOX9 and CYP19A1 gonadal gene expression as sexual markers (Fig. 3A,B,C, Fig. S4A,B).

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. Full size image

Quantitative RT-PCR analysis revealed that two genes (AMH and SOX9) related to testicular differentiation were significantly down-regulated by administration of the AmTRPV4 antagonist RN1734 at MPT conditions in a dose-dependent manner (Fig. 3A,B). Recorded body weights of the embryos were similar among all experimental groups and the differential expressions were not due to delayed embryonic development10 (Fig. S5). Similarly, expression levels assessed by in situ hybridization on the differentiated gonads also reflected the results from quantitative RT-PCR and the lowered gene expression level of AMH was confirmed (Fig. 3B). Administration of an AmTRPV4 agonist, GSK1016790A, at FPT did not result in a significant change in gene expression levels based on quantitative RT-PCR, possibly due to sexually dimorphic AmTRPV4 expression (reduced expression at female generating temperatures) as reported above (Figs. 1B and 3A,C,D). Interestingly, upon closer inspection, the immunohistochemistry revealed an ectopic upregulation of SOX9 in agonist-treated FPT gonads, indicating that AmTRPV4 activation initiated expression of one of the genes required for male sex differentiation (Fig. 3E). It should be noted, however, that administration of GSK1016790A at the higher dosage induced high mortality; necropsy data indicated premature embryo death that occurred shortly after drug administration. Hence, only low dosage results were available for analysis of the TRPV4 agonist exposure group. In contrast to the genes primarily associated with testicular differentiation, expression levels for CYP19A1 was unaffected regardless of altered AmTRPV4 channel activity with differing thermal environments (Fig. 3C). As a result, due to a lack of significantly altered CYP19A1 expression, feminization of RN1734-treated embryos incubated at MPT was very limited: that is, we were not able to induce a feminized gene expression pattern in the gonad following inhibition of AmTRPV4 suggesting that this channel is not effective in the ovarian pathway.

Histological analysis (Fig. 4A,B) revealed that while there were instances of complete feminization following exposure to RN1734, over all both RN1734- and GSK1016790A-treated samples were histologically and morphologically similar to their respective control groups despite significantly lowered AMH and SOX9 gene expression levels in the RN1734-treated groups. However, RN1734-treated groups displayed an increase in prominent Müllerian ducts in a dosage-dependent manner, consistent with lowered AMH expression (Fig. 4A,C). In some of these individuals, the ducts showed remarkable signs of regression, namely the reduction in the mesosalpinx27. Although the AmTRPV4 targeted treatment did not yield statistically significant phenotypic changes, it did result in a rise of individuals with an abnormal sexual phenotype, with both male-like (testis-like gonad) and female-like (developed Müllerian duct) characteristics.

In summary, manipulation of AmTRPV4 activity impeded induction of the testicular differentiation cascade on a molecular level, but had little effect on the ovarian differentiation cascade, suggesting that TRPV4 does not solely account for thermosensitive trigger mechanism in TSD, but rather, may be part of a larger, more complex mechanism in place.