Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Sci Rep
2015 Jan 12;5:18581. doi: 10.1038/srep18581.
Show Gene links
Show Anatomy links
TRPV4 associates environmental temperature and sex determination in the American alligator.
Yatsu R
,
Miyagawa S
,
Kohno S
,
Saito S
,
Lowers RH
,
Ogino Y
,
Fukuta N
,
Katsu Y
,
Ohta Y
,
Tominaga M
,
Guillette LJ
,
Iguchi T
.
???displayArticle.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.
Argentaro,
A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal.
2003, Pubmed
Argentaro,
A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal.
2003,
Pubmed
Barske,
Estrogen represses SOX9 during sex determination in the red-eared slider turtle Trachemys scripta.
2010,
Pubmed
Charnier,
[Action of temperature on the sex ratio in the Agama agama (Agamidae, Lacertilia) embryo].
1966,
Pubmed
Cheng,
Heteromerization of TRP channel subunits: extending functional diversity.
2010,
Pubmed
Cho,
The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons.
2012,
Pubmed
Clark,
Chondroprotective role of the osmotically sensitive ion channel transient receptor potential vanilloid 4: age- and sex-dependent progression of osteoarthritis in Trpv4-deficient mice.
2010,
Pubmed
Cruze,
Towards an understanding of the evolution of the chorioallantoic placenta: steroid biosynthesis and steroid hormone signaling in the chorioallantoic membrane of an oviparous reptile.
2012,
Pubmed
Di-Poï,
Crocodylians evolved scattered multi-sensory micro-organs.
2013,
Pubmed
Ferguson,
Temperature of egg incubation determines sex in Alligator mississippiensis.
1982,
Pubmed
Garcia-Elias,
Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli.
2013,
Pubmed
Gees,
TRP channels.
2012,
Pubmed
Green,
Three crocodilian genomes reveal ancestral patterns of evolution among archosaurs.
2014,
Pubmed
Güler,
Heat-evoked activation of the ion channel, TRPV4.
2002,
Pubmed
,
Xenbase
Hanover,
Calmodulin-driven nuclear entry: trigger for sex determination and terminal differentiation.
2009,
Pubmed
Hayashi,
High temperature causes masculinization of genetically female medaka by elevation of cortisol.
2010,
Pubmed
Kohno,
Potential contributions of heat shock proteins to temperature-dependent sex determination in the American alligator.
2010,
Pubmed
Lance,
Is regulation of aromatase expression in reptiles the key to understanding temperature-dependent sex determination?
2009,
Pubmed
Liedtke,
Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor.
2000,
Pubmed
Masuyama,
TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts.
2008,
Pubmed
Matsumoto,
Epigenetic control of gonadal aromatase (cyp19a1) in temperature-dependent sex determination of red-eared slider turtles.
2013,
Pubmed
Mochizuki,
The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures.
2009,
Pubmed
Moore,
Developmental morphology of the neonatal alligator (Alligator mississippiensis) ovary.
2008,
Pubmed
Muramatsu,
Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation.
2007,
Pubmed
Nagai,
Structure and hibernation-associated expression of the transient receptor potential vanilloid 4 channel (TRPV4) mRNA in the Japanese grass lizard (Takydromus tachydromoides).
2012,
Pubmed
Nakamura,
The mechanism of sex determination in vertebrates-are sex steroids the key-factor?
2010,
Pubmed
Nilius,
The puzzle of TRPV4 channelopathies.
2013,
Pubmed
Parrott,
Differential incubation temperatures result in dimorphic DNA methylation patterning of the SOX9 and aromatase promoters in gonads of alligator (Alligator mississippiensis) embryos.
2014,
Pubmed
Quinn,
Temperature sex reversal implies sex gene dosage in a reptile.
2007,
Pubmed
Rahaman,
TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice.
2014,
Pubmed
Rhen,
Molecular mechanisms of sex determination in reptiles.
2010,
Pubmed
Rzhetsky,
Theoretical foundation of the minimum-evolution method of phylogenetic inference.
1993,
Pubmed
Saito,
Functional diversity and evolutionary dynamics of thermoTRP channels.
2015,
Pubmed
Saito,
Analysis of transient receptor potential ankyrin 1 (TRPA1) in frogs and lizards illuminates both nociceptive heat and chemical sensitivities and coexpression with TRP vanilloid 1 (TRPV1) in ancestral vertebrates.
2012,
Pubmed
,
Xenbase
Saito,
Heat and noxious chemical sensor, chicken TRPA1, as a target of bird repellents and identification of its structural determinants by multispecies functional comparison.
2014,
Pubmed
,
Xenbase
Sengupta,
Sensing temperature.
2013,
Pubmed
Shoemaker,
Analyzing the coordinated gene network underlying temperature-dependent sex determination in reptiles.
2009,
Pubmed
Shoemaker-Daly,
Genetic network underlying temperature-dependent sex determination is endogenously regulated by temperature in isolated cultured Trachemys scripta gonads.
2010,
Pubmed
Sokabe,
The TRPV4 channel contributes to intercellular junction formation in keratinocytes.
2010,
Pubmed
Tamura,
MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.
2011,
Pubmed
Thompson,
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
1994,
Pubmed
Thorneloe,
N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I.
2008,
Pubmed
Urushitani,
Molecular cloning of anti-Müllerian hormone from the American alligator, Alligator mississippiensis.
2011,
Pubmed
Valenzuela,
Transcriptional evolution underlying vertebrate sexual development.
2013,
Pubmed
Vay,
The thermo-TRP ion channel family: properties and therapeutic implications.
2012,
Pubmed
Vincent,
TRPV4 agonists and antagonists.
2011,
Pubmed
Wang,
The draft genomes of soft-shell turtle and green sea turtle yield insights into the development and evolution of the turtle-specific body plan.
2013,
Pubmed
Western,
Temperature-dependent sex determination in the American alligator: AMH precedes SOX9 expression.
1999,
Pubmed
Yamaguchi,
High temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by increasing cortisol levels during gonadal sex differentiation in Japanese flounder.
2012,
Pubmed
Yao,
Temperature, genes, and sex: a comparative view of sex determination in Trachemys scripta and Mus musculus.
2005,
Pubmed
van der Eerden,
TRPV4 deficiency causes sexual dimorphism in bone metabolism and osteoporotic fracture risk.
2013,
Pubmed
