Introduction
The transient receptor potential cation channels (TRPV) subfamily of ion channels is crucial in mediating temperature-sensitive physiological processes in fish species . In teleost , in silico analyses have identified three to four members of the TRPV subfamily: TRPV1 (TRPV1a and TRPV1b in some species), TRPV4 and TRPV5 (Morini et al., 2022) . As global climate change drives elevated water temperatures, concerns have grown over the potential impacts on gonadal development and spermatogenesis. To explore this, we studied how TRPV expression relates to androgens and sperm kinetics in male sea bass during their second year of growth, after being raised in warm water from an early age.
Material and methods
C haracterization of TRPV in European sea bass was made in silico by using BLAST tool from NCBI. Gene-specific primers for TRPV and the reference genes — elongation factor 1-alpha (ef1-α) and ribosomal protein L13 (rpL13) — were designed for quantitative PCR. Juvenile sea bass (average initial weight: 14.0 ± 4.1 g) were divided into two treatment groups, each in triplicate for 21 months: (i) a control group (CG) reared at ambient seawater temperature (40°08′15″ N; 0°10′12″ E), and (ii) a high temperature group (HTG), maintained at 3–4 °C above the control temperature . To assess the reproductive influence of temperature a total of 10 fish (5 males each group) were sampled at three key stages of gonadal development: early recrudescence (October), late recrudescence (December), and final maturation-spermiating testes (February and March). Circulating blood levels of testosterone (T) and 11-k etotestosterone (11-KT) were measured by a conventional enzyme immunoassay (EIA), and TRPV expression from testes were measured monthly by quantitative PCR , while sperm kinetics (MOT, MOTp VAP, VCL, and VSL) were evaluated using Computer-Assisted Sperm Analysis software during the spermiating season, in March.
Results and discussion
Our investigation showed for the first time the presence and expression of four paralog ous genes belonging to the TRPV subfamily in European seabass: TRPV1a, TRPV1b, TRPV4, and TRPV5.
In October, TRPV1b and TRPV4 showed significantly higher expression levels in the HTG (average monthly temperature : 25.8 ºC ) compared to the CG (21.7 ºC) , suggesting the existence of a temperature threshold for these genes in sea bass. Additionally, different expression patterns were noted within the HTG: TRPV1a and TRPV1b showed its maximum expression in October, whereas TRPV4 and TRPV5 exhibited a cyclical expression pattern, reaching its maximum expression at the onset and the end of the reproductive season. A principal component analysis (PCA) revealed a distinct clustering of TRPV and circulating steroids levels under high rearing temperature, suggesting that male reproduction is a temperature-sensitive process involving these receptors. By the end of the reproductive period, correlations between gene expression, circulating steroids levels and sperm kinetics highlighted the involvement of TRPV in reproductive performance: the p aralogs TRPV1a and TRPV1 b displayed an antagonistic role in sperm function, while TRPV1a was associated with boosted sperm performance, high expression levels of TRPV1b were clearly correlated with the reduc tion of sperm kinetics (Fig. 1A, B). Given its correlation with T and 11-KT (Fig. 1B) , we suggest that TRPV1b might be involved during sexual maturation and early stages of spermatogenesis whereas TRPV1a seems to be critical during spermiation. Although a positive correlation was observed between TRPV1a, TRPV4 and VAP in CG (Fig. 1A), the increased temperature modified this interaction, leading TRPV4 to align with TRPV1b, resulting in a negative impact on spermatozoa velocity (Fig. 1B).
Overall, our findings suggest the involvement of TRPV in the reproductive physiology of male sea bass and demonstrate that elevated rearing temperatures can alter their function . Nevertheless, additional studies are required to clarify the specific molecular pathways involved in TRPV-mediated responses and to better understand their impact on reproductive outcomes in a global warming scenario.
Acknowledgments
This study forms part of the ThinkInAzul programme and was supported by MICIU with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (GVA-THINKINAZUL/2021/012; Principal investigator: J.F. Asturiano, Universitat Politècnica de València, and GVA-THINKINAZUL/2021/042; Principal investigator: A. Gómez, CSIC). MM is a recipient of a Seal of Excellence MSCA Post-doctoral Fellowship from Sorbonne University. S.S. was supported by a research contract from Margarita Salas program from Universidad de Las Palmas de Gran Canaria.
References
Morini, M., Bergqvist, C.A., Asturiano, J.F., Larhammar, D., & Dufour, S. (2022). Dynamic evolution of transient receptor potential vanilloid (TRPV) ion channel family with numerous gene duplications and losses. Frontiers in Endocrinology, 13, 1013868. https://doi.org/10.3389/fendo.2022.1013868