The molecular mechanisms underpinning temperaturedependent sex determination (TSD) in reptiles have attracted great biological interest for many years.However, which genetic factors are essential for TSD remain elusive,especially regarding female sex determination.Cytochrome P450 family 19 subfamily A member 1 (Cyp19a1) encodes the endoplasmic reticulum enzyme aromatase, which participates in the catalytic conversion of androgens to estrogens, and is implicated in sexual differentiation in many species.However,whether Cyp19a1 plays a critical role in determining gonadal sexual fate in TSD species remains to be elucidated.In the current study, Cyp19a1 exhibited a temperature-dependent and sexually dimorphic expression pattern, preceding gonadal sex differentiation in a TSD turtle species (Mauremys reevesii).Sexual phenotype of the turtles was successfully reversed by aromatase inhibitor treatment at the femaleproducing temperature (FPT).Furthermore, exogenous estradiol (E2) treatment led to complete male-to-female sex reversal at the male-producing temperature (MPT),accompanied by rapid up-regulation of Cyp19a1.Thus,Cyp19a1 appears to be essential for female sex determination in M.reevesii, suggesting a vital role in the female TSD pathway.

In reptiles, the molecular mechanisms underlying TSD have been researched for several decades.Early studies focused on hormone-dependent mechanisms and the cloning and/or expression of certain classical sex-related genes involved in the genetic sex determination (GSD) system (e.g., Wt1; Amh)in TSD species (Pieau et al., 1999).Later studies demonstrated that several of these genes exhibit temperaturedependent expression patterns during thermo-sensitive periods (TSP), prior to gonadal sex differentiation (Shoemaker et al., 2007).Recent research on the transcriptomes of TSD reptile taxa identified a great number of candidate genes involved in gonadal differentiation (Czerwinski et al., 2016).In addition, genetic engineering (e.g., RNA interference) has been applied to identify the function of sex-related genes in sex determination in reptiles (Ge et al., 2018; Weber et al.,2020).However, despite several decades of research on the potential underlying mechanisms of TSD in reptiles, the role and function of key sex-related genes in the female sex determination process remain unverified.

Cyp19a1 modulates estrogen to promote sexual differentiation and is both necessary and sufficient for ovarian differentiation in GSD species (Bao et al., 2017).Given its vital role in GSD systems, Cyp19a1 may be involved in female TSD.Based on previous transcriptomic analyses, Cyp19a1 is reported to show female-specific expression patterns in early embryonic gonads during late TSP (embryonic developmental stage 19) in TSD turtles (Czerwinski et al., 2016).In contrast,however, Cyp19a1 transcripts have been detected in adrenalkidney-gonad complexes (AKGs) after the sex determination TSP in Alligator mississippiensis and several TSD turtle species (Gabriel et al., 2001; Murdock & Wibbels, 2003),thereby repudiating its role in sex determination.Therefore,the role of Cyp19a1 in TSD remains unresolved in reptiles and may differ among species.Moreover, previous studies have only explored mRNA expression of Cyp19a1 in embryonic gonads and AKGs, which is inadequate for providing unequivocal conclusions regarding Cyp19a1 function.Here,we performed multi-level analysis (e.g., gene expression patterns, gene thermo-sensitivity, hormone/genetic manipulation) of embryonic gonads to determine the function of Cyp19a1 in a TSD turtle species (M.reevesii).

To investigate the expression patterns of Cyp19a1, we analysed expression levels during different developmental embryonic stages (16 to 21) at the male-producing temperature (MPT, 26 °C) and female-producing temperature(FPT, 32 °C).Results showed Cyp19a1 expression in FPTgonads as early as stage 16, i.e., early TSP and clearly prior to gonadal differentiation.Furthermore,Cyp19a1expression increased dramatically with embryonic development.Cyp19a1expression in the FPT gonads was tens of times higher than that in the MPT gonads at stage 16 and thousands of times higher at stages 19 and 25.In contrast, MPT gonads exhibited extremely lowCyp19a1expression throughout embryogenesis(Figure 1A).These results suggest thatCyp19a1is not only involved in sexual differentiation, but is also an early responder for female determination inM.reevesii.

Cyp19a1is reported to show temperature dependence between a temperature range of 26 °C to 32 °C in TSD reptiles (Czerwinski et al., 2016).To date, however, howCyp19a1expression is influenced by temperature is notknown.In the present study, we showed that Cyp19a1 expression in turtle gonads decreased obviously with declining temperature (from 31 °C to 27 °C) at both stage 18 (during TSP) and stage 21 (after TSP) (Figure 1Ba, b).In addition,Cyp19a1 responded rapidly to the temperature shift in the developing gonads when embryos were transferred from MPT to FPT at stage 16.Specifically, Cyp19a1 increased slightly above the MPT-typical level at early stage 17 (2 days after transfer), then increased significantly at subsequent stages.However, Cyp19a1 showed a time lag in response to the temperature shift in gonads after FPT-to-MPT transfer,increasing continuously until stage 19 and then decreasing to the MPT-typical level (Figure 1Bc).These findings confirmed the high thermo-sensitivity of Cyp19a1 in M.reevesii gonadal cells, indicating its vital role in the female pathway by connecting temperature and sex in the TSD system.

Figure 1 Evidence for the role of Cyp19a1 in sex determination in M.reevesii

During the TSP, administration of exogenous estrogen may override the temperature effect to induce feminization of MPT embryos in TSD reptiles (Ramsey & Crews, 2009).Here, we treated MPT embryos of M.reevesii with E2 at stage 16 and found that the gonads exhibited female-like morphology, with a degenerated medulla and a thickened outer cortex at stage 25 (Figure 1Ca–f‘).A significant up-regulation of Foxl2 and Rspo1 and down-regulation of Dmrt1 and Sox9 were also detected in feminized MPT embryos at stage 25 (Figure 1Cg).Correspondingly, the protein expression levels of Sox9 (a Sertoli cell marker) and Vasa (a germ cell marker) in the MPT gonads with estrogen treatment exhibited female-like distribution patterns (i.e., no Sox9 expression, cortical localization of Vasa) rather than male patterns (i.e., high expression of Sox9, medullary cord distribution of Vasa)(Figure 1Ch–m‘‘, Supplementary Figure S3A–F‘‘).Further analysis showed that Cyp19a1 transcripts responded rapidly to estrogen treatment and were significantly up-regulated from stage 17 (2 days after treatment) onwards (Figure 1Cn).Therefore, it is possible that the reversal of Cyp19a1 expression by exogenous estrogen may be responsible for sex-reversal in M.reevesii.These results suggest that Cyp19a1 is required for early ovarian determination in TSD.

In GSD species, aromatase inhibitor or Cyp19a1 loss-offunction treatments can induce permanent female-male sex reversal (Bao et al., 2017; Vaillant et al., 2001).In M.reevesii,when FPT embryos were treated with aromatase inhibitor,gonads became shortened and vascularized and exhibited male-like morphology, including dense medulla with several primordial germ cells and degenerated cortex (Figure 1Da–f).The Sox9 protein was specifically expressed in the nuclei of precursor Sertoli cells in MPT gonads, consistent with the FPT gonads treated by aromatase inhibitor (Figure 1Dg–i‘‘,Supplementary Figure S4A–C‘‘).The distribution of Vasapositive germ cells in FPT gonads following aromatase inhibitor treatment displayed a male-like medulla localization pattern (Figure 1Dj–l‘‘, Supplementary Figure S4D–F‘‘).These findings strongly suggest that Cyp19a1 is necessary for the female determination process in the TSD turtle species M.reevesii.

In vertebrates like fish, amphibians, and birds, Cyp19a1 generally exhibits a sexually dimorphic expression pattern in female gonads during early embryonic stages and is robustly expressed within the cytoplasm of the ovarian medulla (Jin et al., 2020; Mawaribuchi et al., 2014; Nakamoto et al., 2018).In Mus musculus, although Cyp19a1 is not required for sex determination or gonadal differentiation during the embryonic stage, it is essential for maintenance of granulosa cells and ovarian phenotype in postnatal ovaries (Erickso et al., 1979).In this study, we demonstrated that Cyp19a1 is a key female sex-determining gene in M.reevesii turtles.Therefore, the function of Cyp19a1 is highly conserved in the sexual differentiation progress, even in a wider phylogenetic context.Our study provides unequivocal evidence for the critical function of Cyp19a1 in the female pathway in turtles and its importance in female sex determination, thereby shedding light on the elusive molecular mechanism underlying TSD.

SUPPLEMENTARY DATA

Supplementary data to this article can be found online.

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS’ CONTRIBUTIONS

P.F.W., X.F.W., and W.G.D.conceived and designed the study; P.F.W.and X.F.W.performed the experiments; P.F.W.and X.F.W.analyzed the data; and P.F.W., X.F.W., F.G., and W.G.D.cowrote the manuscript.All authors read and approved the final version of the manuscript.

ACKNOWLEDGEMENTS

We thank Hua Ye, Qiong Zhang, Hong-Xin Xie, Chun-Rong Mi, Zi-Han Ding, and Ming-Shuo Qing for their assistance in turtle egg collection and incubation.

Peng-Fei Wu1,3, Xi-Feng Wang1,＊, Fei Gao2, Wei-Guo Du1

1Key Laboratory of Animal Ecology and Conservation Biology,Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

2State Key Laboratory of Stem Cell and Reproductive Biology,Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

3University of Chinese Academy of Sciences, Beijing 100049,China

＊Corresponding author, E-mail: wangxifeng@ioz.ac.cn