Sox9 is indispensable for testis differentiation in the red-eared slider turtle, a reptile with temperature-dependent sex determination

SOX9 (SRY-related HMG box gene 9) is an essential regulator of male sex determination and testis differentiation in many vertebrate species. However, the functional role of Sox9 in testis differentiation has not yet been identified in any reptilian species. Herein, Sox9 knockdown in the red-eared slider turtle (Trachemys scripta) embryos at a male-producing temperature led to complete male-to-female sex reversal, characterized by the formation of an ovary-like structure, disappearance of male marker AMH, and ectopic expression of ovarian regulator FOXL2, as well as a female distribution pattern of germ cells. Conversely, in-ovo overexpression of Sox9 at a female-producing temperature resulted in partial masculinization of putative female embryos, with the coexistence of AMH and FOXL2. Our study provides the first direct evidence that Sox9 is indispensable for testicular differentiation in a reptilian species, further confirming the conserved role of Sox9 in vertebrate sexual development. Many egg-laying reptiles without sex chromosomes exhibit temperature-dependent sex determination (TSD), in which sex is established by the incubation temperature during the temperature-sensitive period (Pewphong et al., 2021). Sox9 is a member of the SRY-related HMG box (SOX) gene family and plays a conserved role in vertebrate sexual development. The expression patterns of Sox9 during embryonic gonadal development have been studied in several TSD species, which suggest the presence of Sox9 expression dimorphism between male-producing (MPT) and female-producing temperatures (FPT) (Díaz-Hernández et al., 2020; MorenoMendoza et al., 2001; Rhen et al., 2007; Shoemaker et al., 2007b; Spotila et al., 1998; Torres-Maldonado et al., 2001; Torres Maldonado et al., 2002; Western et al., 1999). In the red-eared slider turtle (Trachemys scripta), Sox9 expression in embryonic gonads is higher at the MPT than at the FPT in the later temperature-sensitive period (Shoemaker et al., 2007a; Spotila et al., 1998). The expression of Sox9 is significantly down-regulated after exogenous estradiol treatment at the MPT, while inhibition of estrogen synthesis delays Sox9 downregulation at the FPT in T. scripta (Barske & Capel, 2010; Matsumoto et al., 2013). In addition, knockdown of the male sex-determining gene Dmrt1 in T. scripta results in the downregulation of Sox9 expression, while overexpression of Dmrt1 leads to an increase in Sox9 expression (Ge et al., 2017). These studies suggest that Sox9 may have a regulatory effect on sexual differentiation in turtles; however, its functional role has not yet been investigated in any TSD reptilian species. In the current study, we investigated lossand gain-offunction of Sox9 in the red-eared slider turtle using lentivirusmediated genetic manipulation. A lentivirus carrying Sox9 short hairpin RNA (shRNA) or open reading frame (ORF) was injected into T. scripta embryos at stage 15, i.e., very beginning of the temperature-sensitive period (Matsumoto & Crews, 2012). Stage 25 gonads were dissected for quantitative real-time polymerase chain reaction (qRT-PCR), hematoxylin-eosin (H&E) staining, and immunofluorescence. Detailed procedures are described in the Supplementary Materials and Methods. Immunofluorescence analysis of the control group showed that the SOX9 protein was mainly located in the nucleus of Sertoli precursor cells in MPT embryonic gonads, while no SOX9 signal was detected in the FPT embryonic gonads at stage 25 (Figure 1A). In the gonads of MPT embryos treated with LV-Sox9-shRNA, SOX9 decreased to an almost undetectable level, similar to that in the control FPT embryos (Figure 1A), thus indicating a strong inhibitory effect of LV-