Hang-Bo Hui, Ling Xiao, Wei Sun, Ying-Jie Zhou,Hai-Yan Zhang, Chu-Tian Ge,＊

1 Institute of Animal Sex and Development, Zhejiang Wanli University, Ningbo, Zhejiang 315100, China

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 ofSox9in testis differentiation has not yet been identified in any reptilian species.Herein,Sox9knockdown in the red-eared slider turtle (Trachemysscripta) 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-ovooverexpression ofSox9at 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 thatSox9is indispensable for testicular differentiation in a reptilian species, further confirming the conserved role ofSox9in 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).Sox9is a member of the SRY-related HMG box (SOX) gene family and plays a conserved role in vertebrate sexual development.The expression patterns ofSox9during embryonic gonadal development have been studied in several TSD species,which suggest the presence ofSox9expression dimorphism between male-producing (MPT) and female-producing temperatures (FPT) (Díaz-Hernández et al., 2020; Moreno-Mendoza 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 (Trachemysscripta),Sox9expression 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 ofSox9is significantly down-regulated after exogenous estradiol treatment at the MPT, while inhibition of estrogen synthesis delaysSox9downregulation at the FPT inT.scripta(Barske & Capel, 2010;Matsumoto et al., 2013).In addition, knockdown of the male sex-determining geneDmrt1inT.scriptaresults in the downregulation ofSox9expression, while overexpression ofDmrt1leads to an increase inSox9expression (Ge et al., 2017).These studies suggest thatSox9may 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 loss- and gain-offunction ofSox9in the red-eared slider turtle using lentivirusmediated genetic manipulation.A lentivirus carryingSox9short hairpin RNA (shRNA) or open reading frame (ORF) was injected intoT.scriptaembryos 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 LVSox9-shRNA onSox9expression.Histological analysis showed that the MPT gonads had a dense medulla with seminiferous cords and a reduced cortex, while the FPT gonads showed a thickened outer cortex and vacuolated medulla (Figure 1A).In contrast, theSox9knockdown MPT gonads were strongly feminized, characterized by a highly developed cortical region and a significantly degraded medulla region, similar to that of the FPT gonads (Figure 1A).Immunofluorescence labeling showed that VASA-positive germ cells were distributed in the developed medulla of the MPT gonads (Figure 1B).However, afterSox9knockdown,the germ cells were mainly enriched in the outer cortex,exhibiting similar female germ cell distribution (Figure 1B).To confirm activation of the female developmental pathway inSox9-deficient MPT embryos, we analyzed the expression of the male-specific protein AMH and ovarian development regulator FOXL2.Immunofluorescence analysis of the control group showed that AMH was strongly expressed in the Sertoli cells of the control MPT gonads but not in the control FPT gonads, while FOXL2 showed the opposite pattern and

primary expression in the cortex region of the FPT gonads(Figure 1C).AfterSox9knockdown, the expression of AMH in most treated gonads was extremely low, while the expression of FOXL2 was robust, similar to that in normal FPT gonads(Figure 1C).Furthermore, knockdown ofSox9at the MPT led to a high rate (92%, 23/25) of complete male-to-female sex reversal (Figure 1D).

Figure 1 Complete sex reversal induced by Sox9 knockdown

Sox9-overexpressing embryos were generated by an injection of a lentivirus vector carrying theSox9ORF into FTP turtle eggs at stage 15.Immunofluorescence showed strong expression of the SOX9 protein in the medulla of FPT gonads after LV-Sox9-OE treatment (Figure 2A).Morphological analysis indicated that the FTP embryos overexpressingSox9had thicker, shorter, and obviously masculinized gonads(Figure 2A).The H&E staining of gonadal sections showed typical male characteristics in some treated FPT gonads,including a degenerated cortex and well-developed medulla,although some FPT gonads overexpressingSox9still retained a developed cortex (Figure 2A).VASA staining indicated that germ cells were distributed in both the cortex and medulla afterSox9overexpression (Figure 2B).To confirm activation of the male developmental pathway in theSox9-overexpressing FPT embryos, we analyzed the expression levels of testicular differentiation markersDmrt1andAmhand ovarian development regulatorsCyp19a1andFoxl2.The qRT-PCR results indicated that the mRNA expression levels ofDmrt1andAmhwere significantly increased, while the expression levels ofCyp19a1andFoxl2were decreased in response to overexpression ofSox9(Figure 2D).In addition,based on immunofluorescence analysis, the ectopic expression of AMH was detected in the well-developed medulla in almost all cases, while the expression of female marker FOXL2 was markedly reduced (disappeared or limitedly retained) after overexpression ofSox9in the FPT gonads (Figure 2C).Gonads showing both AMH and FOXL2 expression were considered as ovotestes.In addition, 85%(34/40) of FPT embryos overexpressingSox9underwent varying degrees of sex reversal, with eight individuals showing complete sex reversal and 26 individuals showing partial sex reversal (Figure 2E).

Figure 2 Partial female-to-male sex reversal induced by Sox9 overexpression

The sequences of the HMG and C-terminal reactivation domains of SOX9 are highly homologous across humans,chickens, alligators, and fish, suggesting a conserved functional role ofSox9in vertebrate gonadal differentiation(Da Silva et al., 1996; Vining et al., 2021).Sox9is essential for Sertoli cell differentiation and testis formation in mice(Chaboissier et al., 2004; Kent et al., 1996).In XY mice, the deletion or a 77% reduction ofSox9leads to complete maleto-female sex reversal, while 50% reduction causes partial sex reversal (Bagheri-Fam et al., 2020; Gonen et al., 2017; Lavery et al., 2011).Moreover, ectopic expression ofSox9in XX gonads causes male development in the absence ofSry(Huang et al., 1999; Vidal et al., 2001).In addition, the duplication or deletion ofSox9enhancers results in sex reversal in XX or XY individuals, respectively (Croft et al.,2018; Gonen et al., 2018), thus demonstrating its critical role in testis determination in mammals.Sox9is highly expressed in MPT gonads during sex differentiation in red-eared slider turtles (Barske & Capel, 2010; Shoemaker et al., 2007b).This,together with our results showing that knockdown or overexpression ofSox9can lead to partial or complete sex reversal in red-eared turtles, confirms that the conserved role ofSox9in testis differentiation is also present in reptilian species.Interestingly, gonad size decreased in theSox9-deficient embryos, consistent with previous results reported in medaka (Nakamura et al., 2012).This may be due to theSox9gene not only affecting sex determination, but also other biological processes such as cell proliferation and germ cell maintenance.

MammalianSox9is directly regulated by the master sex determining geneSry, and its initial expression occurs immediately after that ofSry(Sekido & Lovell-Badge, 2008).SRY and NR5A1 (nuclear receptor subfamily 5 group A member 1, also named steroidogenic factor 1) together bind to theSox9gonad-specific enhancer and cooperatively upregulateSox9expression (Bagheri-Fam et al., 2012; Sekido &Lovell-Badge, 2008).In mammals,Sox9is reported to activateAmh(anti-Müllerian hormone) during testicular development(Cutting et al., 2013; Da Silva et al., 1996; Yamashita et al.,2019).Unlike mammals,Sox9expression in chickens (Gallus gallusdomesticus) and American alligators (Alligator mississippiensis) occurs later than that ofAmh, indicating thatAmhexpression is not regulated by SOX9, andSox9may be located downstream of the sex differentiation pathway in nonmammalian vertebrates (Hirst et al., 2018; Smith & Sinclair,2004; Western et al., 1999).In teleosts,Sox9plays a role in ovary-testis transition and germ cell maintenance (Nakamura et al., 2012; Sun et al., 2013).These studies suggest thatSox9neofunctionalization likely occurred during vertebrate evolution, such that the specific regulatory position of SOX9 in the male sex differentiation pathway differs among vertebrates with different evolutionary statuses (Cutting et al., 2013).In the red-eared slider turtle, the sexually dimorphic expression ofSox9appears later than that ofDmrt1, and knockdown ofDmrt1in MPT embryos results in down-regulation ofSox9(Ge et al., 2017).In fish, DMRT1 positively regulates the transcription of theSox9bgene (ortholog of tetrapodSox9) by directly binding to a specific cis-regulatory element within theSox9bpromoter (Wei et al., 2019).These results imply thatSox9may be involved in testis differentiation rather than sex determination in red-eared slider turtles and may be directly or indirectly regulated byDmrt1.

Herein, we showed that knockdown ofSox9inT.scriptaled to complete male-to-female sex reversal, while overexpression ofSox9caused partial female-to-male sex reversal, thus implicating the indispensable role ofSox9in testicular differentiation in reptiles.This study confirms the conserved role ofSox9in male sexual development across different vertebrate species.

SUPPLEMENTARY DATA

Supplementary data to this article can be found online.

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS’ CONTRIBUTIONS

C.T.G., H.B.H., and L.X.conceived and designed the study.H.B.H., L.X., W.S., Y.J.Z., and H.Y.Z.collected the samples and analyzed the data.H.B.H., L.X., and C.T.G.wrote the manuscript.All authors read and approved the final version of the manuscript.