Embryonic and Gonadal Development of the Chinese Three-keeled Pond Turtle, Mauremys Reevesii

DOI: https://doi.org/10.21203/rs.3.rs-1345103/v1


Temperature-dependent sex determination (TSD) is a mechanism in which environmental temperature rather than innate zygotic genotype determines the fate of sexual differentiation during embryonic development. The Chinese three-keeled pond turtle, Mauremys reevesii, has TSD and can thus be compared to other TSD species to elucidate the molecular basis underlying this mechanism. Nevertheless, neither embryogenesis nor gonadogenesis has been described in this species. Here, we investigated the chronology of normal embryonic development and gonadal structures in M. reevesii under both female- and male-producing incubation temperatures (FPT or MPT, respectively). External morphology remained indistinct between the two temperature regimes throughout the studied embryonic stages. However, the sexual differentiation of gonadal structures began earlier in the embryos at FPT than at MPT. Moreover, the offset of the thermosensitive period, at which the gonadal structures often remain sexually undifferentiated, did not occur at the same stage at FPT and MPT. In ovo experiments are particularly important for understanding the functional roles of genes involved in TSD, and the present study provides the basis required for designing such developmental studies in M. reevesii.


Sexually dimorphic phenotypes of vertebrates primarily emerge as gonadal sex differentiation during embryonic development. In vertebrates, genetic sex determination is a mechanism of differentiating gonadal sex from a bipotent or undifferentiated state, relying on the dimorphic expression of sex-determining genes usually derived from sexually heteromorphic chromosomes13. In contrast, alternative sex determination mechanisms are governed by environmental cues, among which temperature is one of the most prevalent in reptile sex determination. Temperature-dependent sex determination (TSD) is documented for most turtles, some lizards, all crocodilians, and tuataras, but not for snakes46.

The fate of sexual differentiation in TSD species strongly depends on the environmental temperatures that individuals experience during a critical developmental period, the temperature sensitive period (TSP)79. The TSP has historically been derived from experiments that shift the egg incubation temperatures during the embryonic development and analyze the resulting sex of the individuals. Interestingly, the TSP in turtles and alligator is closely associated with the sexual differentiation of gonadal structures8,9. In vertebrates, the coelomic epithelium thickens and develops into the genital ridges on the mesonephros at early embryonic stages10. The red-eared slider turtle Trachemys scripta shows this general gonadal development pattern, and the beginning of its TSP corresponds to the early stage at which the genital ridges develop, i.e., stage 16 (or earlier)8. The TSP in T. scripta then lasts until the sexually bipotential gonads acquire the ovarian or testicular structures, corresponding to stages 18–19 or 20–21 when incubated under 31°C or 26°C, respectively8. After the sexually dimorphic gonads develop, the sex is no longer reversible regardless of the changes in incubation temperature.

Studies on the molecular basis of TSD often investigate the expression of gonadal genes throughout embryonic stages, particularly focusing on the TSP. Recent advents in sequencing techniques have revealed genes presumably involved in gonadal sex differentiation or sexually distinct transcription in TSD species1116, which were further investigated in Alligator mississippiensis and T. scripta embryos using techniques, such as pharmacological treatments and genetic manipulations via viral infections, respectively12,17,18. These manipulations were used to evaluate whether the sex expected from a female- or male-producing incubation temperature (FPT or MPT, respectively) could be reversed, assuming that gonadal sex was structurally undifferentiated at the time of these manipulations. Understanding the structural differentiation of gonads throughout embryonic stages is a prerequisite for these studies, and this information is already available for A. mississippiensis and T. scripta, which makes these species the best models for TSD studies.

Our main objective in the present study was to describe the differentiation of gonadal structures throughout the normal embryonic stages of the Chinese three-keeled pond turtle, Mauremys reevesii, a freshwater species that prevalently inhabits East Asia19. Although wild populations of M. reevesii are reportedly under threat in China because of habitat change and human exploitation, cultured individuals are commercially available in Japan and China20. Du et al. (2007) reported the TSD of M. reevesii, with a heavily female- and male-biased sex ratio of hatchlings at incubation temperatures above 30°C and below 26°C, respectively. This TSD pattern is Ia (i.e., the proportion of females increase with incubation temperature), as in T. scripta5. Thus, M. reevesii can be compared to T. scripta for better understanding of the temperature dependency of sex determination. As the normal embryonic development and the differentiation of gonadal structures in M. reevesii have not yet been described, the present study investigated the following in M. reevesii: (1) normal embryonic stages, (2) developmental rate under both MPT and FPT, and (3) differentiation of gonadal structures at each embryonic stage. The present study provides a basis for effective sampling of embryos and the design of in ovo experiments using M. reevesii.


We described the external morphology of M. reevesii embryos at stages 13–24 and their gonadal structure at stages 16–24. Eye pigmentation was first clearly recognized by candling at stage 13; thus, our analysis began at stage 13. The key characteristics used for staging M. reevesii are summarized in Table 1. The eggs of M. reevesii at the time of arrival weighed between 7.72 and 15.8 g (mean 12.6 g) and remained nearly constant in weight throughout embryonic development (Fig. 1a, Table S1). The embryos grew faster at FPT than at MPT (Fig. 1b, Table S1) but reached similar body mass at each stage regardless of the incubation temperature (Fig. 1c, Table S1). No sexual dimorphic features were observed in respect of the external morphology during the embryonic development either at FPT (31°C) or MPT (26°C). The individuals representative of each stage are shown in Figures 2, 3, and 4 in lateral, ventral, and dorsal view, respectively. Histology sections of representative gonads for both FPT and MPT are shown in Figure 5.

Table 1

The key characteristics used for staging Mauremys reevesii.




Pigmented over retina


Pigmentation reaches pupil


Lateral margin of carapacial ridge is evident


Anterior end of carapacial ridge is evident


Posterior margin of plastron is evident


Digits II–IV protrude, and the thickness at the web is greater than the protrusion


Digits II–IV protrude as much as or more than the thickness at the web


Digits II–IV protrude approximately twice their thickness at the web


The scales on the dorsal forelimb are slightly visible, but do not reach the proximal border of the webbing


The scales on the dorsal forelimb extend to the ranges from the proximal border of the webbing to the distal region of the digits


The palmar surface of the forelimb is fully covered by small circular scales


The translucent sheath on the ungual phalanx recede toward its distal end


Embryonic development

Stage 13

In the lateral view, the anterior edge of the occipital protuberance extends anteriorly beyond the posterior edge of the eye. In the same view, the maxillary process encroaches anteriorly beyond the optic fissure. Pigmentation of the eye is dense, but the pupil and its margin are unpigmented. The optic fissure appears as an unpigmented “triangular” slit at the ventral region of the eye. The forelimb buds are longer than wide.

Stage 14

In the lateral view, the occipital protuberance recedes posteriorly as its anterior edge is near the level of the posterior edge of the eye. The area between the maxillary and lateral nasal processes—where the external nares are later formed—is weakly marked by shallow creases. The pigmentation of the iris reaches the margin of the unpigmented white pupil. The pharyngeal arches are evident. The anterior end of the mandible extends anteriorly beyond the level of the posterior edge of the eye. The forelimb bends in caudal orientation. The body is more flexed ventrally and the tail is longer and more curled than at stage 13. The genital protuberance is evident, with a crease at its posterior margin.

Stage 15

The pharyngeal arches disappear at this stage. The anterior end of the mandible extends to the level of the center of the eye. The carapacial ridge becomes visible in lateral view but its anterior edge remains absent and smooth. The digital plate is well formed. The posterior border of the genital protuberance is evident by a crease between the genital protuberance and the tail. The urogenital papilla is slightly distinguished from the entire genital protuberance by a shallow crease. The dorsal surface is slightly pigmented from the anterior edge of the carapace to the nostril and at the base of the tail.

Stage 16

The optic fissure is evident but becomes narrower, from a “triangular” to “teardrop” shape. An incipient tympanum is barely visible in lateral view. The digital plate has a smooth periphery. The carapacial ridge becomes thicker and its anterior edge is distinct in the lateral view. The pigmentation becomes denser on the dorsal surface of the neck and head regions and at the base of the tail.

Stage 17

The optic fissure is visible but becomes a narrow slit. The caruncle is visible as a small white mark on the rostral tip of the upper jaw. Each of the five digits can be distinguished by a ridge as the digital plate is slightly serrated. The posterior border of the plastron is evident. A crease on the posterior border of the genital protuberance extends more laterally. The urogenital papilla becomes larger and protrudes as it occupies a large proportion of the genital protuberance. Ribs can be slightly seen through the carapace in dorsal view.

Stage 18

The optical fissure disappears at this stage, and scleral papillae are evident. The caruncle is more evident. The mandible and the upper jaw make an almost complete closure as the mandible extends anteriorly to the level of the anterior end of the eyes in the lateral view. Digits II–IV protrude along the periphery of the digital plate but the thickness at the web is greater than the protrusion. Carapace pigmentation begins at this stage 18, such that the dorsolateral margins of the carapace are slightly pigmented, exposing the marginal scutes with faint borders. The plastron remains unpigmented, but it weakly shows the borders of the plastral scutes. The lateral sides of the tail are slightly pigmented, forming lines of pigments. A fold is evident surrounding the urogenital papilla.

Stage 19

The pigmentation on the head region becomes denser and the external nares are emphasized by the round, unpigmented areas. The lower eyelid is slightly formed. Scleral papillae are distinct. The mandible extends anteriorly, the ending of which is between the level of the anterior end of the eye and that of the nostril in the lateral view. The mandible and the upper jaw make a complete closure. Digits II–IV protrude as much as or more than the thickness at the web along the periphery of the digital plate. The carapace becomes more pigmented, evidencing the borders of all scutes. The three keels on the carapace are barely visible; one runs along the midline of the vertebral scutes and the other two run along the costal scutes.

Stage 20

The lower eyelid reaches the scleral papillae. The protrusion of digits II–IV proceeds and reaches approximately twice their thickness at the web. The digits are weakly pigmented. Rows of cutaneous papillae are slightly visible on the dorsal surface of the neck. The entire carapace is pigmented, but the vertebral scutes are more pigmented than the other scutes. The cutaneous papillae on the vertebral scutes are barely visible in some specimens. The urogenital papilla is evident and remains prolapsed from the vent.

Stage 21

The scleral papillae disappear or become unclear at this stage. The entire body becomes more evidently pigmented. The pigmentation of the digits becomes denser at the thinner distal ends than the thicker proximal regions on most of specimens, vaguely indicating the border of digits and claws. The scales are slightly visible on the dorsal forelimb, but do not reach the proximal border of the webbing. The cutaneous papillae on the vertebral scutes are evident. The urogenital papilla withdraws into the vent.

Stage 22

The iris becomes distinct and the lower eyelid reaches its outer margin. The ungual phalanx enclosed in a translucent sheath is clearly visible including its tip. Pigmentation becomes heavier on the dorsal forelimb. The scales on the dorsal forelimb extend distally to the ranges of the proximal border of the webbing to the distal region of the digits. The palmar surface of the forelimb is slightly covered by small circular scales, and in some specimens, these scales overlap adjacent scales. Four large circular scales are more evident and aligned along the anteroposterior axis of the palmar surface. Each of these four scales slightly overlaps or projects over the surrounding palmar surface. The claws are homogeneously opaque and white-colored with evident pigmentation. The carapace becomes darker so that the presence of cutaneous papillae on the entire carapace is emphasized. The intestinal loop is withdrawn into the body.

Stage 23

The ungual phalanx is evident in a translucent sheath. The palmar surface of the forelimb is fully covered by small circular scales, and their overlap to adjacent scales is evident. The density of the cutaneous papillae increased on the vertebral and pleural scutes. The plastron is more pigmented so that the whitish regions are less than the darker pigmented regions.

Stage 24

The embryos resemble the hatchlings. The translucent sheath on the ungual phalanx recedes toward its distal end, exposing the ventral surface of the ungual phalanx. The individual scales on the forelimb, hindlimb, and tail are evident and overlap one another. The pigmentation becomes darker throughout the body; the pigmentation of the carapace is dark brown in color but that of the plastron and skin is blackish brown. The color contrast increases on the head, exposing a discernible pattern on the lateral and ventral surface of the head.

Gonadal development

At stage 16, the gonadal ridges are present on the mesonephros of embryos. The gonads have an inner medulla consisting of sex cords and outer cortical layers with 1–2 cells in thickness. The germ cells, with large spherical nuclei, and the medullary sex cords become more evident at stage 17. At stage 18, the sex cords begin to degenerate in some specimens at FPT. By stage 20 at FPT, the sex cords become disorganized and the medullary region shows the homogeneous structures, while at MPT the sex cords become more distinct, developing into the seminiferous tubules. As the seminiferous tubules develop at stages 20 and 21, the germ cells are enclosed within the cords. During stages 20 and 21, the cortex becomes slightly enlarged and thicker at FPT; conversely, the cortex becomes a thin one-cell-thick layer at MPT. From stage 21, the tissue area connecting gonads and the mesonephros begins to decrease, with the gonads becoming merely connected by a narrow stalk of the connective tissues by stage 25. At stages 24 and 25 at FPT, the distinction between cortex and medullary region is more evident and the medullary region becomes vacuolated.


The sex of TSD species is determined during embryonic development, particularly during the TSP, depending on thermal environmental conditions5. Sex determination is primarily recognized by the differentiation of gonadal structures toward testicular or ovarian developmental pathways. While M. reevesii has been reported to be a TSD species, the chronology of the sexual differentiation of its gonadal structures has not been investigated. In the present study, we analyzed the gonadal development of M. reevesii under both MPT and FPT according to the chronology of its external embryonic morphogenesis.

The embryonic development of M. reevesii regarding its external morphology was analyzed following criteria developed for C. serpentina and with a particular reference to the testudinoid species, T. scripta and M. japonica2123. Several features of M. reevesii were distinct from those of both or either of the latter two species. The claw development is a key to discerning later stages in T. scripta and particularly in M. japonica, but we recognized the characteristics of the claw development at distinct times between M. reevesii and T. scripta/M. japonica, obscuring the equivalent stages across the species. At stage 23, the ungual phalanx is vaguely present in the translucent sheath of T. scripta while that of M. japonica shows a homogeneous structure in the lateral view. At stage 24, the ungual phalanx is evident in the translucent sheath, but its tip remains unclear in the lateral view in both T. scripta and M. japonica. The ungual phalanx (including its tip) becomes evident at stage 25 in these species. Nevertheless, the presence of the ungual phalanx (including its tip) is recognized at an earlier stage in M. reevesii equivalent to stage 22 in T. scripta. Thus, the detection of the ungual phalanx in the translucent sheath hardly distinguishes the following stages in M. reevesii. Instead, we defined stage 23 in M. reevesii by referring to the development of the palmar scales in T. scripta at stage 23 when its palmar surface becomes fully covered by small circular scales. The translucent sheath begins to recede from the base of the claw toward its distal end during stage 25 until hatching in T. scripta. The recession occurs within a few days of stage 23 in M. reevesii, and thus, we investigated stage 24 based on the recession of the translucent sheath as the last embryonic stage in M. reevesii.

The scleral papillae also appear at different times and duration among species. Mauremys reevesii present the scleral papillae at stages 18–20, while T. scripta and M. japonica present those at stage 18 and 19–20, respectively2223. These variations also occur in other turtles in Yntema’s (1968) stages 16–2121,24. The scleral papillae induce the development of the scleral ossicles that are present in all birds and turtles25. Although controversial, the functional roles of scleral ossicles are presumably adaptive (e.g., to prevent the eyes from physical deformation or visual accommodation)26. Therefore, the interspecific variation in the development of scleral papillae may be involved in species-specific visual adaptation.

One of the most evident differences in external morphology between T. scripta and M. reevesii/M. japonica is the carapacial keel that appears as only one ridge in the former species but as three ridges in the latter two. The three keels appear from stage 19 in M. reevesii. Although there is only a single keel running at the midline of the vertebral scute in T. scripta, its formation begins at stage 19. The three keels also occur in distantly related turtle species, such as Sternotherus odoratus (Chelyroidea, Chelyroidae), which also shows primary evidence of keel development at stage 2027. Interestingly, this suggests that the embryonic chronology of the carapacial diversity in turtles is conserved, when staged according to Yntema (1968). Nevertheless, embryos of different species at the same Yntema (1968) stage do not necessarily have the same degree of embryonic development. Thus, the development of certain characters should ideally be discussed based on relative timing throughout development, as in Tokita and Kuratani (2001)28. Further studies on carapace development around stage 19–20 could reveal whether the genetic mechanisms regulating keel development are the same among species and will elucidate the genetic basis underlying carapacial diversity. The present results show that the development of the external morphology of M. reevesii is more similar to that of M. japonica than that of T. scripta, as expected based on their phylogenetic distance29.

We then described the gonadal development of M. reevesii from stages 16 to 24 using the histology of the gonads. Although TSP generally begins at early stages, when the sexually undifferentiated gonadal ridge is formed, TSP ends at different stages depending on the incubation temperature8,9. In T. scripta, the sex cords begin to degenerate at stages 18–19 at 31°C, but they develop into seminiferous tubules at stages 20–21 at 26°C8. Structural differentiation arises as the first evidence of gonadal sex differentiation in T. scripta and corresponds to the end of TSP8,30. According to the chronology of sexual differentiation of gonadal structures, the results suggest that TSP in M. reevesii occurs at stage 16 or earlier and lasts until stages 18–19 and 20–21 at FPT and MPT, respectively. The development of gonads in M. reevesii followed similar patterns and chronology to that of T. scripta.

The development of external morphology in M. reevesii proceeds at a higher rate at FPT than at MPT but remains indistinct between the two temperature regimes throughout the studied developmental stages. Interestingly, gonadal sex differentiation is strongly associated with the stages. We calibrated the initial date of the analysis using egg candling and showed that the incubation date provides clues to predict each stage for both MPT and FPT (Fig. 3). While the egg weight seems to change independently of the embryonic stage, the embryonic weight increases as the embryonic development proceeds and can also be a parameter to support staging (Fig. 3). These results facilitate the effective sampling of embryos at target stages in M. reevesii.

The current study described the development of external and gonadal morphology in M. reevesii under two temperature regimes, i.e., FPT and MPT and recorded the time required to reach each stage. The external morphology follows developmental trajectory indistinct between FPT and MPT but the gonadal structures sexually differentiate in temperature-dependent manner after stages 18–19 and 20–21 at FPT and MPT, respectively. We showed that the embryonic stages and the associated state of the gonadal differentiation in M. reevesii are well predictable as a function of the incubation days and temperatures. Stages at which gonadal structures remain sexually undifferentiated are particularly important as often be the target for in ovo experiment in the studies of sex determination. Thus, the current study establishes the basis for designing in ovo functional analysis of target molecules using pharmacological treatments and/or genetic manipulations via viral infections. Further intra- and interspecific comparisons of TSD species including M. reevesii would lead to a comprehensive understanding of how temperature stimuli regulate genetic responses towards sex determination.

Materials And Methods

Turtle eggs were purchased from Kondo farm in Maniwa, Okayama, Japan from June to August 2020 and from June to July 2021. The eggs were collected by farmers 1–3 days from oviposition, but some older eggs may have been accidentally included. The eggs were then transported to the laboratory at Tokyo University of Science, where they were immediately divided into two groups and incubated in environmental chambers (Panasonic MIR-554-PJ, Sanyo MIR-253, or Sanyo MIR-153) at a constant temperature of 26°C (MPT) or 31°C (FPT). The incubation containers (length × width × height = 23.5 cm ×16.5 cm × 4 cm) were half-filled with vermiculite:water (1:1 ratio) (Setogahara Kaen, Kidori, Gunma, Japan). Each container housed up to 20 uniformly arranged eggs and was placed in a ziplocked plastic bag to maintain humidity. Every three or four days, the containers were ventilated, rehydrated, and rotated in the chamber to reduce the influence of potential thermal heterogeneity. Although the exact date of oviposition was unknown, the appearance of the pigmented eye, observed using the egg candling technique (pigmentation becomes denser with embryonic development), was used to standardize the ages of the eggs. Thus, the initial date at which eye pigmentation was observed was recorded as Day 0 of the investigation.

Embryonic and gonadal development was investigated using 198 embryos of M. reevesii. Egg weight was recorded on the day of arrival and of dissection. Five to six embryos were dissected every sampling day for each temperature treatment in phosphate-buffered saline and under a microscope (Olympus SZ61), and then weighed without the extraembryonic membranes. Gonads of some of embryos were excised for histological analysis. Whole body and excised gonads were fixed in 4% paraformaldehyde overnight at 4°C and then in methanol at increasing concentrations (25%, 50%, 75%, and 100%). The fixed embryos were photographed (Olympus TG-6), examined, and staged referring to the criteria described for Chelydra serpentina and the phylogenetically closely related species, T. scripta and M. japonica2123, 29. In T. scripta and M. japonica, stages 13–25 were defined primarily based on forelimb morphology; therefore, forelimb morphology was also used herein as a primary criterion for staging in M. reevesii. In the present study, forelimb morphology between adjacent stages was sometimes indistinct (see Results). In such cases, other characteristics, such as eye or carapace development, were referenced in combination with forelimb morphology as the primary criterion. For clarity when describing morphological features, the embryos were rotated, and the lateral view was fixed. In other words, for describing stages 13–16, the lateral view is set as the optic fissure is at the bottom of the microscopic field. For describing stages 17–18, the lateral view is set as the line of the upper jaw lies horizontally in the microscopic field. All graphs were produced using R (version 4.1.0; https://cran.r-project.org).

Histological analysis of the excised gonads was conducted for each of the embryonic stages at both incubation temperatures. The fixed tissues were embedded in histological paraffin and cut into 8-µm thick sections. Each section was stained with hematoxylin and eosin (HE) using a standard procedure and photographed under a microscope (Olympus BX53) with a mounted digital camera (Olympus DP72). Images and graphs were edited and formatted using programs Preview 11.0 (Apple, Inc.), Adobe Photoshop (version 23.1.1), and Adobe Illustrator (version 26.0.2). The description and identification of the gonadal structures followed the terminology proposed for T. scripta by Wibbels et al. (1991)8.



We are grateful to K. Ueda, T. Inuzuka, M. Ishikawa, S. Yanagawa, I. Hirayama, and S. Masuda for the maintenance of turtle eggs in captivity at the Tokyo University of Science. This work was partly supported by Grant-in-Aid for JSPS Fellows [Grant Number JP19J01186 (H.A.)], Fostering Joint International Research B [Grant Number JP19KK0184 (H.A.)], Grant-in-Aid for Early-Career Scientists [Grant Number JP21K15166 (H.A.) and JP20K15835 (G.Y.)], Grant-in-Aid for Scientific Research B [Grant Number JP21H02522 (S.M.)], and Grant-in-Aid for Scientific Research on Innovative Areas [Grant Number JP17H06432 (S.M.)] from Japan Society for the Promotion of Science (JSPS).

Author Contributions

H.A. conceived and designed the study. H.A., M.K., H.Y., and K.M. fixed the embryos and investigated their external morphology. M.K. and H.Y. conducted histological sectioning. All authors investigated the external and gonadal morphology. H.A. wrote the original draft, prepared tables and figures, and all authors contributed to review, comment, and edit the manuscript.

Data availability

Raw pictures in Figures 2–5 are available upon request. Parameters used to produce Figure 1 are listed in Supplementary Table 1 and R scripts utilized herein are available upon request.

Additional Information

Competing financial interests

The authors declare no competing financial interests.


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