Chronology of Gonadal Development in the Malayan Snail-eating Turtle Malayemys macrocephala

© 2020 Academia Sinica, Taiwan Open Access Chronology of Gonadal Development in the Malayan Snail-eating Turtle Malayemys macrocephala Rangsima Pewphong1, Jirarach Kitana1,2, and Noppadon Kitana1,2,* 1Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. *Correspondence: E-mail: [email protected] (NK) E-mail: [email protected] (Pewphong); [email protected] (JK) 2BioSentinel Research Group (Special Task Force for Activating Research), Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Received 6 July 2019 / Accepted 8 May 2020 / Published 17 June 2020 Communicated by Benny K.K. Chan The snail-eating turtle, Malayemys macrocephala, is a common freshwater turtle that can be used as an animal model for developmental biology. However, a thorough investigation of its development is needed before this species can be used as a model. Thus, this study aimed to examine the gonadal development of M. macrocephala. Turtle eggs were collected from rice fields in Phra Nakhon Si Ayutthaya Province, Thailand, and transported to the laboratory. Eggs were incubated in microprocessor-controlled incubators and randomly dissected on a weekly basis to reveal the developing embryos, then their developmental stage was identified according to Yntema (1968). Primordial germ cells and gonad structure were processed through the paraffin method. Moreover, the dynamics of germ cell proliferation and apoptosis were examined by immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively. Examination of the gonad revealed four main stages of gonadal development: (i) germ cell migration, (ii) genital ridge appearance, (iii) testicular formation, and (iv) ovarian formation. In the male turtle (incubated at 26°C), gonad developed into the testis with medullary sex cords starting at Yntema stage 17. In the female turtle (incubated at 32°C), these sex cords then degenerated, followed by cortical development into an ovarian structure starting at Yntema stage 19. Subsequently, testicular and ovarian development occurred independently, and distinct sex organs were apparent at Yntema stage 25. In addition, the presumptive testis showed germ cell proliferation in the medulla at Yntema stages 17, 19, and 25 and germ cell apoptosis in the cortex at Yntema stages 19 and 25. The presumptive ovary showed germ cell proliferation in the cortex at Yntema stages 19 and 25, and germ cell apoptosis in the medulla at Yntema stages 19 and 25. Key words: Apoptosis, Embryos, Freshwater turtle, Microscopic examination, Proliferation. Citation: Pewphong R, Kitana J, Kitana N. 2020. Chronology of gonadal development in the Malayan snail-eating turtle Malayemys macrocephala. Zool Stud 59:16. doi:10.6620/ZS.2020.59-20. BACKGROUND Sex determination in reptiles is mediated via temperature-dependent sex determination (TSD), which provides an ideal model system to test predictions concerning the biological importance of global temperature change (Girondot and Kaska 2014). The first study on gonadal development in freshwater turtles showed qualitative differences in response to different incubation temperatures (Ewert 1985). In many turtles, gonad sex differentiation is controlled by the incubation temperature of the egg during a critical period of embryonic development (Pieau and Dorizzi 1981). The time during embryonic development when Zoological Studies 59:20 (2020) doi:10.6620/ZS.2020.59-20 1 © 2020 Academia Sinica, Taiwan the temperature influences the sexual phenotype of the gonad is referred to as the temperature sensitive period (TSP) (Girondot et al. 2018). The somatic development of turtle embryos is generally established based on morphological changes in 26 stages starting from gastrulation to hatching (Yntema 1968). Gonadal development is one of the important landmarks in organogenesis and can be divided into four phases. The first is the germ cell migration phase, when the germ cells in the posterior germinal crescent at the end of gastrulation migrate through the dorsal mesentery to reach the gonad anlagen (Pieau et al. 1999). A prior study on the mechanism by which the primordial germ cells (PGCs) migrate was evaluated by light microscopy in the turtle (Ewert 1985), in which the PGCs were located in the posterior part of the extra embryonic area and then migrated by ameboid movement, similar to germ cell migration in other vertebrates (Ewert 1985). The second phase is the bipotential gonad phase, when the bipotential genital ridge forms and both sexes develop primitive medullary sex cords (Yntema stages 14 and 15). The third phase is the sex determination phase, when the gonad is differentiated but the sexual trend of the gonadal primordium is still flexible (Yntema stages 16– 19). The fourth is the sex differentiation phase, when the final gonadal morphology develops and the sex is fully determined (testicular or ovarian structures form; Yntema stages 20–26) (Yao et al. 2004). In the slider turtle (Trachemys scripta), the gonad develops from the genital ridge and can be divided into two zones: an inner medulla and outer cortical layers. Primitive sex cords are found in the medullary region of the bipotential gonad, regardless of the environmental temperature. In the developing ovary, these primitive sex cords degenerate during stages 18–20, while in the testis they develop into the seminiferous tubules starting at stage 18 and become very evident by stage 20 (Ramsey and Crews 2009). Interestingly, the cords develop into the testis while the sex is still labile, making this process reversible until about stages 20–21 (Ramsey and Crews 2009). For gonad development, germ cells are initially located in the outer germinal epithelium of the gonad. In the ovary, the germ cells remain in the cortical region, where they proliferate along with differentiating granulosa cells to form follicles. In the developing testis, the germ cells migrate into the medullary region beginning at stage 18, where they are enveloped by the developing seminiferous tubules (Wibbels et al. 1991; Yao et al. 2004). Although 16 species (two families) of freshwater turtles can be found in Thailand (Pipatsawasdikul et al. 2010; Chan-ard et al. 2015; Ihlow et al. 2016), their susceptibility to temperature change is unknown due to a lack of information on the chronology of their gonadal development pattern. The snail-eating turtle, Malayemys macrocephala, is a native freshwater turtle commonly found in rice fields in central Thailand. Previous histological analysis of this species suggested that TSD occurs in males at 26°C and females at 32°C (Pewphong et al. 2016). This study aimed to examine the chronology of gonadal development in M. macrocephala using histological analysis during the germ cell migration, bipotential, sex determination, and sex differentiation phases. Moreover, germ cell dynamics were followed by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) to detect cell proliferation and apoptosis, respectively, in M. macrocephala. MATERIALS AND METHODS Turtle egg collection All egg collection and handing procedures were approved by the Chulalongkorn University Animal Care and Use Committee (Protocol Review Number 1723005), and eggs were collected in accordance with the Wild Animals Reservation and Protection Act B.E. 2546 of Thailand and approved by the Department of Fisheries, Ministry of Agriculture and Cooperatives (Permit Number 17/2559). Turtle eggs were collected from an agricultural area of Bang Ban District, Phra Nakhon Si Ayutthaya Province, central Thailand (UTM Zone 47P 0650143-0663244 and 1586924-1599393), which have large areas of M. macrocephala habitat and nesting sites (Keithmaleesatti 2008). Turtle nests were found by visual searching from late November 2016 to April 2017, and a total of 570 eggs from 95 clutches were located. Only freshly laid eggs without white spot development were collected. These eggs were labeled individually before being transported to a laboratory at Chulalongkorn University, Bangkok, Thailand. Turtle egg incubation Eggs from each clutch were randomly assigned to covered boxes containing moistened vermiculite, which were then divided into two groups to be incubated at either 26°C, the male producing temperature (MPT), or 32°C, the female-producing temperature (FPT). The egg trays were rotated twice a week within the incubator to avoid small temperature-gradient effects. Three eggs per group of the same age, laid on the same day, were randomly selected and dissected on a weekly basis. After dissection, the developmental stage of the turtle page 2 of 11Zoological Studies 59:20 (2020) © 2020 Academia Sinica, Taiwan embryo was determined according to their external morphology. The series of normal developmental stages of the snail-eating turtle M. macrocephala used in this study is comparable to the widely used developmental stages of Chelydra serpentina (Yntema 1968) and Pelodiscus sinensis (Tokita and Kuratani 2001), with a minute difference in that M. macrocephala lacks the early stages (Yntema stages 1–5) (Pewphong 2012). Histology of gonadal development To study the germ cell migration, embryos from MPT and FPT rearings were collected from Yntema stages 10 to 14 and their gonad structure was histologically examined from Yntema stages 14 to 25. Embryos were immersed in neutral buffer formalin fixative (3–6 embryos per stage per temperature) for 24 h and then preserved in 70% (v/v) ethanol. Fixed embryos (Yntema stages 10–19) and dissected gonads (Yntema stages 20–25) were embedded in paraffin, sectioned at 5 µm thickness, and periodic acid Schiff stained before being observed using light microscopy. Gonadal development and sex differentiation in M. macrocephala were evaluated with reference to previous reports on the snapping turtle Trachemys scripta and the Magdalena river turtle, Podocnemis lewyana (Wibbels et al. 1991; Sánchez et al. 2014). Proliferating cell detection (PCNA immunohistochemical assay) Malayemys macrocephala embryos (stages 15, 17, 19) and gonads (stage 25) were processed by the paraffin method and sectioned in a transverse plane at 5 µm thickness and immunohistochemically stained for proliferating cell nuclear antigen (PCNA), a marker for cell division (Wolf and Dittrich 1992). The PCNA is a ring-like protein that provides the DNA polymerase processivity for DNA replication (Kelman 1997). The sections were deparaffinized, hydrated, and incubated in two changes of 3% (v/v) H2O2 for 10 min. After rinsing in distilled water, slides were pretreated with 1% (w/v) zinc sulfate in a 800 W microwave oven at 100% power setting for 15 min to retrieve the antigenic determinants. Sections were washed in distilled water, followed by 0.05% (v/v) Tween-20 in phosphate buffered saline (PBS-T). Nonspecific binding was blocked by adding 3% (v/v) normal horse serum to each slide and incubating at room temperature for 1 h. Slides were washed between each incubation step in PBS-T. Sections were incubated with mouse monoclonal antibody against PCNA (Bioscience International) overnight at 4°C, then washed and incubated with biotinylated horse anti-mouse IgG (Millipore Corporation) for 1 h. After washing, they were incubated for 10 min with 0.03% (w/v) 3,3’-diamionobenzidine-4 HCl (DAB; SigmaAldrich), 3% (v/v) H2O2, and 0.2% (w/v) ammonium nickel sulfate in PBS, then rinsed with distilled water, dehydrated, cleared, and mounted with PermountTM. Apoptosis detection (TUNEL assay) Malayemys macrocephala embryos (stages 15, 17, and 19) and gonads (stage 25) were processed by the paraffin method and sectioned in a transverse plane to 5 µm thick. Apoptotic cells in the gonads were then detected by the TUNEL assay (Gavrieli et al. 1992) using a DNA fragmentation kit (Merck Millipore, catalog number S7107). TUNEL is based on the ability of TdT to label blunt ends of double-stranded DNA breaks independent of a template (Kyrylkova et al. 2012). Each paraffin section of the gonad was deparaffinized, hydrated, and incubated in proteinase K for 15 min at room temperature to permeabilize the specimen, followed by 3% (v/v) H2O2 in methanol for 15 min to block endogenous peroxidase activity. After incubating with equilibration buffer for 10 s, fragmented DNA was labeled by incubating with the TdT enzyme in the TdT reaction mixture at 37°C for 1 h. The reaction was terminated by submerging the sections in 10 mM EDTA solution. The sections were then incubated with blocking buffer for 10 min, and labeled DNA was detected by incubating the sections with peroxidase streptavidin conjugate for 30 min, then washed and developed in 0.03% (w/v) DAB and 3% (v/v) H2O2 in PBS. Slides were then rinsed in distilled water, counterstained with 0.5% (w/v) methyl green, dehydrated, cleared, and mounted with PermountTM. RESULTS Chronology of gonadal development The chronology of gonadal development is summarized in table 1. The embryonic developmental rate, as assessed by morphological stages, was faster when the embryo was incubated at the FPT (32°C) than at the MPT (26°C), as expected. Microscopic examination of the gonad revealed four main stages of gonadal development: germ cell migration (Fig. 1), genital ridge appearance, testicular formation, and ovarian formation (Fig. 2). Early gonad development (Yntema stages 10 to 14) was examined to follow the germ cell migration, and the histological study of PGCs in M. macrocephala was based on a previous report on the loggerhead turtle (Caretta caretta; Fujimoto et al. 1979). The PGCs page 3 of 11Zoological Studies 59:20 (2020) © 2020 Academia Sinica, Taiwan appeared at stage 10 (day 14 and 24 at the FPT and MPT, respectively) outside the genital ridge or a ridge of embryonic mesoblast developing from the mesonephros and giving rise to the gonad on either side of the body (Fig. 1a). They were observed migrating from the yolk sac into the connective tissue of the hindgut at stage 11 (day 17 and 28 at the FPT and MPT, respectively; Fig. 1b). At stage 12 (day 18 and 32 at the FPT and MPT, respectively), the PGCs migrated from the hindgut to the mesonephric duct (Fig. 1c), and then higher in the mesonephric duct at stage 13 (day 19 and 36 at the FPT and MPT, respectively) (Fig. 1d). Similarly, the transverse section of M. macrocephala embryos at stage 14 (day 22 and 40 at the FPT and MPT, respectively) Table 1. Embryonic stage correlated with the age (days) of embryos incubated at the FPT or MPT, and the degree of gonadal differentiation Yntema stage Approximate age (d) Degree of gonadal differentiation 26°C (MPT) 32°C (FPT) 10 24 (n = 3) 14 (n = 3) Germ cell migration 11 28 (n = 3) 17 (n = 3) Germ cell migration 12 32 (n = 5) 18 (n = 3) Germ cell migration 13 36 (n = 5) 19 (n = 3) Germ cell migration 14 40 (n = 5) 22 (n = 4) Germ cell migration/ Genital ridge 15 47 (n = 5) 25 (n = 4) Genital ridge appearance 17 57 (n = 4) 31 (n = 4) Differentiating gonads/ male 19 70 (n = 5) 37 (n = 5) Differentiating gonads/ female 21 85 (n = 3) 47 (n = 3) Differentiating gonads/ male, female 25 115 (n = 4) 92 (n = 4) Differentiated MPT = male producing temperature and FPT = female producing temperature. Fig. 1. Location of the PGCs in M. macrocephala embryos. The red oval line indicates the location of the PGCs at the current stage, while the black oval dashed line indicates their previous location in embryos at stage (a) 10, (b) 11, (c) 12, (d) 13, and (e) 14, plus the (f) area and (g) character of the PGCs, which can be identified by its large size, round nuclei, and pale cytoplasm (red arrow). page 4 of 11Zoological Studies 59:20 (2020) © 2020 Academia Sinica, Taiwan Fig. 2. Gonadal development during Yntema stages 15, 17, 19, and 25 when incubated at the MPT (26°C) or FPT (32°C). Location of the gonad at Yntema stages (a, b) 15 (black arrows), (c) 17 (MPT), (d) 17 (FPT), (e) 19 (MPT), (f) 19 (FPT), (g) 25 (MPT), and (h) 25 (FPT). C = cortical region or outer layer of tissue immediately below the epithelium; F = ovarian follicle; M = medullar region or the inner region of the gonad which is distinguishable from the outer region; SC = medullary sex cord; ST = seminiferous tubule; an asterisk indicates an area with germ cell division; a star indicates an area with cortical development. page 5 of 11Zoological Studies 59:20 (2020) © 2020 Academia Sinica, Taiwan revealed that the PGCs were still in the mesonephric duct and moved to a higher location than in stage 12 or 13 (Fig. 1e). Note that the PGCs of M. macrocephala are large with a round nucleus and pale cytoplasm, making them relatively easy to detect (Fig. 1f and g). The genital ridge was apparent as an indifferent gonad starting from Yntema stage 14 (Fig. 2a and b), and could be divided into two zones: inner medulla and outer cortical layers. The indifferent gonad then developed into testis with medullary sex cords starting at stage 17 (day 31 and 57 at the FPT and MPT, respectively; Fig. 2c) and the level of germ cell division was distinctly higher in the medullary sex cords from stage 19 (day 70 at the MPT; Fig. 2e). In the female turtle, the gonad was identified as an undifferentiated gonad at stage 17 (day 31 at the FPT; Fig. 2d). Then the presumptive sex cords degenerated, followed by cortical development into the ovarian structure starting from stage 19 (day 37 at the FPT; Fig. 2f). Subsequently, testicular (day 115 at the MPT; Fig. 2g) and ovarian (day 92 at the FPT; Fig. 2h) development occurred independently and distinct sex organs became apparent at stage 25. Detection of proliferating cells The dynamics of germ cell proliferation were identified immunohistologically using PCNA staining. There were essentially no positive PCNA reactions in the indifferent gonad from embryos reared at the MPT (stage 15; Fig. 3a), but germ cell proliferation was found in the medullar at stage 17 (Fig. 3b). Afterwards, testicular development was strongly PCNA positive at stages 19 (Fig. 3c) and 25 (Fig. 3d). The cell proliferation (as PCNA positive staining) in embryos reared at the FPT was not detected in the indifferent gonad (stage 15; Fig. 4a) or at stage 17 (Fig. 4b), but it was found in the cortex at stage 19 (Fig. 4c), and weak PCNA signals were identified at stage 25 (Fig. 4d). Detection of apoptotic cells The TUNEL assay revealed no evidence of apoptosis at stages 15 (Fig. 5a) or 17 (Fig. 5b) in the embryos reared at the MPT, but germ cell apoptosis (strong TUNEL staining) was found in the cortex at stages 19 (Fig. 5c) and 25 (Fig. 5d). In embryos reared at the FPT, there was also no evidence of apoptosis at stages 15 (Fig. 6a) or 17 (Fig. 6b), but strong TUNEL signals were found in the medullar sex cord at stage 19 (Fig. 6c) and weak signals were found at stage 25 (Fig. 6d). Strong cell proliferation and apoptosis were evident at stage 25 based on the immunohistochemical detection of proliferating (PCNA positive) and apoptotic (TUNEL positive) cells in the presumptive testis, whereas in the presumptive ovary both cell proliferation and apoptosis were weak at this stage. DISCUSSION An increase in the average global temperature can change both the physical and biological environment and affect the survival of organisms. Organisms that cannot adapt well to the new environment will decline and may become extinct (Walther et al. 2002). In many reptiles, gonadal differentiation is controlled by TSD. In most freshwater turtles, the sexual differentiation of gonads is influenced by the incubation temperature of the egg during the critical TSP of embryonic development (Valenzuela 2004). A previous histological analysis suggested that M. macrocephala undergoes TSD (Pewphong et al. 2016), so this research aimed to examine the chronology of gonadal development at the presumed MPT and FPT. Prior studies have been conducted on the chronology of gonadal development in turtles using light microscopy (Ewert 1985), electron microscopy (Wibbels et al. 1991), and gene expression and synthesis (Ramsey et al. 2007). This study evaluated the germ cell migration of M. macrocephala from the extraembryonic area to the genital ridge (Ewert 1985), since the PGCs in turtle species are initially located in the lateral posterior region before moving to the gut and dorsal mesentery, then the gonad (Bachvarova et al. 2009). This is similar to other vertebrates, in which the PGCs originate outside the embryo and later migrate to the genital ridge (Gilbert and Barresi 2016). Note that the PGCs of reptiles are large, spherical, and PAS positive and have a distinct nucleolus (Raynaud and Pieau 1985), making them relatively easy to study. An understanding of the chronology of gonadal differentiation is essential for designing studies on how temperature affects the development and physiology of turtles, especially endangered ones (Wibbels et al. 1991; Rafferty and Reina 2014). A similar chronology of gonadal differentiation in M. macrocephala was inferred from the freshwater turtle Emys orbicularis, in which testis development was distinguishable at stage 17 and ovary development at stage 19 (Pieau et al. 1998). In this study, the male turtle gonads at Yntema stage 17 incubated at the MPT were PCNA positive (proliferating cells) in the medullary sex cords, and TUNEL positive (apoptotic) germ cells were observed in the cortex, especially at Yntema stage 25. In the female turtle (incubated at the FPT), the presumptive ovary was strongly and weakly PCNA positive in the page 6 of 11Zoological Studies 59:20 (2020) © 2020 Academia Sinica, Taiwan Fig. 3. Immunostaining of proliferating cells in the presumed male gonad of the M. macrocephala embryo at Yntema stages (b) 17, (c) 19, and (d) 25. Black arrows indicate PCNA-positive cells. Fig. 4. Immunostaining of proliferating cells in the presumed female gonad of the M. macrocephala embryo at Yntema stages (a) 15, (b) 17, (c) 19, and (d) 25. Black arrows indicate PCNA-positive cells. page 7 of 11Zoological Studies 59:20 (2020) © 2020 Academia Sinica, Taiwan Fig. 5. Immunostaining of apoptotic cells in the presumed male gonad of the M. macrocephala embryo at Yntema stages (a) 15, (b) 17, (c) 19, and (d) 25. Black arrows indicate PCNA-positive cells. Fig. 6. Immunostaining of apoptotic cells in the presumed female gonad of the M. macrocephala embryo at Yntema stages (a) 15, (b) 17, (c) 19, and (d) 25. Black arrows indicate PCNA-positive cells. page 8 of 11Zoological Studies 59:20 (2020) © 2020 Academia Sinica, Taiwan cortex at Yntema stages 19 and 25, respectively, while strongly and weakly TUNEL positive germ cells were expressed in the medullar sex cord at Yntema stages 19 and 25, respectively. However, in the red-eared slider turtle, the sexual fate of males became fixed prior to that of females (Mork et al. 2014). The faster differentiation of male gonads than female gonads is well exhibited in vertebrates (Ramsey et al. 2007), and the sexual differentiation of gonads is associated with the growth rate (morphological stage) of embryonic development (Deeming and Ferguson 1988). Based on sex determination in vertebrates, it is reasonable to suggest that sex factors control gonadal differentiation (Trukhina et al. 2013). Such sex factors in reptiles likely include sex steroids (Crews 1996), steroidogenic enzymes (e.g., aromatase; Matsumoto and Crews 2012), and even heat shock proteins (Kohno et al. 2010). Using molecular techniques, Matsumoto et al. (2013) found that temperature may influence sex determination in freshwater turtles through epigenetic control of gene expression, including DNA methylation and histone modification (Matsumoto et al. 2016). In further studies, it would be interesting to evaluate the gonadal development of M. macrocephala using both hormonal and molecular approaches like those mentioned in the aforementioned studies, as well as other assays including functional assays (Schroeder et al. 2016) and genome-wide transcriptomic analyses (Liu et al. 2019). CONCLUSIONS The chronology of gonadal development in M. macrocephala can be split into four main gonadal development stages: germ cell migration, genital ridge appearance, testicular formation, and ovarian formation. The PGCs arise outside the genital ridge and from Yntema stages 10 to 14, and they can be observed migrating from the yolk sac into first the connective tissue of the hindgut, then the mesonephric duct, and finally the adjacent genital ridge. The genital ridge was apparent as an indifferent gonad starting from Yntema stage 14, and could be divided into the two zones of inner medulla and outer cortical layers. This gonad started developing into the testis with medullary sex cords at Yntema stage 17. In the female turtle, these sex cords then degenerated, followed by cortical development into an ovarian structure from Yntema stage 19. Subsequently, testicular and ovarian development occurred independently in male and female embryos, respectively, and distinct sex organs were apparent at Yntema stage 25. These results are summarized in figure 7 as a chronological diagram of the developmental stages in embryos of M. macrocephala. In addition, the Fig. 7. Summary diagram of the chronology of developmental stages in embryos of the snail-eating turtle M. macrocephala. page 9 of 11Zoological Studies 59:20 (2020)