Steroidogenic potential of the gonad during sex differentiation in the rice field frog Hoplobatrachus rugulosus (Anura: Dicroglossidae).

Prior studies demonstrated that gonadal differentiation in the rice field frog, Hoplobatrachus rugulosus, was of an undifferentiated type since all individuals had ovaries at complete metamorphosis. However, the steroidogenic potential of the gonad is still unknown. In this study, H. rugulosus were obtained by stimulating fertilization in the laboratory under natural light and temperature conditions. The gonads were collected and their steroidogenic potential was evaluated by determining the expression level of messenger RNA (mRNA) encoding for cytochrome P450 17-hydroxylase/C17-20 lyase (CYP17) and cytochrome P450 aromatase (CYP19) using quantitative real-time RT-PCR and the localization of CYP17 mRNA in tissues by in situ hybridization. The CYP17 mRNA levels in males at 4-11 weeks postmetamorphosis were higher than in female and intersex gonads. This corresponded to their localization in the gonadal tissues, where CYP17 signals were specifically detected in the Leydig cells of the testis at 5-16 weeks postmetamorphosis but was undetectable in all ovary samples. The CYP19 mRNA levels in females at 4-11 weeks postmetamorphosis was higher than in male and intersex gonads, which corresponded with gonadal development, indicating the potential steroidogenic function of the ovary. Based on the present results, the role of CYP17 and CYP19 mRNA in sex differentiation in H. rugulosus may occur after gonadal sex differentiation and the steroidogenic potential of the gonads exhibited a sexual dimorphic pattern. These results provide a crucial basis for further research on the developmental biology in anuran species.

Thermosensitive period for sex determination of the tropical freshwater turtle Malayemys macrocephala.

Many egg-laying reptiles possess temperature-dependent sex determination (TSD) in which outcome of gonadogenesis is determined by incubation temperature during a temperature-sensitive period of development. Prior studies on Malayemys macrocephala showed that incubation temperatures influence gonadal development and suggested that M. macrocephala exhibits TSD. However, information on the temperature-sensitivity period in this species was unknown until the current study. Turtle eggs were collected from rice fields in central Thailand from December 2016 to February 2017. In the laboratory, eggs were incubated at male-biased temperature (26 °C) and shifted to female-biased temperature (32 °C), or vice versa. Single shift experiments were performed systematically during embryonic stages 13-21. After hatching, sex of individual turtles was determined by histological analysis. We found that the sex determination of M. macrocephala is affected by temperature up to stage 16 of embryonic development.

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

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.

Pattern of Gonadal Sex Differentiation in the Rice Field Frog Hoplobatrachus rugulosus (Anura: Dicroglossidae).

Sex differentiation during gonadal development is diversified among anuran amphibian species. In this study, the anuran experimental species Hoplobatrachus rugulosus was examined. The pattern of gonadal sex differentiation was observed by morphological and histological approaches. The gonad was observed morphologically at Gosner stage 33, while distinct testis and ovary were evident from 3-4 weeks after metamorphosis ended. Histological analysis showed that genital ridge formation began at stage 25 and ovarian differentiation began at stage 36. The developing ovary appeared with numerous primary oogonia, which developed into oocytes, while the medulla regressed to form an ovarian cavity. During metamorphosis, only an ovary was observed. Testicular differentiation seemed to begin later, during the first week after metamorphosis, and occurred via an intersex condition. The intersex gonads contained developing testicular tissue with both normal and atretic oocytes. The fully developed testis was first identified at 6 weeks after metamorphosis. Comparing the times of gonadal differentiation and somatic development revealed that the ovary exhibited a basic rate of differentiation while the testis exhibited a retarded one. These results establish that males of this species develop later than do females, and the testis develops through an intersex gonad, as is evident from its seminiferous cord formation, the presence of testis-ova, and atretic oocytes in the tissue. Thus, the pattern of gonadal sex differentiation in H. rugulosus is an undifferentiated type, in which only female gonads are observed during metamorphosis and intersex and male gonads are observed later. These results are crucial for further research on the sexual development of anurans.

Hepatic metallothionein and Glutathione-S-Transferase responses in two populations of rice frogs, Fejervarya limnocharis, naturally exposed to different environmental cadmium levels.

Glutathione-S-Transferase (GST) and metallothionein are important biomarker endpoints in studying the effect of Cd exposure. The purpose of this research was to study the correlation between hepatic GST and metallothionein with hepatic Cd in wild Fejervarya limnocharis exposed to environmental Cd. Results showed that frogs from contaminated sites had significantly higher hepatic metallothionein (3.58 mg/kg wet weight) and GST activity (0.259 µmol/min/mg total protein) than those from the reference site (2.36 mg/kg wet weight and 0.157 µmol/min/mg total protein respectively). There was a significantly positive correlation between hepatic Cd and GST activity (r = 0.802, p = 0.009) but not between hepatic Cd and metallothionein (r = 0.548, p = 0.139). The results concluded that while frogs from the contaminated site had higher GST and metallothionein, only GST showed significant positive correlation with hepatic Cd levels, indicating that hepatic GST activity may be used as a biomarker endpoint.

Reproductive Mode of Fejervarya limnocharis (Anura: Ranidae) Caught from Mae Sot, Thailand Based on Its Gonadosomatic Indices.

Amphibians show a variety of reproductive modes and strategies. The cyclicity and continuity of reproduction can often be predicted from the annual gonadosomatic index trends in a species specific manner. This research aims to document the female and male gonadosomatic index profiles and to ascertain the reproductive cyclicity or continuity of Fejervarya limnocharis (Anura: Ranidae) caught in Mae Sot, Tak Province, Thailand. Frogs were collected monthly between November 2007 and October 2009 in rice fields and their surrounding areas in the study site. For each frog, total weight and ovarian/testicular weight were measured to obtain the female and male gonadosomatic index. The number of female individuals with mature eggs (postvitellogenic eggs) was also counted. The results showed that female frogs had two main surges of increased GSI in March and September of 2008. Alternatively, male frogs showed a more gradual increase and decrease in gonadosomatic index, thus the index remaining high throughout the year. This study concluded that while F. limnocharis in this area is essentially a continuous breeder, it is more optimized for a cyclic reproduction mode with two breeding cycles during the rainy season.

Cadmium accumulation in two populations of rice frogs (Fejervarya limnocharis) naturally exposed to different environmental cadmium levels.

Contaminant accumulation analysis is important in the study of sentinels. This research determined cadmium accumulation and bioconcentration factors of whole organism, liver, kidney, ovary and testis of Fejervarya limnocharis exposed to different environmental cadmium levels. Frogs from contaminated sites had significantly higher hepatic (1.939 mg/kg), renal (7.253 mg/kg) and testicular (1.462 mg/kg) cadmium than those from the reference sites (0.205, 0.783 and 0.379 mg/kg, respectively). Cadmium accumulation was the highest during the late dry and early rainy seasons. If this species is used as a sentinel for cadmium accumulation, the utilization of its whole organism, liver, kidney and testis is appropriate.