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.

Testing of the estrogenic activity and toxicity of Stephania venosa herb in ovariectomized rats.

Stephania venosa Spreng is a traditional herb which has been used for cancer treatment as well as an aphrodisiac. The scientific literature strongly supports its in vitro antiproliferative effects on cancer cell lines and has suggested developing this plant as a potential anticancer drug. However, the in vivo steroidogenic activity and toxicity of this plant have never been tested. We analyzed the levels of five key isoflavones in the plant extract by quantitative HPLC and then evaluated the in vivo estrogenic activity and toxicity in ovariectomized rats, in comparison with the phytoestrogen-rich plant, Pueraria mirifica. Twenty rats were first ovariectomized, and then seven days later divided into four groups and gavaged daily with 0, 10 and 100 mg/kg body weight/day of S. venosa, or 100 mg/kg body weight/day of P. mirifica for 28 days. A trace amount of puerarin, daidzin and daidzein with a subtle amount of genistein and genistin were isolated from the S. venosa tuber extract. S. venosa tuber powder, at both doses, did not exhibit any detectable estrogenic activity in ovariectomized rats, as assessed by the vaginal cytology and uterotropic assays, whilst P. mirifica induced a remarkable vaginal and uterine proliferation. S. venosa induced a toxicological effect on the hematological values and histopathological appearance of metabolic organs. Taken together, these results suggest that S. venosa has no discernable estrogenic activity but that it is toxic, at least to ovariectomized rats. Thus, the use of this plant for anticancer treatment needs to be reassessed or used with caution.