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.

Pesticide use in Thailand: Current situation, health risks, and gaps in research and policy.

Agriculture in Thailand, which employs over 30 percent of the workforce and contributes significantly to the country's gross domestic product, is a key sector of its economy. Import and use of pesticides has increased over the past decade due to Thailand's major role as a leading exporter of food and agricultural products. The widespread and poorly regulated use of pesticides presents a potential risk to the health of farmers, farm families, the general population including children and the environment. This article is a result of the Southeast Asia GEOHealth Network Meeting of February 2019. It summarizes the current situation on pesticide use and regulation in Thailand and reports research findings on the potential health and environmental impacts of pesticide use, as well as highlighting gaps in research that could play an important and influential role in future policy initiatives on pesticides. Although Thailand has made remarkable progress in improving agricultural health and safety and similarly strong research and policy programs are being developed in other countries in the region, there are still significant gaps in research and policy that need to be filled.

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.

Effect of cadmium on gonadal development in freshwater turtle (Trachemys scripta, Chrysemys picta) embryos.

Prior studies on painted turtle (Chrysemys picta) sub-populations near the Massachusetts Military Reservation (MMR), a Superfund site on Cape Cod, Massachusetts, USA, suggest several reproductive deficits which may be related to xenobiotics. Several heavy metals, including cadmium, have been detected in Cape Cod surface water and sediments. The present study was carried out to investigate the effect of an environmentally relevant dose of cadmium on gonadal development during the end of the germ cell migration phase and post-natal gonadal maturation in freshwater turtles. Comparison of cadmium concentration in eggs of C. picta from Cape Cod showed that eggs from the impacted site animals had significantly higher cadmium in yolk than eggs from the reference site animals (7.23 +/- 1.95 ng/g vs. 1.31 +/- 0.50 ng/g). Gonadal structure and the number of proliferating germ cells of neonates derived from eggs of adult females from these sites showed no marked difference between sites. However, apoptosis of oocytes was significantly increased in neonate C. picta from the impacted pond compared to the reference pond. The effect of an administered environmentally relevant dose of cadmium on germ cell number and oocyte apoptosis was subsequently assessed in lab-reared Trachemys scripta, a closely related freshwater turtle species. Assessment of isotopic cadmium transmission showed that 6.29% of cadmium applied to the eggshell was transmitted through the eggshell to the yolk. The results showed that the total number of germ cells in cadmium-treated (1 microg/g) embryos was less than half that found in control embryos. The reduced germ cell number in Cd-treated embryos suggests that cadmium may reduce proliferation and/or delay migration of germ cells to the genital ridge. The effects of cadmium on turtle gonadal development were found to extend into 3 months post hatch. Proliferation of oocytes was not influenced by exposure to cadmium in ovo. In contrast, apoptosis of oocytes was significantly increased in cadmium exposed T. scripta. Since a lesion at the gonial stages will lead eventually to the depletion of more mature germ cells, the results for neonate turtles suggest that an environmental impact due to a xenobiotic mixture may enhance the rate of apoptosis, thus resulting in the reduction in follicle number seen in adult turtles from the impacted site. Overall, the data provide evidence that environmentally relevant doses of cadmium may affect gonadal developmental processes of freshwater turtles during embryonic and post-natal stages that may result in disruption of reproductive processes later in life.

Stimulating effects of quercetin on sperm quality and reproductive organs in adult male rats.

AIM: To investigate effects of quercetin on weight and histology of testis and accessory sex organs and on sperm quality in adult male rats. METHODS: Male Sprague-Dawley rats were injected s.c. with quercetin at the dose of 0, 30, 90, or 270 mg/kg body weight/day (hereafter abbreviated Q0, Q30, Q90 and Q270, respectively), and each dose was administered for treatment durations of 3, 7 and 14 days. RESULTS: From our study, it was found that the effects of quercetin on reproductive organs and sperm quality depended on the dose and duration of treatment. After Q270 treatment for 14 days, the weights of testes, epididymis and vas deferens were significantly increased, whereas the weights of seminal vesicle and prostate gland were significantly decreased, compared with those of Q0. The histological alteration of those organs was observed after Q270 treatment for 7 days as well as 14 days. The sperm motility, viability and concentration were significantly increased after Q90 and Q270 injections after both of 7 and 14 days. Changes in sperm quality were earlier and greater than those in sex organ histology and weight, respectively. CONCLUSION: Overall results indicate that quercetin might indirectly affect sperm quality through the stimulation of the sex organs, both at the cellular and organ levels, depending on the dose and the duration of treatment. Therefore, the use of quercetin as an alternative drug for treatment of male infertility should be considered.

Reproductive deficits in male freshwater turtle Chrysemys picta from Cape Cod, Massachusetts.

Contaminated groundwater plumes have formed on the Massachusetts Military Reservation (MMR), a Superfund site on Cape Cod, Massachusetts, as a result of chemical waste disposal. The plumes are of concern to the local people who rely on groundwater as a drinking water source. We used the freshwater turtle as a sentinel species to monitor the reproductive effects of exposure and, by inference, the potential for impact on human health. Our observations of male Chrysemys picta field-trapped from Moody Pond (an impacted site) and Washburn Pond (a reference site) on Cape Cod extended and supported prior observations of reproductive deficits. Morphometric comparison of precloacal length (PCL), which is a sexually dimorphic trait in the turtle, showed that Moody Pond males had a significantly longer PCL than Washburn Pond males. Moody Pond turtles showed reduced testicular weight, which was associated with significantly smaller seminiferous tubule diameter. Epididymal sperm counts were also markedly reduced in Moody Pond animals compared to Washburn Pond animals. Testicular histology and gonial proliferation, as determined by PCNA, were similar in both male populations, while the Moody Pond males had significantly higher germ cell apoptosis than the animals in Washburn Pond. These results suggest that a low-level mixture of xenobiotic contaminants impairs the reproductive functions of turtles exposed to the impacted site but not to the reference site environment.

Gonadotropin and estrogen responses in freshwater turtle (Chrysemys picta) from Cape Cod, Massachusetts.

As a result of chemical waste disposal on the Massachusetts Military Reservation, a Superfund site on Cape Cod, MA, contaminated groundwater plumes have formed. These plumes are of concern due to the widespread use of groundwater wells as a drinking water source by the local population. Prior observations on a sentinel species Chrysemys picta field-trapped from ponds on Cape Cod suggested deficits in reproductive processes including lower levels of vitellogenin, estradiol-17beta, oviduct weights, and oocyte numbers in females and lower testicular weight and sperm count in males. Possible loci in the hypothalamic-pituitary-gonadal-liver axis at which xenobiotics may act were determined in turtles trapped from Moody Pond (a test site) and Washburn Pond (a reference site). Specifically, gonadotropin and estrogen responses were assessed using plasma steroids and vitellogenin as markers. Basal vitellogenin levels were significantly lower in Moody Pond females; however, vitellogenin responses to estradiol-17beta were the same in both groups, indicating a normal hepatic response to estrogen. In contrast, estradiol-17beta secretion was not stimulated by gonadotropin in Moody Pond females, compared to Washburn animals. Basal plasma testosterone and the response to gonadotropin in males were similar, although steroid levels in Moody Pond animals were slower to return to baseline after gonadotropin injection. The results suggest that a low-level mixture of xenobiotic contaminants may interfere with the steroid metabolic pathways in turtles exposed to the test site, but not the reference site, environment.