Reproductive maturation of the tropical clawed frog: Xenopus tropicalis (2023)

Pesticides are a major cause of reduction in the global population of amphibians. This study investigates the effect of varying concentrations of cyproconazole (1 and 10 mg/L) on Rana nigromaculata during a chronic 90 days exposure period. High levels of cyproconazole (10 mg/L) induced declined body weight, short snout–vent length, slow metamorphic development and abnormal behavioral endpoints in R. nigromaculata tadpoles. Tadpoles exposed to 10 mg/L did not survive beyond 42 days. Abnormal behaviors were observed more frequently with exposure to the highest concentration of cyproconazole. Compared with controls, the concentrations of dismutase (SOD), catalase (CAT) and glutathione (GSH) were significantly increased in tadpoles exposed to 1 mg/L cyproconazole. However, when the concentration of cyproconazole increased to 10 mg/L, concentrations of SOD, GSH and CAT activity began to decline. In addition, thyroid and gonad development were also affected at the gene and hormone level, with varied effects observed with different exposure levels and days. Exposure to cyproconazole at the lower level of 1 mg/L induced damage to histological structures of the thyroid gland. Stereoselective tissue distribution and bioaccumulation of cyproconazole was observed in tadpoles. The ranked order of bioaccumulation was: enantiomer -4 > 3> 2 > 1, with the level of cyproconazole highest in the gut. These findings reflect the toxicity of cyproconazole to R. nigromaculata and further our understanding of the effects of pesticide exposure on global amphibian population declines.

The Larval Amphibian Growth and Development Assay (LAGDA) is an internationally harmonized testing guideline for evaluating effects of chronic chemical exposure in amphibians. In order to evaluate the effects of chronic exposure to an antiandrogenic chemical in an amphibian model, prochloraz was tested using a variation of the LAGDA design. Exposure was initiated with <1d post-fertilization embryos at nominal concentrations of 0, 6.7, 20, 60 and 180 μg/L (0, 18, 53, 159, 478 nM) and continued in flow-through conditions until two months following the median time that controls completed metamorphosis. Growth, developmental rate, circulating thyroid hormone and thyroid gland histopathology were evaluated in a subsample at completion of metamorphosis. There were no effects on growth or development at this stage, but circulating thyroid hormone was elevated in the 20, 60 and 180 μg/L treatments and minimal to mild thyroid follicular cell hypertrophy was observed histologically in the 180 μg/L treatment. Growth, overt toxicity, and reproductive development were evaluated at test termination. There were no effects on growth in either gender, but livers and kidneys exhibited treatment-related pathologies consistent with organ toxicity related to metabolism and presumably impaired excretion of prochloraz metabolites. Histological assessments of female ovaries resulted in minimal pathologies only in the 180 μg/L treatment while male testes exhibited numerous treatment-related pathologies that are consistent with previously reported antiandrogenic effects of prochloraz in other species. The most severe testis pathologies occurred in the 180 μg/L treatment; however, incidences of treatment-related pathologies occurred in all prochloraz treatments. Müllerian duct regression in males was inhibited by prochloraz exposure while Müllerian duct maturation in females was accelerated, characteristic of a feminizing effect. Gene expression levels of potential biomarkers of testis function were also measured. Relative abundance of cyp17a1 transcripts was generally unaffected by prochloraz exposure whereas the Insl3 orthologue, rflcii, was elevated by 3 and >5-fold in the 60 and 180 μg/L treatments, respectively, indicating impaired Leydig cell maturation and testosterone signaling. Overall, prochloraz exposure caused effects characteristic of an antiandrogenic mode of action, which is consistent with previously reported results in other species and supports the utility of the LAGDA design for chemical testing.

Perchlorate (ClO₄⁻) contamination has been reported in ground and surface waters across North America. However, few studies have examined the effects of prolonged exposure to this thyroid hormone disrupting chemical, particularly at environmentally relevant concentrations in lower vertebrates, such as amphibians. The aim of this study was to examine the effects of a yearlong chronic exposure to ClO₄⁻ in adult male and female Western clawed frogs (Silurana tropicalis). Frogs were spawned and raised from fertilized embryo until sexual maturity in potassium perchlorate (KClO₄)-treated water at different concentrations (0, 20, 53, and 107 μg/L). Developmental and reproductive indices – including adult morphology, androgen plasma levels, gonadal thyroid hormone- and sex steroid-related transcript levels, and sperm motility – were evaluated in male and female adult frogs. Female growth (e.g., body mass, snout-vent length, and hind limb length) was significantly reduced following chronic exposure to environmentally relevant concentrations of KClO₄ resulting in females with morphometric indices similar to those of control males – indicating potential sex-specific sensitivities to KClO₄. Changes to reproductive indices (i.e., plasma androgen levels, gonadal thyroid hormone- and sex steroid-related transcript levels, and sperm motility) were also observed in both sexes and suggest that KClO₄ exposure may also have indirect secondary effects on the reproductive axes in male and female adult frogs. These effects were observed at concentrations at or below those reported in surface waters contaminated with ClO₄⁻ suggesting that this contaminant may have developmental and reproductive effects post-metamorphosis in natural amphibian populations.

Sperm count is an important quality assessment tool in farming programs and stock improvement in crustaceans. However, this procedure is still little used in caridean shrimps and standardization of appropriate techniques for the group is lacking. In this study, we propose a simple protocol adapted to determine sperm count in carideans using as model the Amazon River prawn Macrobrachium amazonicum. Males of dominant morphotypes (GC1 and GC2) of this species with amphidromous and hololimnetic life cycles were collected and carefully dissected. The ejaculatory duct was removed from the vas deferens and dissociated in a solution of distilled water (9μl) and methylene blue (1μl). Subsequently, 1μl of this new solution was added to distilled water (9μl), and then 1μl was pipetted and quantified in a Neubauer chamber. The feasibility of this technique was also evaluated in animals preserved (5–480days) in 70% ethanol from collections and the structural morphology of spermatozoa (spz) was examined. Despite morphometric differences observed between different types of males, the mean sperm count was similar for the species. In amphidromous animals, 60,258spz/μl were registered for GC1 and 65,308spz/μl for GC2, while in hololimnetic prawns, 48,950spz/μl were registered in GC1 and 53,850spz/μl in GC2. The variation in sperm count among animals preserved for different periods of time was small and very similar to those of fresh animals. Also, no microscopic changes in the structures of spz were observed. This technique can be applied to obtain a spermiogram in fresh as well as preserved animals, being especially important in studies with animals examined in population studies and deposited in collections or laboratories. This protocol can be used as a general model for spermiogram in caridean shrimps due to the great similarity of male reproductive systems within the group.

Sexual maturity involves the differentiation of the reproductive system, the maturation of germ cells, and the development of secondary sexual characteristics. Even though this topic has received much attention, little is known about the sequence of events that encompass reproductive maturation in anurans and what it could reveal about the developmental basis of life cycle evolution. The discovery of froglets of Pseudis minuta with incipient vocal sacs calling in breeding pools alongside several larger adult specimens with fully developed vocal sacs raised the question of the timing of sexual maturity in this species. Here we describe the sequence and timing of differentiation, development and maturation of the vocal sac apparatus and the testes in P. minuta (Anura, Hylidae), in order to establish a timeline of events leading to sexual maturity. Differentiation of the vocal sac apparatus begins at the final metamorphic stages, earlier than reported for other species, and the vocal sac acquires its final shape during the early postmetamorphic period. These modifications occur after gonadal differentiation, which begins early during the larval period and proceeds with a highly accelerated rate of development (e.g., secondary spermatids appear well before metamorphic climax), a situation reported previously for other anuran species only in the genus Pseudis. These results, together with a skeletochronological analysis showing that some calling specimens presented no lines of arrested growth, indicate acceleration in the timing of sexual maturity in Pseudis, and raise questions about the interdependence/decoupling during the development of the different components involved in reaching the adult stage.

Amphibian gonadal differentiation involves the action of sex steroids. Recent research indicates that the anti-Müllerian hormone (AMH) is involved in testicular development in some lower vertebrate species. For amphibians there is a lack of data on ontogenetic expression of the AMH receptor AMHR2/amhr2 and of progesterone receptors (PGRS/pgrs). Here we expand the knowledge on amphibian sex differentiation by characterizing ontogenetic mRNA levels of amh, amhr2, intracellular and membrane pgrs (ipgr and mpgr beta) and cytochrome P450 19a1 (cyp19a1) (ovarian marker) in the urogenital complex of the model species Xenopus (Silurana) tropicalis. Furthermore, we characterized the ontogenetic development of the Müllerian ducts (precursors of the female reproductive tract) histologically. The developmental period investigated spanned from beginning of gonadal differentiation, Nieuwkoop and Faber (NF) stage 51, to 4weeks post-metamorphosis. The Müllerian ducts were first observed at NF 64 in both sexes. Male-enhanced amh mRNA levels from NF 53/54 to 6days post-metamorphosis and female-enhanced cyp19a1 levels from NF 53 to 4weeks post-metamorphosis were noted. The sexually dimorphic mRNA level profile was more distinct for amh than for cyp19a1. The pgrs mRNA levels increased over the studied period and showed no sex differences. At later developmental stages, the amhr2 mRNA level was increased in putative females compared with males. Our findings suggest that AMH has a role in gonadal differentiation in X. tropicalis. We propose relative gonadal amh mRNA level as a testicular marker during early gonadal development in amphibians.

General and Comparative Endocrinology, Volume 220, 2015, pp. 70-77

In amphibians, although genetic factors are involved in sex determination, gonadal sex differentiation can be modified by exogenous steroid hormones suggesting a possible role of sex steroids in regulating the process. We studied the effect of testosterone propionate (TP) and estradiol-17β (E₂) on gonadal differentiation and sex ratio at metamorphosis in the Indian skipper frog, Euphlyctis cyanophlyctis with undifferentiated type of gonadal differentiation. A series of experiments were carried out to determine the optimum dose and sensitive stages for gonadal sex reversal. Our results clearly indicate the importance of sex hormones in controlling gonadal differentiation of E. cyanophlyctis. Treatment of tadpoles with 10, 20, 40, and 80μg/L TP throughout larval period resulted in the development of 100% males at metamorphosis at all concentrations. Similarly, treatment of tadpoles with 40μg/L TP during ovarian and testicular differentiation resulted in the development of 90% males, 10% intersexes and 100% males respectively. Treatment of tadpoles with 10, 20, 40, and 80μg/L E₂ throughout larval period likewise produced 100% females at all concentrations. Furthermore, exposure to 40μg/L E₂ during ovarian and testicular differentiation produced 95% females, 5% intersexes and 91% females, 9% intersexes respectively. Both TP and E₂ were also effective in advancing the stages of gonadal development. Present study shows the effectiveness of both T and E₂ in inducing complete sex reversal in E. cyanophlyctis. Generally, exposure to E₂ increased the larval period resulting in significantly larger females than control group while the larval period of control and TP treated groups was comparable.

Zoology, Volume 122, 2017, pp. 63-79

Spermatogenesis in frogs was for the first time divided into two phases: prespermatogenesis, when gonocytes proliferate in developing tadpole testes, and active spermatogenesis when spermatogonial stem cells (i.e. descendants of gonocytes), either self-renew or enter into meiotic cycles within cysts formed by Sertoli cells. We argue that amphibian larval gonocytes are homologues to mammalian gonocytes, whereas spermatogonial stem cells (SSCs) in adult frogs are homologous to mammalian single spermatogonia (A_s). Gonocytes constitute sex cords, i.e. the precursors of seminiferous tubules; they are bigger than SSCs and differ in morphology and ultrastructure. The nuclear envelope in gonocytes formed deep finger-like invaginations absent in SSCs. All stages of male germ cells contained lipid droplets, which were surrounded by glycogen in SSCs, but not in gonocytes. Mitochondria in gonocytes had enlarged edges of cristae, and in SSCs also lamellar mitochondria appeared. Minimal duration of prespermatogenesis was 46days after gonadal sex differentiation, but usually it lasted longer. SSCs give rise to secondary spermatogonia (equal to mammalian A, In, and B). Their lowest number inside a cyst was eight and this indicated the minimal number of cell cycles (three) of secondary spermatogonia necessary to enter meiosis. We sorted them according to the number of cell cycles (from 8 to 256 cells). This number is similar to that recorded for mammals as the result of a single A_s proliferation. The number of secondary spermatogonia correlates with the volume of a cyst. The general conclusion is that spermatogenesis in amphibians and mammals follows basically the same scheme.

General and Comparative Endocrinology, Volume 233, 2016, pp. 63-72

Knowledge about sensitivities and responses of amphibian larvae to sex steroids and the chemicals alike is the first step towards understanding and assessing the effect of diverse chemicals that interfere with gonadal development and other endocrine functions. Herein, we used Microhyla ornata to determine the role of sex steroids on its gonad differentiation and sex ratio. Our results show that the exposure to increasing concentrations of estradiol-17β throughout larval development did not affect gonad differentiation resulting in 1:1 sex ratio at metamorphosis. But, females emerging from estradiol-17β treatment had larger ovaries with larger sized follicles. Further, testes of some males contained lumens, the number of which was dose dependent. Similarly, exposure to testosterone propionate had negligible effects on gonad differentiation. However, the mean diameter of the largest follicles was smaller in treated ovaries. Treatment of tadpoles with tamoxifen had no effect on gonad differentiation and ovary development while testicular development was accelerated at the highest concentration. Similarly, treatment of tadpoles with cyproterone acetate had little effect on gonad differentiation as well as development, hence the sex ratios at the end of metamorphosis. Further, in tadpoles exposed to increasing concentrations of formestane, gonad differentiation was normal resulting in 1:1 sex ratio. Thus, in M. ornata, both estradiol and testosterone are essential for the development of ovaries and testes respectively but, they are not critical to gonadal differentiation. Hence, the effects of sex steroids and other endocrine disrupting chemicals could be species-specific; different species may have differential sensitivities to such chemicals.

General and Comparative Endocrinology, Volume 203, 2014, pp. 232-240

The objectives of this study were to examine the effects of potassium perchlorate (KClO₄) treatment on androgen- and thyroid hormone (TH)-related transcript levels during gonadogenesis in the frog Silurana tropicalis. Androgen- and TH-related gene expression was examined in gonad–mesonephros complex (GMC) and liver tissues at stage NF 56 and stage NF 60. These stages of development coincide with the period of sexual differentiation. Real-time RT-PCR analyses revealed that androgen- and TH-related transcript levels in the GMC and liver of stage NF 56 and NF 60 frogs are responsive to KClO₄ exposure during prometamorphosis. An increase in srd5α2 mRNA levels in hepatic tissues of KClO₄-treated NF 56 tadpoles suggests an important role for hepatic tissues in androgen metabolism. Gene transcript differences highlight possible stage- and tissue-specific sensitivities to KClO₄. A greater number of TH- and androgen-related transcriptional changes were discerned in the hepatic tissues compared to the gonads, and overall fewer transcriptional changes were observed in stage NF 60 tadpoles compared to stage NF 56 larvae. Perchlorate suppressed somatic and hind-limb development during the 96-d exposure period. Treatment with KClO₄ had no significant effect on sex ratios, however a notable reduction in the percentage of males (33.3% M: 66.7% F) in the highest KClO₄ concentration (107μg/L) was observed. Overall, these findings suggest that KClO₄ has secondary androgenic disrupting properties in addition to its known primary thyroid hormone-disrupting role.

Chemosphere, Volume 112, 2014, pp. 348-354

An immunoassay for leopard frog (Rana pipiens) vitellogenin was developed for studying endocrine disruption. Male frogs were injected with estradiol-17β to stimulate vitellogenin for purification. SDS–PAGE revealed high amounts of a 170–180kDa protein, which was confirmed to be vitellogenin by Western blotting. Vitellogenin was purified by DEAE chromatography and used to generate a polyclonal antibody. A competitive ELISA was developed for leopard frog vitellogenin with a detection limit of 6.0ngmL⁻¹ and a working range of 20–1000ngmL⁻¹. The intra-assay coefficient of variation averaged 5.47% for control sera and 9.71% for estrogen-treated sera. The inter-assay coefficient of variation averaged 8.21% for control sera and 9.93% for estrogen-treated sera. Recovery of purified vitellogenin averaged 95.2%. Vitellogenin was measured in male frogs immersed in the estrogenic compound diethylstilbestrol (DES) for various times and doses. Serum vitellogenin was detected within five days after immersion in 1.0mgL⁻¹ DES and levels continued to increase through 20d. In a 20-day dose–response experiment, serum vitellogenin was detected in frogs immersed in 0.01mgL⁻¹ DES and vitellogenin concentration increased with dose. Immersion of frogs in one of several xenobiotic estrogens (nonylphenol, octylphenol, bisphenol-A) for 20d did not increase vitellogenin for any treatment, suggesting that this frog may be less sensitive than fish to endocrine disruptors. Vitellogenin induction in R. pipiens may be a useful amphibian model system for field studies of endocrine disruption, due to its broad geographic range.

General and Comparative Endocrinology, Volume 203, 2014, pp. 137-145

Investigations of endocrine disrupting chemicals found in aquatic ecosystems with estrogenic and androgenic modes of action have increased over the past two decades due to a surge of evidence of adverse effects in wildlife. Chemicals that disrupt androgen signalling and steroidogenesis can result in an imbalanced conversion of testosterone (T) into 17β-estradiol (E2) and other androgens such as 5α-dihydrotestosterone (5α-DHT). Therefore, a better understanding of how chemicals perturb these pathways is warranted. In this study, the brain, liver, and testes of Silurana tropicalis were exposed ex vivo to the human drug finasteride, a potent steroid 5α-reductase inhibitor and a model compound to study the inhibition of the conversion of T into 5α-DHT. These experiments were conducted (1) to determine organ specific changes in sex steroid production after treatment, and (2) to elucidate the transcriptomic response to finasteride in testicular tissue. Enzyme-linked immunosorbent assays were used to measure hormone levels in media following finasteride incubation for 6h. Finasteride significantly increased T levels in the media of liver and testis tissue, but did not induce any changes in E2 and 5α-DHT production. Gene expression analysis was performed in frog testes and data revealed that finasteride treatment significantly altered 1,434 gene probes. Gene networks associated with male reproduction such as meiosis, hormone biosynthesis, sperm entry, gonadotropin releasing hormone were affected by finasteride exposure as well as other pathways such as oxysterol synthesis, apoptosis, and epigenetic regulation. For example, this study suggests that the mode of action by which finasteride induces cellular damage in testicular tissue as reported by others, is via oxidative stress in testes. This data also suggests that 5-reductase inhibition disrupts the expression of genes related to reproduction. It is proposed that androgen–disrupting chemicals may mediate their action via 5-reductases and that the effects of environmental pollutants are not limited to the androgen receptor signalling.