Phenotypic Sex Research Articles

Function of Foxl2 and Dmrt1 proteins during gonadal differentiation in the olive flounder Paralichthys olivaceus

Study on fish sex differentiation is important both from academic and practical aspects. Foxl2 and Dmrt1 are important transcription factors that should be involved in fish gonadal differentiation, but there is still no direct evidence to clarify their protein functions. Olive flounder Paralichthys olivaceus, an important mariculture fish in China, Japan, and Korea, shows sex-dimorphic growth. In this study, the Foxl2 and Dmrt1 proteins were detected in granulosa cells of the ovary and Sertoli cells of the testis, respectively, showing significant sex-dimorphic expression patterns. Then, bioactive high-purity Foxl2 and Dmrt1 recombinant proteins were obtained in vitro. Furthermore, effects of the recombinant Foxl2 and Dmrt1 during gonadal differentiation period were evaluated by intraperitoneal injection in juvenile fish. Compared with the control group, the male rate in the Dmrt1 group increased from 0 % to 82 %, showing for the first time in fish that the recombinant Dmrt1 could alter the sex phenotype. In addition, transcription levels of cyp19a and its transcription factors also changed after the recombinant Foxl2 and Dmrt1 injection. These findings reveal that Foxl2 and Dmrt1 are vital regulators for fish gonadal differentiation by regulating cyp19a expression, and also provide a new approach for sex control in fish aquaculture.

X chromosome agents of sexual differentiation.

Understanding sex differences in physiology and disease requires the identification of the molecular agents that cause phenotypic sex differences. Two groups of such agents are genes located on the sex chromosomes, and gonadal hormones. The former have coherent linkage to chromosomes that form differently in the two sexes under the influence of genomic forces that are not related to reproductive function, whereas the latter have a direct or indirect relationship to reproduction. Evidence published in the past 5 years supports the identification of several agents of sexual differentiation encoded by the X chromosome in mice, including Kdm5c, Kdm6a, Ogt and Xist. These X chromosome agents have wide pleiotropic effects, potentially influencing sex differences in many different tissues, a characteristic shared with the gonadal hormones. The identification of X chromosome agents of sexual differentiation will facilitate understanding of complex intersecting gene pathways underlying sex differences in disease.

Sex reversal explains some, but not all, climate-mediated sex ratio variation within a viviparous reptile.

Evolutionary transitions in sex-determining systems have occurred frequently yet understanding how they occur remains a major challenge. In reptiles, transitions from genetic to temperature-dependent sex determination can occur if the gene products that determine sex evolve thermal sensitivity, resulting in sex-reversed individuals. However, evidence of sex reversal is limited to oviparous reptiles. Here we used thermal experiments to test whether sex reversal is responsible for differences in sex determination in a viviparous reptile, Carinascincus ocellatus, a species with XY sex chromosomes and population-specific sex ratio response to temperature. We show that sex reversal is occurring and that its frequency is related to temperature. Sex reversal was unidirectional (phenotypic males with XX genotype) and observed in both high- and low-elevation populations. We propose that XX-biased genotypic sex ratios could produce either male- or female-biased phenotypic sex ratios as observed in low-elevation C. ocellatus under variable rates of XX sex reversal. We discuss reasons why sex reversal may not influence sex ratios at high elevation. Our results suggest that the mechanism responsible for evolutionary transitions from genotypic to temperature-dependent sex determination is more complex than can be explained by a single process such as sex reversal.

Host-dependent impairment of parasite development and reproduction in the acanthocephalan model

BackgroundA central question in parasitology is why parasites mature and reproduce in some host species but not in others. Yet, a better understanding of the inability of parasites to complete their life cycles in less suitable hosts may hold clues for their control. To shed light on the molecular basis of parasite (non-)maturation, we analyzed transcriptomes of thorny-headed worms (Acanthocephala: Pomphorhynchus laevis), and compared developmentally arrested worms excised from European eel (Anguilla anguilla) to developmentally unrestricted worms from barbel (Barbus barbus).ResultsBased on 20 RNA-Seq datasets, we demonstrate that transcriptomic profiles are more similar between P. laevis males and females from eel than between their counterparts from barbel. Impairment of sexual phenotype development was reflected in gene ontology enrichment analyses of genes having differential transcript abundances. Genes having reproduction- and energy-related annotations were found to be affected by parasitizing either eel or barbel. According to this, the molecular machinery of male and female acanthocephalans from the eel is less tailored to reproduction and more to coping with the less suitable environment provided by this host. The pattern was reversed in their counterparts from the definitive host, barbel.ConclusionsComparative analysis of transcriptomes of developmentally arrested and reproducing parasites elucidates the challenges parasites encounter in hosts which are unsuitable for maturation and reproduction. By studying a gonochoric species, we were also able to highlight sex-specific traits. In fact, transcriptomic evidence for energy shortage in female acanthocephalans associates with their larger body size. Thus, energy metabolism and glycolysis should be promising targets for the treatment of acanthocephaliasis. Although inherently enabling a higher resolution in heterosexuals, the comparison of parasites from definitive hosts and less suitable hosts, in which the parasites merely survive, should be applicable to hermaphroditic helminths. This may open new perspectives in the control of other helminth pathogens of humans and livestock.

Whole genome sequencing identifies a missense polymorphism in PADI6 associated with testicular/ovotesticular XX disorder of sex development in dogs

Disorders of sex development (DSDs) are congenital malformations defined as discrepancies between sex chromosomes and phenotypical sex. Testicular or ovotesticular XX DSDs are frequently observed in female dogs, while monogenic XY DSDs are less frequent. Here, we applied whole genome sequencing (WGS) to search for causative mutations in XX DSD females in French Bulldogs (FB) and American Staffordshire Terries (AST) and in XY DSD Yorkshire Terries (YT). The WGS results were validated by Sanger sequencing and ddPCR. It was shown that a missense SNP of the PADI6 gene, is significantly associated with the XX DSD (SRY-negative) phenotype in AST (P = 0.0051) and FB (P = 0.0306). On the contrary, we did not find any associated variant with XY DSD in YTs. Our study suggests that the genetic background of the XX DSD may be more complex and breed-specific.

Sex determination mechanisms and sex control approaches in aquaculture animals.

Aquaculture is one of the most efficient modes of animal protein production and plays an important role in global food security. Aquaculture animals exhibit extraordinarily diverse sexual phenotypes and underlying mechanisms, providing an ideal system to perform sex determination research, one of the important areas in life science. Moreover, sex is also one of the most valuable traits because sexual dimorphism in growth, size, and other economic characteristics commonly exist in aquaculture animals. Here, we synthesize current knowledge of sex determination mechanisms, sex chromosome evolution, reproduction strategies, and sexual dimorphism, and also review several approaches for sex control in aquaculture animals, including artificial gynogenesis, application of sex-specific or sex chromosome-linked markers, artificial sex reversal, as well as gene editing. We anticipate that better understanding of sex determination mechanisms and innovation of sex control approaches will facilitate sustainable development of aquaculture.

Screening and characterization of sex-linked DNA markers in Mozambique tilapia (Oreochromis mossambicus)

The Mozambique tilapia (Oreochromis mossambicus) is an important aquaculture fish. It shows sexual dimorphism in growth, with males growing much faster than females. Thus, production of all-male fingerlings of Mozambique tilapia would improve aquaculture efficiency. However, the lack of accurate sex-specific markers has hampered the mono-sex breeding and the study of the molecular mechanisms of sex determination in this species. Recently, we performed genome sequencing and re-sequencing of female and male Mozambique tilapia and identified the sex chromosome and sex determining region. In the present study, two sex-linked SNP markers were identified and validated. Male specific contigs were assembled from the unmapped reads after aligning male re-sequencing data against the published XX genome. Based on these male-specific contigs, three sex-linked DNA markers, Marker-1, Marker-2 and Marker-3, were developed and correctly identified the genetic sex of 16 fish. The universality of these three male-specific markers was then tested in a panel of 144 individuals, which showed a discrepancy of 5.5% (8/144) between genotypic sex and phenotypic sex. In conclusion, we developed three sex-linked markers to identify different sex genotypes in Mozambique tilapia. This study lays a foundation for the establishment of MAS-GMT (marker-assisted selection of genetically male tilapias) technique and sex-controlled breeding of Mozambique tilapia, as well as for the identification of the sex determining gene.

Asymmetrical sex reversal: Does the type of heterogamety predict propensity for sex reversal?

Sex reversal, a mismatch between phenotypic and genetic sex, can be induced by chemical and thermal insults in ectotherms. Therefore, climate change and environmental pollution may increase sex-reversal frequency in wild populations, with wide-ranging implications for sex ratios, population dynamics, and the evolution of sex determination. We propose that reconsidering the half-century old theory "Witschi's rule" should facilitate understanding the differences between species in sex-reversal propensity and thereby predicting their vulnerability to anthropogenic environmental change. The idea is that sex reversal should be asymmetrical: more likely to occur in the homogametic sex, assuming that sex-reversed heterogametic individuals would produce new genotypes with reduced fitness. A review of the existing evidence shows that while sex reversal can be induced in both homogametic and heterogametic individuals, the latter seem to require stronger stimuli in several cases. We provide guidelines for future studies on sex reversal to facilitate data comparability and reliability.

Possible stochastic sex determination in Bursaphelenchus nematodes

Sex determination mechanisms evolve surprisingly rapidly, yet little is known in the large nematode phylum other than for Caenorhabditis elegans, which relies on chromosomal XX-XO sex determination and a dosage compensation mechanism. Here we analyze by sex-specific genome sequencing and genetic analysis sex determination in two fungal feeding/plant-parasitic Bursaphelenchus nematodes and find that their sex differentiation is more likely triggered by random, epigenetic regulation than by more well-known mechanisms of chromosomal or environmental sex determination. There is no detectable difference in male and female chromosomes, nor any linkage to sexual phenotype. Moreover, the protein sets of these nematodes lack genes involved in X chromosome dosage counting or compensation. By contrast, our genetic screen for sex differentiation mutants identifies a Bursaphelenchus ortholog of tra-1, the major output of the C. elegans sex determination cascade. Nematode sex determination pathways might have evolved by “bottom-up” accretion from the most downstream regulator, tra-1.

Sex-specific markers undetected in green sunfish Lepomis cyanellus using restriction-site associated DNA sequencing.

We used restriction-site associated DNA sequencing for SNP discovery and genotyping of known-sex green sunfish Lepomis cyanellus DNA samples to search for sex-diagnostic single nucleotide polymorphisms (SNPs) and restriction-site associated sequences present in one sex and absent in the other. The bioinformatic analyses discovered candidate SNPs and sex-specific restriction-site associated sequences that fit patterns of male or female heterogametic sex determination systems. However, when primers were developed and tested, no candidates reliably identified phenotypic sex. The top performing SNP candidate (ZW_218) correlated with phenotypic sex 63.0% of the time and the presence-absence loci universally amplified in both sexes. We recommend further investigations that interrogate a larger fraction of the L.cyanellus genome. Additionally, studies on the effect of temperature and rearing density on sex determination, as well as breeding of sex-reversed individuals, could provide more insights into the sex determination system of L.cyanellus.

Masculinization of Adult Gambusia holbrooki: A Case of Recapitulation of Protogyny in a Gonochorist?

Simple SummaryAlthough gonochoristic fish express one sex or the other in adulthood, some adults display traits similar to those of their opposite sex, suggesting plasticity similar to hermaphrodites. To fully test this potential in the eastern mosquitofish, a gonochorist, two adult stages of females were fed 17α-Methyltestosterone incorporated feed (0–200 mg/kg diet) for 50 days. The hormone (particularly at 50 mg/kg diet) stimulated the formation of complex male copulatory structures and testicular tissue as well as upregulated expression of Anti-Müllerian Hormone gene and altered the behavior from females to males. Collectively, we infer that Gambusia holbrooki retains potential for sex reversal at adulthood, similar to what occurs naturally in hermaphroditic fish and can provide an ideal system to investigate these mechanisms in a regulated fashion.17α-Methyltestosterone (MT) is a synthetic steroid that has been widely used to masculinize many fish species when administered early during larval development, however, reports on its efficacy on adults is limited. To this end, this study investigated the efficacy of MT in the masculinization of the eastern mosquitofish (G. holbrooki) at two adult stages (maiden and repeat gravid females). The treated females were fed control or respective MT incorporated feed (0–200 mg/kg diet) for 50 days. Effects of the hormone on secondary sexual characteristics, internal gonad morphology, expression of the Anti-Müllerian Hormone (amh) gene and sexual behavior of the treated females were investigated. The results showed that MT at the dose of 50 mg/kg feed stimulated secondary sexual character development, upregulated expression of amh, formation of testicular tissue and a shift in the behavior similar to those of normal males, prominently so in treated maiden gravid females. Post-treatment, long-term observations indicated that only two masculinized females reverted back to being females and gave birth to young. Induction of masculinizing effects in most individuals suggests that the sexual phenotype of this species appears to be highly plastic with potential to sex reverse at adulthood. This in combination with its small size and short reproductive cycle could provide an ideal system to explore the mechanisms of sequential hermaphroditism in fish and contribute to genetic control of this pest fish.

Copulatory behaviour and seminal parameters of rapid ejaculator rats during competition and female pacing

Axioms of sperm competition theory (risk and intensity model) predict that males increase sperm expenditure when ongoing competitive copulating encounters with other males. Particularly, the intensity model that considers a real male confrontation has not been experimentally tested in laboratory rats. Furthermore, no study has explored the active role of the female (that occurs in natural conditions) in regulating the timing of copulation on the effects of competition on sexual behaviour and seminal parameters. Thus, the goal of this study was to analyse the copulatory behaviour and seminal parameters under male–male competition and female pacing. In order to control for possible variations due to the sexual behaviour phenotype, all experiments were done in male rats with short ejaculation latencies (rapid ejaculators). The results show that competition decreased by half the ejaculation latency and increased sperm quantity, but substantially decreased sperm motility. On the contrary, when competition is tested under conditions where the female regulates the timing of copulation (paced mating), the ejaculation latency increased to double, the sperm count did not change, and the sperm motility significantly decreased. The variations in the parameters of the ejaculate depend on the context, i.e., the confrontation with a competitor, and the active participation of the female during copulation. Probably, when there is more than one male during mating, the female – through cryptic choice – promotes mechanisms that interfere temporally with sperm motility.

Variability of Flowering Sex and Its Effect on Agronomic Trait Expression in White Guinea Yam.

White Guinea yam (Dioscorea rotundata) is mainly a dioecious tuberous crop that produces flowers of varying sex phenotypes. Agronomic traits in Guinea yam differ according to the sex phenotype, but the precise interaction between the traits and sex phenotype is not clearly understood. This might be due to the high heterozygosity of yam where cultivars with different flowering sex have different genetic backgrounds, which mask the sole effect of sex phenotype on the agronomic traits. This study used F1-derived clonal progenies from a bi-parental cross to minimize the impact of different genetic backgrounds among the plants with different sex phenotypes. The impact of plant sex on agronomic traits, specifically tuber yield, was evaluated through field trials conducted for four years. The results showed that only plants with a female genotype exhibited varying sex phenotypes even within the clones of same accession grown in the same experimental field. The significant effects of sex genotype and phenotype on agronomic traits were detected. Our results revealed that the flowering date was delayed in the plants with female genotypes compared to male genotypes, even when compared only among the plants with male phenotypes. The flowering date is the most important reason for the sexual differences in tuber yield. A high tuber yield was obtained when plants with the female phenotype flowered before tuber enlargement. This result can be attributed to the fact that the low flowering intensity in female plants increases the availability of carbon resources for leaf development. Female plants also showed a large negative effect of late flowering on tuber yield owing to resource competition between flowering and tuber enlargement. These findings demonstrate the feasibility of yield improvement by controlling the flowering time, with a higher effectiveness achieved in female than in male plants.

The ornamental fighting fish is the next model organism for genetic studies

AbstractThe fighting fish Betta splendens is a freshwater species from Thailand and other Southeast Asian countries. This fish has been domesticated for 1000 years and bred for fighting, various colours, body size and fin types for 600 years. It is one of the most important fish species cultured for the world ornamental fish market. This fish is easy to culture, highly fecund and displays great morphological diversity. Its biology has been studied for over 100 years. Recently, its compact genome and transcriptomes have been sequenced. Genome editing with CRISPR/cas9 has been applied to knock out genes in this fish. Its diverse phenotypes, including colours, colour patterns, fin types, and aggressive behaviour, are complementary to those of other model animals. Therefore, this fish could be the next important model organism for studying phenotypic variation and aggressive behaviour. In this paper, we synthesized knowledge about its aquaculture, biology, genetics, genomic tools, phenotypes and novel insights on phenotypic variation, sex determination and aggression. We hope that the information described in this paper will facilitate genetic studies on phenotypic variations in aquaculture and aggressive behaviour in other species, including humans.

“Heat waves” experienced during larval life have species-specific consequences on life-history traits and sexual development in anuran amphibians

Extreme temperatures during heat waves can induce mass-mortality events, but can also exert sublethal negative effects by compromising life-history traits and derailing sexual development. Ectothermic animals may, however, also benefit from increased temperatures via enhanced physiological performance and the suppression of cold-adapted pathogens. Therefore, it is crucial to address how the intensity and timing of naturally occurring or human-induced heat waves affect life-history traits and sexual development in amphibians, to predict future effects of climate change and to minimize risks arising from the application of elevated temperature in disease mitigation. We raised agile frog (Rana dalmatina) and common toad (Bufo bufo) tadpoles at 19 °C and exposed them to a simulated heat wave of 28 or 30 °C for six days during one of three ontogenetic periods (early, mid or late larval development). In agile frogs, exposure to 30 °C during early larval development increased mortality. Regardless of timing, all heat-treatments delayed metamorphosis, and exposure to 30 °C decreased body mass at metamorphosis. Furthermore, exposure to 30 °C during any period and to 28 °C late in development caused female-to-male sex reversal, skewing sex ratios strongly towards males. In common toads, high temperature only slightly decreased survival and did not influence phenotypic sex ratio, while it reduced metamorph mass and length of larval development. Juvenile body mass measured 2 months after metamorphosis was not adversely affected by temperature treatments in either species. Our results indicate that heat waves may have devastating effects on amphibian populations, and the severity of these negative consequences, and sensitivity can vary greatly between species and with the timing and intensity of heat. Finally, thermal treatments against cold-adapted pathogens have to be executed with caution, taking into account the thermo-sensitivity of the species and the life stage of animals to be treated.

Sex-Related Effects on Cardiac Development and Disease.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality. Interestingly, male and female patients with CVD exhibit distinct epidemiological and pathophysiological characteristics, implying a potentially important role for primary and secondary sex determination factors in heart development, aging, disease and therapeutic responses. Here, we provide a concise review of the field and discuss current gaps in knowledge as a step towards elucidating the “sex determination–heart axis”. We specifically focus on cardiovascular manifestations of abnormal sex determination in humans, such as in Turner and Klinefelter syndromes, as well as on the differences in cardiac regenerative potential between species with plastic and non-plastic sexual phenotypes. Sex-biased cardiac repair mechanisms are also discussed with a focus on the role of the steroid hormone 17β-estradiol. Understanding the “sex determination–heart axis” may offer new therapeutic possibilities for enhanced cardiac regeneration and/or repair post-injury.

Liver fat in adult survivors of severe acute malnutrition

The association between severe acute malnutrition (SAM) in early childhood and liver fat in adults is unknown. We hypothesized that exposure to SAM, especially severe wasting, is associated with fatty liver later in life. In this observational study, abdominal CT was used to quantify mean liver attenuation (MLA) and liver:spleen attenuation ratio (L/S). Birth weight (BW), serum lipids, insulin resistance (homeostatic model assessment), anthropometry and intrabdominal fat were collected. Mean differences between diagnostic groups were tested and hierarchical regression analysis determined the best predictors of liver fat. We studied 88 adult SAM survivors and 84 community participants (CPs); age 29.0 ± 8.4 years, BMI 23.5 ± 5.0 kg/m2 (mean ± SDs). SAM survivors had less liver fat than CPs (using L/S) (p = 0.025). Severe wasting survivors (SWs) had lower BW (-0.51 kg; p = 0.02), were younger, thinner and had smaller waist circumference than oedematous malnutrition survivors (OMs). In the final regression model adjusting for age, sex, birth weight and SAM phenotype (i.e., oedematous malnutrition or severe wasting), SWs had more liver fat than OMs (using MLA) (B = 2.6 ± 1.3; p = 0.04) but similar liver fat using L/S (p = 0.07) and lower BW infants had less liver fat (MLA) (B = -1.8 ± 0.8; p = 0.03). Greater liver fat in SWs than OMs, despite having less body fat, supports our hypothesis of greater cardiometabolic risk in SWs. Other postnatal factors might influence greater liver fat in survivors of severe wasting, suggesting the need to monitor infants exposed to SAM beyond the acute episode.

Genotypic confirmation of a biased phenotypic sex ratio in a dryland moss using restriction fragment length polymorphisms.

PremiseIn dioicous mosses, sex is determined by a single U (female, ♀) or V (male, ♂) chromosome. Although a 1 : 1 sex ratio is expected following meiosis, phenotypic sex ratios based on the production of gametangia are often female‐biased. The dryland moss Syntrichia caninervis (Pottiaceae) is notable for its low frequency of sex expression and strong phenotypic female bias. Here we present a technique to determine genotypic sex in a single shoot of S. caninervis, and report results of a case study examining genotypic and phenotypic sex ratios.MethodsWe reanalyzed 271 non‐expressing gametophyte shoots from a previous study on S. caninervis sex expression across microhabitats using a restriction fragment length polymorphism (RFLP) method.ResultsWe recovered a genotypic sex ratio in non‐expressing shoots of 18.4♀ : 1♂, which exceeds the female bias of the phenotypic ratio (5.3♀ : 1♂; P = 0.013). We also found that the distribution of male and female genotypes across microsites with different levels of sun exposure was not predicted by patterns of sex expression in these microsites.DiscussionThese findings contribute to our understanding of how the environment may modulate sex ratios in S. caninervis, either through its direct influence on sex expression or through selection on genotypes with particular sex expression phenotypes.

Evolutionary stability inferred for a free ranging lizard with sex-reversal.

The sex of vertebrates is typically determined genetically, but reptile sex can also be determined by developmental temperature. In some reptiles, temperature interacts with genotype to reverse sex, potentially leading to transitions from a chromosomal to a temperature‐dependent sex determining system. Transitions between such systems in nature are accelerated depending on the frequency and fitness of sex‐reversed individuals. The Central Bearded Dragon, Pogona vitticeps, exhibits female heterogamety (ZZ/ZW) but can have its sex reversed from ZZ male to ZZ female by high incubation temperatures. The species exhibits sex‐reversal in the wild and it has been suggested that climate change and fitness of sex‐reversed individuals could be increasing the frequency of reversal within the species range. Transitions to temperature‐dependent sex determination require low levels of dispersal and high (>50%) rates of sex‐reversal. Here, we combine genotype‐by‐sequencing, identification of phenotypic and chromosomal sex, exhaustive field surveys, and radio telemetry to examine levels of genetic structure, rates of sex‐reversal, movement, space use, and survival of P. vitticeps in a location previously identified as a hot spot for sex‐reversal. We find that the species exhibits low levels of population structure (FST ~0.001) and a modest (~17%) rate of sex‐reversal, and that sex‐reversed and nonsex‐reversed females have similar survival and behavioural characteristics to each other. Overall, our data indicate this system is evolutionary stable, although we do not rule out the prospect of a more gradual transition in sex‐determining mechanisms in the future in a more fragmented landscape and as global temperatures increase.

Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats.

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable because environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex‐ratio distortion and population decline. Despite these implications, there is scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex‐reversing stimuli more frequently, which may lead to higher sex‐reversal rate or, alternatively, these populations may adapt to resist sex reversal. We developed PCR‐based genetic sex markers for the common toad (Bufo bufo) to assess the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous oestrogenic pollutants in a common garden experiment. We found negligible sex‐reversal frequency in free‐living adults despite the presence of various endocrine‐disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg L−1 17α‐ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng L−1 EE2 and a glyphosate‐based herbicide with 3 µg L−1 or 3 mg L−1 glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the extremely low sex‐reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species‐specific resistance to sex reversal.