Androgen receptor agonism leading to male-biased sex ratio

• Dan Villeneuve
• Kelvin Santana Rodriguez
• Gerald Ankley

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Life Stage

The life stage applicable to this AOP is developing embryos and juveniles prior to- or during the gonadal developmental stage. This AOP is not applicable to sexually differentiated adults. 

Sex

The molecular initiating event for this AOP occurs prior to gonad differentiation. Therefore, this AOP is only applicable to sexually undifferentiated individuals.

Taxonomic

Most evidence for this AOP is derived from fish in the class Osteichthyes. Both phylogenetic analysis and evaluation of protein sequence conservation via SeqAPASS (https://seqapass.epa.gov/seqapass/) has shown that the structure of the AR is  well conserved among most jawed vertebrates (Gnathostomata). This AOP is not expected to apply to mammals, birds, or other jawed vertebrates with genetic sex determination. However, it may be applicable to fishes, amphibians, and reptiles with environmentally-dependent sex determination, although outcomes may differ across physiologically different taxa. The present AOP is not considered relevant to Agnathans since the AR appears not to be present in jawless fishes (Thornton 2001; Hossain et al 2008). 

Support for the essentiality of several of the key events in the AOP is provided by both in vivo and in vitro studies with chemicals acting as AR agonists and antagonists. Important in vivo studies typically have been conducted during the critical period of sexual differentiation.

Golan & Levavi-Sivian (2014) exposed genetic females of Nile tilapia (Oreochromis niloticus) to 17α-methyltestosterone (MT) and dihydrotestosterone (DHT), and showed that the two  AR agonists  induced a  male biased sex ratio in a dose-dependent manner. However, in combined exposures with the AR antagonist flutamide (a pharmaceutical), the sex inversion potency of MT and DHT was reduced in a dose-dependent manner.  This provides direct evidence that activation of the AR is required for the subsequent key events to occur.

Crowder et al. (2018) generated zebrafish (Danio rerio) with a mutation in the AR gene (ar^uab105/105). Most mutants developed ovaries and displayed female secondary sexual characteristics. The small percentage of mutants that developed as males displayed female secondary sexual characteristics with structurally disorganized testes, and were unable to produce normal levels of sperm. This demonstrates that the AR is required for proper testis development and fertility and supports the essentiality of AR activation in testis differentiation.

In a similar study with zebrafish, Yu et al. (2018) generated an AR gene mutant line using CRISPR/Cas9 technology. The number of female offspring was increased and the resulting AR-null males had female secondary sex characteristics and were infertile due to defective spermatogenesis. This study again supports the essentiality of AR agonism for the development of testis and how inappropriate (increased) signaling in the pathway could result in a male biased sex ratio.

Biological Plausibility

The biological plausibility linking AR activation to increased differentiation to testis is very strong. Actions of androgens are mediated by the AR which is part of the nuclear receptor superfamily. ARs are ligand-dependent transcription factors (Hossain et al., 2008). Steroidal androgens act by entering the cell and forming a complex with the AR, resulting in conformational change (Bohen et al., 1995; Pratt and Toft, 1997). The ligand-AR complex is translocated to the nucleus where it binds to specific short DNA sequences (Androgen Responsive Elements), thereby activating transcripton of androgen regulated genes (Harbott et al., 2009). During sexual development, endogenous androgen can therefore induce the upregulation of many genes involved in the male developmental pathway.

The direct link between increased differentiation to testis leading to a male biased sex ratio is highly plausible. If the conditions that favored a male producing phenotype (in this case, exposure to AR agonsts) overlap with the critical period of sex differentiation in a given population, it is reasonable that more phenotypic males will be produced (Orn et al., 2003; Seki et al., 2004; Bogers et al., 2006; Morthorst et al., 2010; Baumann et al., 2014; Golan & Levavi-Sivian 2014). Therefore, androgen exposure for repeated or prolonged periods of time conceptually will result in a male-biased population. Empirical evidence supporting the direct link between male-biased sex ratio and reduced population growth rate in fish species is lacking. However, altered sex ratios have the potential to affect fish populations (Marty et al. 2017). For example, a male-biased sex ratio suggests that the number of breeding females would be reduced. If the male-biased sex ratio persists and/or increases over time, the offspring produced per reproductive cycle would decrease, eventually leading to a diminished population size, assuming other demongraphic parameters remained largely constant (Brown et al. 2015; Grayson et al. 2014; Miller et al. 2022).

Concordance of Dose Response Relationships

The concentration-dependence of the key event responses with regard to the concentration of exogenous AR agonists has been established in vivo for some key events in the AOP. In general, effects on downstream key events occurred at concentrations equal to or greater than those at which upstream events occurred. However, binding to the androgen receptor (the MIE) was not directly measured in any of the in vivo studies. Nonetheless, AR binding of several of the agonists tested in vivo has been documented with in vitro studies with fish ARs (e.g., Wilson et al. 2004). In fish, phenotypic masculinization of female secondary sex characteristics has been used as an indirect measurement of in vivo AR agonism. However, in the of case this AOP specifically, AR agonism is occurring prior to sexual differentiation and the resultant “phenotypic measurement” for the in vivo study (gonad differentiation) is a discrete downstream key event. Consequently, in vitro evidence can reliably be used to identify specific chemicals as AR agonists (i.e., able to activate the MIE). That is, dependence of the severity of the downstream in vivo responses on concentration and potency of chemicals activating the AR in vitro can be used as indirect evidence of dose-response concordance between the MIE and downstream key events.

1. Concentration dependent androgen receptor agonism (in vitro)

• COS Whole Cell Binding Assay with fathead minnow AR (fhAR) were used in competitive binding experiments testing several natural and synthetic steroids, some of which are environmental contaminants, such as R1881, 17beta-trenbolone , and 17alpha-trenbolone. All showed a concentration dependent displacement of [3H]R1881 binding proving to be high affinity ligands for the fmAR. (Wilson et al., 2004).
  •  The synthetic steroids, R1881 and methytestosterone, had the highest affinities of all the chemicals tested with IC50 values of about 1.6 nM, followed by the synthetic steroids 17α- and 17β-trenbolone with IC50 values of about 8 and 16 nM, respectively.
  • Of the natural steroids, dihydrotestosterone was the strongest competitor with an IC50 of about 20 nM. The IC50 for the fish specific androgen, 11- ketotestosterone, was approximately 40 nM, followed by both testosterone and androstenedione at about 100 nM
• Important to note that all of the above steroids tested were used in the in vivo studies that were selected to support this AOP demonstrating that all bound to the fhAR with a higher affinity than 11- ketotestosterone.

2. Concentration dependent increased differentiation to testes:

• Studies with zebrafish and Japanese medaka (Oryzias latipes) exposed to 17β-trenbolone during development resulted in masculinization in a concentration-dependent manner as evidenced from a significantly increased maturity of testes (Orn et al., 2006; Morthorst et al., 2010; Baumann et al., 2014) for some studies, this was determined ether by the abundance of spermatozoa and/or by the area of the testis.

3. Concentration dependent increased male biased sex ratio:

• Exposure of developing zebrafish to different concentrations of 17β-trenbolone and dihydrotestosterone led to an increased number of males in a dose-dependent fashion (Orn et al., 2003; Holbech et al.,2006; Morthorst et al., 2010; Baumann et al., 2013; Baumann et al., 2014)

4. Concentration dependent decline in population trajectory:
   • Modeled population trajectories show a concentration-dependent reduction in projected population size (Brown et al 2015, Miller et al. 2022) based on the ratio of male to female. Population-level effects exposure of fish to AR agonists have not been measured directly. 

Dose Concordance Table

Temporal concordance

Because this AOP involves actions during a specific development transition from an undifferentiated to differentiated gonad, the temporal concordance of the events is implicit. A male biased sex ratio cannot be observed until the population has undergone sexual differentiation. Likewise, reproduction and associated population growth rate cannot be assessed until the animals achieve sexual maturity.

Consistency

We are aware of no cases where substantial exposure of susceptible teleost fish species during sexual differentiation to known AR agonists  did not impact male sex ratios (for reviews see Pandian and Sheela 1995 and Leet et al. 2011). There are cases however, in which exposure to aromatizable androgens such as methyltestosterone can lead both to masculinization and feminization of fish (e.g., Piferrer et al. 1993; Orn et al., 2003); this most likely is due to conversion of the androgen to its corresponding estrogen analogue (i.e., methylestradiol; e.g., Hornung et al. 2004 ). In other instances, non-aromatizable androgens (e.g., dihydrotestosterone) have been reported to feminize fish exposed during early development (e.g., Davis et al. 1992; Bogers et al. 2006). The mechanism underlying this is uncertain, but plausibly could involve binding to the estrogen receptor which is known to interact with a variety of steroids, including androgens at comparatively test concentrations.

The adverse outcome is not wholly specific to this AOP.  Key events included overlap with AOPs linking other MIEs (e.g., aromatase inhibition, AOP 346) that can lead  to male biased sex ratios.

Overall, there is strong empirical support for the proposed AOP. We did not find notable inconsistencies in the literature reviewed as part of this AOP development. However, there were several notable data gaps which could potentially be addressed through further research:

(1) The detailed gene expression and signaling mechanisms via with AR activation induces differentiation to testes is not well understood or defined. If key steps in this process could be defined, one or more additional key events could potentially be inserted between Event 25 (agonism, androgen receptor) and Event 1790 (differentiation to testes, increased).

(2) Population-level consequences of a male biased sex ratio are based on modeling. At present, we found no empirical studies that establish the effect of a male-biased sex ratio on population growth rates. Current models assume that other demographic variables such as predation rates, prey availability, habitat availability, etc. are unaffected by sex ratio. This may or may not be the case in real-world populations.

At this time available data are insufficient to develop a quantitative AOP linking AR activation to male biased fish populations.