Reproductive viability of paradoxically masculinised Gambusia holbrooki generated following diethylstilbestrol (DES) treatment

Abstract

Hormonal sex reversal can produce monosex fish stocks and provide insights into their gamity and reproductive physiology. However, paradoxical effects have been reported in several fish species that remain largely ignored as anomalies, particularly those of masculinisation. As a first step, this study examined reproductive viability of paradoxically masculinised Gambusia holbrooki produced following oral administration (20–100 mg/kg feed) of a feminizing hormone diethylstilbestrol (DES). Contrary to expectation, all treatment groups produced 100% male populations. Survival, mating behaviour, gamete production, breeding output as well as expression of anti-Mullerian hormone (amh), ovarian (cyp19a1a) and brain (cyp19a1b) aromatase of masculinised fish were also examined. Survival (≤ 54.1 ± 7.3%) at termination of DES treatment was significantly lower compared with controls (88.6 ± 4.3%) but remained unaffected post treatment. Gonopodium thrusting frequency (33 ± 9.8 per 10 min) was not significantly different to untreated males just as sperm abundance (3.9 ± 1.5 × 10⁸/male) and their motility (88.6 ± 29.1%). Importantly, paradoxically masculinised fish mated with virgin females and produced clutch sizes (22 ± 4) and progeny survival (87.0 ± %) that were comparable to that of untreated males. Masculinised testes showed high amh and low cyp19a1a expression, a pattern resembling those of untreated males. Production of paradoxically sex-reversed males with a capability to produce viable offspring has not been reported previously in this or other fish species. The outcomes support a feed-back regulation of oestrogenic pathways in this viviparous fish and could be useful for ecological applications such as controlling invasive fish populations.

Introduction

In teleosts, exogenous administration of sex steroids is a common practice to generate monosex populations for improving aquaculture production (Pandian and Sheela, 1995; Singh, 2013), generate desired aquarium traits (Piferrer and Lim, 1997) or to manipulate natural sex reversal in sequential hermaphrodites (Abduh et al., 2020). Further, progeny sex ratio of such hormonally sex reversed individuals, can facilitate inferring/verifying the gamity (i.e. sex determining mechanisms) such as in the channel catfish (Ictalurus punctatus; Davis et al., 1990) and Nile tilapia (Oreochromis niloticus; Mair et al., 1991) among others. More recently, its use to control pest populations via Trojan Y approach (Gutierrez and Teem, 2006), including those of the Gambusia holbrooki, a notorious pest fish (Patil, 2012) are gaining popularity.

Typically, both natural and synthetic sex steroids are administered either orally or via immersion at effective dose and duration during early life stages, when the sex is still labile (Pandian and Sheela, 1995). These exogenously administered estrogens and androgens are known to mimic their endogenous analogues, overriding the genetic sex, resulting in all female or male populations respectively (Pandian and Sheela, 1995). At molecular level, this parallels the observations that overexpression of ovarian aromatase gene (cyp19a1a) and concomitant increase in aromatase activity and thus endogenous estrogen, leads to feminisation of genetically male fish, for example, in medaka (Oryzias latipes; Iwamatsu et al., 2006), zebrafish (Danio rerio; Andersen et al., 2003) and several tilapia species (Oreochromis spp.;Rosenstein and Hulata, 1994). Conversely, ablation of cyp19a1a activity results in masculinisation, such as in zebrafish (Lau et al., 2016).

Of the many oestrogenic compounds, diethylstilbestrol (DES), a synthetic estrogen first used to treat miscarriages in humans (Veurink et al., 2005), has been widely used in the aquaculture industry for feminizing fish due to its proven potency (Pandian and Sheela, 1995; Piferrer, 2001). The first successful usage of DES to feminise fish was reported in medaka (Oryzias latipes; Yamamoto, 1953) and since then DES has been successfully used to feminise other fish species such as the Nile (Oreochromis niloticus; Tayamen and Shelton, 1978) and Mozambique (O. mossambicus; Varadaraj, 1989) tilapias, guppy (Poecilia reticulata; Kavumpurath and Pandian, 1993) and black molly (P. sphenops; George and Pandian, 1995). The doses usually range between 50 and 100 mg DES /kg of feed for egg lying teleosts, with livebearing fish requiring much higher (300–500 mg/ kg feed) doses (Kavumpurath and Pandian, 1993; George and Pandian, 1995; Piferrer, 2001). However, there are no prior studies that have demonstrated sex reversal in G. holbrooki. Instead, most reports in this species are restricted to partial effects of enviornmental pollution i.e. endocrine disrupting chemicals on juvenile and adult life stages (e.g. Brockmeier et al., 2013; Game et al., 2006; Midgley et al., 2014).

Interestingly, DES along with other feminizing hormones such as 17β-estradiol and spironolactone has been shown to paradoxically produce masculinisation effect in Western mosquitofish (G. affinis; Howell et al., 1994), Nile tilapia (Alcantar-Vazquez, 2018) and fathead minnow (Pimephales promelas; Uguz, 2017), suggesting the effect of estrogenic hormone is not always predictable in certain species or circumstances. Paradoxical masculinisation is often viewed sceptically because it is believed that there is no apparent mechanism for such an effect (Piferrer, 2001) at least based on the simplistic understanding of uni-directional conversion of androgen to estrogen by aromatase (cyp19a1) gene. As raised by Warner et al. (2014), such reservation may have encouraged overlooking the ‘anomalies’ and hence ignored. Thus far, no compelling evidence has been put forward but it has been thought to be due to several factors including repression of cyp19a1a by exogenous estrogen which results in accumulation of androgen (Paul-Prasanth et al., 2011), non-specific binding of exogenous estrogen to androgen receptors (Richter et al., 2007; Warner et al., 2014) and interference of exogeneous estrogen in reducing mitotic activity of germ cells (Paul-Prasanth et al., 2011).

In view of its rareness, paradoxical masculinisation may offer a unique opportunity to investigate plasticity of sex differentiation in teleosts. Also, paradoxical sex reversal as a technique to produce monosex progenies is not entirely undesirable especially if these fish could grow and reproduce normally. However, the assessment of paradoxically masculinised individuals is limited, particularly with no reports of breeding and progeny viability. Taking advantage of the unexpected and persistent recurrence of paradoxical masculinisation caused by DES in G. holbrooki, this study investigated mating behaviour, gamete viability and reproductive output of masculinised individuals. The responses of anti-Mullerian hormone (amh), ovarian (cyp19a1a) and brain (cyp19a1b) aromatase genes as surrogates for subtle end-point reproductive trade-offs were also investigated. In teleosts, while the amh plays a central role in testicular differentiation and function (Kwan and Patil, 2019; Poonlaphdecha et al., 2011), the cyp19a1 isoforms, cyp19a1a (Fernandino et al., 2008; Patil and Gunasekera, 2008) and cyp19a1b (Patil and Gunasekera, 2008) are critical for ovarian and brain sex functions, respectively.