Tilapia male urinary pheromone stimulates female reproductive axis
Introduction
The use of waterborne chemical signals to control physiological process and behaviour has been observed in several fish (Chung-Davidson et al., 2011, Hara, 1994, Rosenthal and Lobel, 2005, Stacey and Sorensen, 2005). Among this group of chemicals, sex pheromones are involved in reproduction by mediating location of suitable partners, evoking appropriate behavioural and endocrine responses, and improving synchronisation of gametogenesis, spawning, fertility and paternity (see reviews by Burnard et al., 2008, Stacey et al., 2003). The identification and characterisation of these compounds is important for understanding fish reproductive physiology (Stacey, 2011) and as a potential tool for population management (e.g., aquaculture and species invasions) (Johnson and Li, 2010, Sorensen and Stacey, 2004).
Pheromonal responses can occur at different levels, scales and contexts. Behavioural responses to a given stimulus occur usually within seconds, (Liley et al., 1986, Rouger and Liley, 1993, Stacey et al., 1989) but relatively rapid physiological changes, e.g. sex steroid synthesis and metabolism, are also possible within minutes or hours (Bayunova et al., 2011, Dulka et al., 1987, Scott et al., 1994).
Sex steroids are produced by the gonads through gonadotrophic stimulation and released into the blood to be transported to target organs. Therefore, steroid production is traditionally determined by analysis of blood samples in vertebrates. However, handling and sampling induce acute stress responses that may mask any response (Scott and Ellis, 2007, Scott et al., 2008). Consequently, there is increasing interest in the use of non-invasive procedures, including determination of steroid content in water and faeces, in fishes (Scott et al., 2008). The fish gill is considered to be the main route for release of free steroids at a rate that reflects largely their plasma concentrations. Indeed, several studies suggest that free steroids are preferentially released via the gills and are found at much lower concentrations in the urine or faeces compared to conjugated metabolites (Ellis et al., 2005, Miguel-Queralt and Hammond, 2008, Scott et al., 2008, Siefkes et al., 2003, Vermeirssen and Scott, 1996). Therefore, changes of steroid concentration in water samples can parallel those in blood, taking into account the dilution effect, and estimation of sex steroids in the water is a reliable indicator of fish endocrine status (Scott and Sorensen, 1994, Scott et al., 2008, Sebire et al., 2009). In cases of lack of correlation between blood plasma concentrations and release rates of steroids this has been ascribed to several factors including steroid characteristics and metabolism, and the presence of steroid binding globulins in blood with differing affinity for steroids (Scott and Sorensen, 1994, Scott et al., 2008).
The Mozambique tilapia (Oreochromis mossambicus; hereafter ‘tilapia’) is a polygynous maternal mouth-brooding African cichlid. In nature, males aggregate in breeding arenas (leks) and dominant males defend small territories centred on pits (nests) that they dig in the sand, adopting a typical black colouration; visiting females spawn in the territory of a dominant male and then move away from the males to mouth brood the eggs (Almeida et al., 2005, Barata et al., 2007, Oliveira and Almada, 1998, Russell et al., 2012, Turner, 1986). Furthermore, signalling of male dominance via controlled urination has been demonstrated. Unlike subordinate males and females, dominant tilapia males store urine which is a vehicle for potent odorants actively released during aggressive disputes and mating behaviour (Almeida et al., 2005, Barata et al., 2007, Barata et al., 2008, Miranda et al., 2005). Moreover, the urinary bladders of dominant males are larger and more muscular than those of subordinate males or females, suggesting an adaptation facilitating storage of larger urine volumes for longer and more frequent urination in the appropriate social context, which may modulate aggression between opponent males (Keller-Costa et al., 2012). Males can discriminate the sexual status of females using olfactory cues (Miranda et al., 2005) and when in the presence of ovulated females, their urination rate increases (Almeida et al., 2005, Barata et al., 2008). The olfactory potency of male urine depends on the social status of the donor (Barata et al., 2007). These observations indicate that tilapia males release a pheromone via the urine which influences female spawning (Barata et al., 2008). However, the physiological and/or behavioural effect of the male pheromone on females is still unknown.
To determine the possible priming effect of the sex pheromonal compound present in male urine on female tilapia we measured their response, in terms of sex steroids, to male urine. We chose to measure steroids secreted mainly during the secondary growth phase (testosterone and 17β-estradiol; E2) and during final maturation of the oocytes (17,20β-P). Testosterone is produced by the theca cells surrounding the follicle under gonadotrophic stimulation and has positive feedback effects in the pituitary (Nagahama et al., 1995). Testosterone is the precursor of E2 produced in the granulosa cells, which promotes ovarian growth through the stimulation of synthesis and secretion of vitellogenin and egg shell proteins in the liver (Lubzens et al., 2010, Nagahama et al., 1995, Senthilkumaran et al., 2004, Young et al., 2005). After oocyte growth, a shift in steroidogenesis leads to the production of 17,20β-dihydroxypregn-4-en-3-one (17,20β-P) also in the granulosa cells which induces the resumption of meiosis and final oocyte maturation (Nagahama, 1997, Senthilkumaran et al., 2004). In goldfish 17,20β-P is also released to the water and is perceived by males thereby stimulating the endocrine system, spermiation as well as increased fertility and paternity (Dulka et al., 1987, Scott and Sorensen, 1994, Zheng et al., 1997).
Here we establish that urine from tilapia males contains a pheromone that primes the female’s reproductive system by increasing the production and release rates of the maturation-inducing steroid 17,20β-P. We also show that these changes can only be detected in the water, possibly because the tilapia plasma sex steroid binding globulin has low affinity for 17,20β-P.
Section snippets
Experimental animals
Adult Mozambique tilapia (O. mossambicus) of both sexes were taken from a stock population established at the Centre of Marine Sciences, University of the Algarve. Fish were tagged and 8 groups of one male and four females were setup in tanks of 250 L with sand substratum and kept at 27 ± 1 °C under a photoperiod of 12 h light/12 h dark (lights on at 7.30 a.m.) and fed twice daily. Spawning occurred spontaneously in each tank, producing viable offspring. After each spawning, as soon as the females
Effect of urinary pheromone on blood and urine steroid levels
Exposure to male urine had no apparent effect both on plasma and urine steroid concentrations (Fig. 1). However, the possibility that rapid, temporary changes may have occurred within the duration of the experiment cannot be excluded. Pre-ovulated females had significantly higher levels of blood plasma testosterone (17.1 ± 2.39 ng ml−1) than post-spawn females (3.9 ± 1.06 ng ml−1), but not of any of the other steroids, irrespective of being exposed to urine or not (Fig. 1). The mean levels of plasma E2
Discussion
A pheromone present in urine from dominant tilapia males stimulates the release of 17,20βP in pre- and post-ovulated tilapia females. Considering the established role of 17,20βP as maturation-inducing steroid (Nagahama, 1997) and the suggested role in primary follicles (Zapater et al., 2012), the stimulation of 17,20βP production and release demonstrates a specific priming effect for the male urine pheromone to accelerate reproduction. The involvement of urine in several communication processes
Conclusions
In conclusion, urine from male tilapia contains a pheromone that dramatically affects 17,20βP metabolism in females, less than 1 h after exposure; the first time a primer effect of male pheromone on females has been reported in a cichlid. Furthermore, this pheromonal effect was manifest using non-invasive measurement of steroids in the water, but may not have been so obvious using conventional blood sampling. This method may prove to be a simple and rapid bioassay for the study of pheromone
Acknowledgments
Funded by European Union-FSE/FEDER and Ministério da Ciência e do Ensino Superior, Portugal through Fundação para a Ciência e Tecnologia, Portugal (Grant numbers POCI/BIA-BDE/55463/2004 and SFRH/BPD/26339/2006). The authors are grateful to Elsa Couto for her help with the steroid radio-immunoassays.
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