The Role of Nonapeptides in Male Reproduction in Two Cyprinid Species, the Zebrafish (Danio rerio) and the Goldfish (Carassius auratus)
Creators: name: Altmieme, Zeinab
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2019
Subjects: Vasotocin
Courtship behaviour
Isotocin
Pheromone
Androgen
Reproductive endocrinology
Neuroendocrinology

description: Two distinct nonapeptide systems, consisting of the vasotocin- and oxytocin-related peptides have evolved in vertebrates, and their role in male reproduction is well-described in mammals. In contrast, their comparative role in reproduction in basal vertebrate species, and teleost fishes in particular, has not been investigated in great detail. Using two cyprinid species, the zebrafish (D. rerio) and the goldfish (C. auratus), I address the hypothesis that the teleost nonapeptides vasotocin and isotocin stimulate male cyprinid reproductive physiology by affecting central neuronal and/or peripheral endocrine pathways. To test this hypothesis in zebrafish, an indeterminate breeder, I conducted pharmacological inhibition experiments employing vasotocin and isotocin-specific antagonists in males, a treatment predicted to inhibit reproductive success in mating trials. Because nonapeptides can act both as central peptide neuromodulators and as secreted hormone, I further quantified indices of male courtship behavior (nudging, circling and chasing) and major androgens (testosterone and 11-keto-testosterone) as key endocrine indices of the male reproductive axis. Together, these experiments revealed a dose-dependent, differential inhibition of spawning success, with significant reductions (-65%) in egg fertilization rate observed in pairs in which males had been i.p. injected with 5 ng/g vasotocin and significant reductions (-79%) observed at 500 ng/g i.p injected isotocin. In either case, these partial inhibitions of reproductive success were correlated with significant decreases in specific indices of male courtship behavior, but not endocrine indices, suggesting that individual nonapeptides mediate their effects via central modulation of behavioural neurocircuits. Interestingly, a co-administration of vasotocin and isotocin antagonists completely abolished reproductive success, however this effect was neither correlated with decreases in male courtship behavior, nor endocrine indices, suggesting a separate mode of action, possibly at the level of male pheromone release. To further probe the role of nonapeptides in male zebrafish reproduction, I subsequently tested the hypothesis that nonapeptide systems are acutely activated by key reproductive cues, specifically the releaser pheromone PGF2α, which serves as a chemoattractant and acutely stimulates male reproductive behavior in male cyprinids. Using a chemoattractant choice assay in conjunction with immunohistochemistry and gene expression approaches, I determined whether male zebrafish are attracted to pheromonal cues and acutely activate isotocinergic neurons in the short term and/or regulate nonapeptide gene expression in the longer term. My results show that individual male zebrafish are attracted to PGF2α in an acute choice test. Furthermore, an increase in p-ERK immunoreactivity, a marker of neuronal activation, was observed in the olfactory bulb 10 min following exposure, suggesting a specific response to the pheromone compared to EtOH vehicle. However, no co-localization of p-ERK and IT-positive perikarya was observed in the preoptic area (POA), refuting the hypothesis that PGF2α exposure acutely activates isotocinergic neurons in zebrafish. Analysis of whole brain relative mRNA transcript abundance revealed that PGF2α exposure time-dependently regulates whole brain isotocin, but not vasotocin transcript abundance, suggesting secondary longer-term effects of PGF2α exposure on the isotocinergic system. Using an analogous experimental approach, I further tested the hypothesis that nonapeptides stimulate male reproductive physiology in goldfish, a determinate breeder. Sexually mature male goldfish pretreated with saline or vasotocin or isotocin antagonists were exposed to saline or PGF2α-injected stimulus females and male courtship behavior (chasing, circling), endocrine indices (circulating testosterone) and milt release were quantified. Both nonapeptide antagonists reduced strippable male milt quantity in response to PGF2α-injected females, suggesting a neuronal or hormonal action of both nonapeptides on goldfish milt release. Together, I show that nonapeptides contribute to male reproductive physiology in two species of cyprinids with different reproductive tactics. However, the mode of action may differ from one species to another, with evidence suggesting that nonapeptides play a role in the regulation of reproductive behavior and, possibly, male pheromone, release in zebrafish, while effects on male goldfish seem to be exclusively related to the release of milt. Future studies should compare other teleost species with specific reproductive biology and focus on the gonadal roles of nonapeptides in sperm maturation and/or release.