Regulatory mechanisms associated with egg production and egg activation in Oreochromis niloticus.

Abstract

This study investigated several important aspects of reproductive physiology in the Nile tilapia (Oreochromis niloticus). Although tilapias have been cultured for several decades, there has been a rapid increase in production by aquaculture over recent years. Tilapia have thus become one of the main teleosts contributing significantly to world aquaculture. However, significant problems exist that constrain the efficient management of reproduction in these fish. These include low fecundity, and the asynchronous, and hence unpredictable, nature of spawning cycles. Manipulation of photoperiod has proved to be a powerful tool in the control of reproductive cycles in various other fish species, and has become an established aspect of the culture of certain species. We know very little about the ways in which photoperiod might be involved in the control of reproduction in tilapiine species, especially the hormonal rhythms associated with this regulatory mechanism. In addition, manipulation of environmental parameters such as photoperiod has often been reported to influence fertilization rates in some teleosts. Spawning induction using the application of exogenous hormones, a technique often used in aquaculture to produce predictable spawning patterns, is also associated with problems concerning fertilization. It is clear from the literature that there is very little information available concerning the precise mechanisms involved with fertilization and egg activation in fish. Thus, the research described in this Thesis falls into two main sections. The first section investigates how photoperiod may impart a regulatory role over reproduction in O. niloticus broodstock, how circulating levels of the hormone melatonin vary in this species, and describes the isolation and partial characterization of a melatonin receptor from this species. In the second part of this Thesis, I have made a preliminary investigation of the precise mechanisms that might be involved at egg activation in fish using tilapia as a research model; these studies were then extended to three other commercially-important fish. Tilapia are now a major aquaculture species with production levels of over a million tonnes annually. The hatchery production of fry however, remains very inefficient due to relatively low fecundity and lack of spawning synchrony. Any methodology that enables farmers to synchronise the reproductive cycles of their broodstock would have immense practical advantages. Light is already known to play an important role in the initiation of gonad maturation in other fish species. In this investigation the reproductive performance of 32 sibling Nile tilapia was evaluated under four different photoperiods: short daylength (6L:18D), normal daylength (12L:12D), long daylength (18L:6D), and continuous illumination (24L:0D). Significantly larger eggs (P < 0.05) were produced under normal daylength (12L:12D) compared to other treatment groups. Fish reared under long daylength (18L:6D) exhibited significantly higher (P < 0.05) total fecundity (2408 ± 70 eggs spawn-1) and relative fecundity (7.2 ± 0.2 eggs g-1 body weight) concomitant with a significant reduction in inter-spawn-interval (ISI, 15 ± 1 days) in comparison with the rest of the trials. This investigation shows that long daylength (18L:6D) helps improve some important reproductive traits in Nile tilapia, and suggests that such methodology may be used to alleviate the production problems caused by low fecundity and poor spawning synchrony, and thus play a valuable future role in tilapia culture. Plasma melatonin levels were determined by radioimmunoassay (RIA) in fish kept under controlled photo-thermal conditions to investigate how the hormone melatonin varies with environmental change. Six melatonin profiles were described, the first over a 24 hour period (diel cycle), the second describing only changes in melatonin during the night-time, and four further profiles describing the melatonin levels of fish under four different experimental light regimes: short daylength (6L:18D), normal daylength (12L:12D), long daylength (18L:6D), and continuous illumination (24L:0D). Results showed that in tilapia, melatonin is produced in a rhythmic way; melatonin profiles showed that maximal levels of melatonin were reached as soon as the light went off, then these levels remain high throughout the dark phase and just after the onset of the light phase, melatonin levels were suppressed to base levels. Studies also demonstrated that melatonin levels were very low in O. niloticus (50 - 100 pg/ml) compared with salmonids and other species, in which much higher production of melatonin (600 - 1000 pg/ml) has been reported. Experiments showed that a negative correlation exists between melatonin levels and reproduction in tilapia. Those fish exposed to long daylength (18L:6D) exhibited the lowest melatonin levels but highest fecundity, and lowest ISI; fish reared under short daylength (6L:18D) exhibited the highest melatonin levels but much reduced fecundity and longer ISI. Although, it appears that photoperiod thus seems to play an important role in the reproduction of O. niloticus, and is certainly known to impart a strong regulatory effect upon reproduction in other fish, the present investigation also shows that those fish reared under continuous illumination produced the second highest fecundity and exhibited reduced Inter-Spawning-Interval (ISI). However, melatonin levels in these fish were kept constant at very low levels (20 - 30 pg/ml). Interestingly, these observations might suggest that melatonin may not be exerting a strong effect upon reproduction in O. niloticus. Results also showed that the role of melatonin in the reproduction of O. niloticus is not as well-defined as in other species of fish. There was clearly a negative relationship between melatonin level and reproductive activity in our experiments; although the results of the continuous illumination treatment produced data that suggested that melatonin might not play a major role in the regulation of reproduction in this species. It was clearly important therefore, to further elucidate the role and function of melatonin in this species. In a further series of experiments, the melatonin receptor from O. niloticus was successfully cloned (Mella), and a partial sequence of this receptor was obtained. This partial sequence was generated using primers based upon known sequence information for the melatonin receptor in rainbow trout. The tilapia melatonin receptor was highly expressed in the brain. However, no expression was found in either gonadal or somatic tissues other than brain after 25 cycles of PCR amplification. In the present study, photoperiod was shown to enhance various reproductive parameters in tilapia, melatonin profiles were defined throughout known periods of light:dark, and a melatonin receptor isolated and partially characterised. However, further research is required to fully characterise the precise function of melatonin in the regulation of tilapia reproduction, especially in terms of its interaction with other endogenous factors, and its relationship with exogenous factors other than photoperiod. Studies in a variety of organisms including amphibians, fish, ascidians, nemerteans, echinoderms, mammals, and even a species of flowering plant, clearly demonstrate that an increase in intracellular egg calcium is crucial to the process of egg activation at fertilization. Mammalian studies suggest that calcium is released from internal egg stores at fertilization by a sperm-derived cytosolic protein factor. Recent studies in the mouse have identified this sperm-derived factor as being a novel sperm-specific phospholipase C (PLC) isoform (PLCQ. Homologues of PLCζ have since been isolated from human and monkey sperm. In addition, sperm factor activity has been detected in non-mammalian species including chicken, Xenopus, and a flowering plant. In this thesis, I report novel evidence for the existence of a similar sperm-derived factor in a commercially important species of teleost fish, the Nile tilapia. Using an established bioassay for calcium release, the sea urchin egg homogenate, it was clearly demonstrated that protein extracts obtained from O. niloticus spermatozoa exhibited PLC activity similar to that seen in mammalian sperm extracts, and also induce calcium release when added directly to the homogenate. Further, sperm extracts prepared from O. niloticus induced calcium oscillations when injected into mouse oocytes, suggesting that O. niloticus sperm contained a similar calcium-mobilizing molecule to that found in mammalian sperm. The same bioassay was used to assay the calcium-releasing properties of sperm extracts prepared from three further commercially important aquaculture species: Atlantic halibut (Hippoglossus hippoglossus), African catfish (Clarias gariepinus), and rainbow trout (Onchorhynchus mykiss). All three of these species exhibited the ability to release calcium in the bioassay, suggesting that the four species of teleost tested in this Thesis appear to use a similar mechanism of egg activation as that reported for mammalian species. However, it was not possible to identify the specific sperm-specific molecule involved, nor the precise cell signalling system used, although present data would support the involvement of a PLC molecule. Several attempts were undertaken to isolate a possible PLC£ homologue from O. niloticus, using molecular techniques such as the Polymerase Chain Reaction (PCR) and screening of a tilapia bacteria artificial chromosome (BAC) library. However, within the time frame imposed by this Thesis, I was unable to successfully isolate a PLC£ homologue from O. niloticus, although research effort in this area is gathering pace and now involves cDNA library screening, and genomic technology. Nevertheless, the results presented herein have provided a valuable insight into the process of egg activation in fish and should provide a stable foundation for future research. Further elucidation of this crucial biological process in fish may help in the reduction of commercial loss post-fertilization, and during early embryonic development.