Expression pattern of genes of hormones that control growth in fish

Felix G. Ayson¹, Evelyn Grace T. de Jesus-Ayson¹ and Akihiro Takemura²

¹Southeast Asian Fisheries Development Center, Aquaculture Department (SEAFDEC AQD), Tigbauan 5021, Iloilo, Philippines
²Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, 3422 Sesoko, Motobu, Okinawa 905-0227, Japan

In aquaculture or the farming of fish in a controlled environment like in fishponds, in land-based tanks, and in net cages in the sea, growth is highly dependent on food availability. Whether the culture system used is extensive, where the fish stocks rely mainly on the presence of natural food like algae and other aquatic organisms in the culture environment such as in fishponds, or intensive, where the stocks are largely, if not totally dependent on artificial feeds like in high density culture of milkfish in open sea cages, the food factor is a major determinant of success. Because giving excess feeds is mistakenly equated with faster growth, fish stocks tend to be overfed, as usually is the case in intensive culture conditions. Since feed cost is a major cost item in intensive aquaculture operations constituting roughly 60% of the production cost, any means of reducing this without affecting the growth of the fish will certainly help the fish farmers.

Growth in fish, as in other mammals, is a complex process. Genetics, nutrition, environment, and many other physiological factors are involved. Several hormones in the “brain-pituitary-liver axis” or the “growth axis” play a key role in this process. These hormones include the growth hormone releasing hormone (GHRH) and the growth hormone release inhibiting factor (GHRIF) in the brain, growth hormone (GH) in the pituitary gland, and insulin-like growth factor I and II (IGF-I and IGF-II) in the liver. Upon stimulation by GHRH from the brain, the pituitary gland secretes GH into the bloodstream, and GH in turn stimulates the liver to secrete IGFs, mainly IGF-I. IGF-I then binds to its receptors in many organs in the body and this binding triggers a series of cellular events that result in processes like cell proliferation and differentiation, and ultimately results in growth.

All these hormones act in concert to effect growth and their secretion patterns follow a rhythm according to a given set of environmental and physiological factors. One of these factors is nutrition. This most recent study of ours tries to look at whether the gene expression patterns of these hormones (GH, prolactin (PRL), somatolactin (SL), IGF-I and IGF-II) change with a shift in the feeding schedule as well as during times of food restriction or starvation. This is as a prelude to future work of developing a feeding strategy that is applicable in aquaculture systems that would reduce the amount of feeds given, thereby reducing the feed cost, without affecting the fish’ growth. PRL and SL were included in the investigation since these two hormones are members of the growth hormone family of proteins. PRL and SL share structural similarities with GH and as such they also share some biological properties and have overlapping functions.

Using rabbitfish as the experimental fish, a series of experiments were done to address 2 basic questions. The first was whether the expression pattern of these important hormones changes with a shift in the feeding time. The hypothesis was, if the gene expression follows a rhythm in relation to the feeding time then an expression pattern may be observed in fed fish with feeding time as reference, and that this rhythm may be non-existent in starved fish. Three series of experiments were done to address this question with feeding time as the variable. In all 3 experiments, fish were first acclimatized to the experimental tank conditions for 3 days during which food was given in the morning at about 10:00 AM. After the acclimatization period, the fish were reared for another 7 days during which the feeding time was set at 10:00 AM (Experiment 1), 3:00 PM (Experiment 2), and no feeding (Experiment 3). After 7 days of feeding at the prescribed feeding time (for experiments 1 and 2) and 3 days of no feeding (for experiment 3), representative samples of fish were sacrificed every 3 hours for 24 hours and the pituitary glands and the liver were removed for measurement of the gene expression levels of GH, PRL and SL, and IGF-I and II, respectively.

No pattern was observed in GH gene expression in relation to the feeding time. However, a diurnal pattern with high GH levels during night time and low levels during day time is observed which is similar in other mammals like in humans. Gene expressions for PRL and SL were also irregular and did not follow a pattern with feeding time. SL gene expression, were low during day time especially during mid day which is consistent with the established inverse relationship between SL’s physiological activity and illumination levels in fish. While the expression of the three pituitary hormones do not seem to follow a pattern with feeding time, IGF-I gene expression in the liver showed a trend. IGF-I gene expression pattern was influenced by feeding time where the highest levels were recorded consistently 5-6 hours after feeding. This pattern in IGF-I gene expression was not observed in the group of fish that were not fed. This observation suggests a particular need for IGF-I some time after feeding probably to aid or hasten the chemical breakdown of food by inducing the secretion of key digestive enzymes and facilitate the absorption of nutrients. Indeed, there is evidence that IGF-I can induce the activity of lipase, an enzyme that catalyzes the breakdown of lipids (Tremblay et al., 2001). While IGF-I gene expression consistently showed a rhythm with feeding, IGF-II gene expression was irregular and did not follow a pattern with feeding time. A common observation in all 3 experiments, however, is the consistently high level of IGF-II during early morning (06:00 AM). The physiological significance of this high IGF-II level early in the morning is presently unknown, although it may be related to the onset of light as the photic cue that synchronizes the daily secretion patterns of some hormones as observed in other fish species like rainbow trout (Boujard and Leatherland, 1992).

The second question was whether the hormone expression pattern changes when food is limiting or during periods of short-term starvation. A 15-day starvation experiment was conducted. Two groups of fish were acclimatized for 7 days in experimental tank conditions and feed daily at 10:00 AM. On the 8th day, food was withheld in one group and this continued for another 15 days (starved group). After 15 days of no feeding, the starved group was re-fed for 6 days. The control group was given food for the entire duration of the experiment. In both groups, representative samples of fish were sacrificed at the start of the experiment (for initial samples), at days 3, 6, 9, 12, and 15 of starvation, and at days 3 and 6 of re-feeding. During every sampling, the pituitary glands and livers were removed for the determination of gene expression levels of GH, PRL and SL in the pituitary gland, and IGF-I and II in the liver.

GH gene expression was significantly elevated starting on the 9th day of starvation and remained high until the last day of starvation. The levels returned back to normal when the starved fish were re-fed, a clear indication of GH response during times when food is limiting. Although metabolism is one of the many functions of PRL, very little is known about its involvement during fasting or starvation in fish. In our study, a very clear reduction in PRL gene expression was observed with starvation. The level started to decrease on the 6th day of starvation and it became more pronounced as starvation progressed. Interestingly, the level returned back to normal after re-feeding. Our results are the first report on a very clear reduction of PRL gene expression during starvation in fish. This finding about PRL is not totally surprising since in mammals PRL is known to affect metabolic homeostasis by regulating key enzymes and transporters that are associated with glucose and lipid metabolism (Ben-Jonathan et al., 2006). Since there is a need to mobilize the fat reserves to supply the energy requirement during period of starvation, the enzymes involved in fatty acid mobilization and synthesis need to be activated. Because of the inhibitory role of PRL on these enzymes, it makes sense that during period of energy deficit such as during starvation, PRL level will be reduced. In the case of SL, there is some evidence for its involvement in energy homeostasis in sea bream. Like in other fish species studied to date, however, we did not observe significant changes in the expression of SL genes in fish that were fed and starved. This may indicate that unlike GH and PRL, SL may not be critically involved or play an active metabolic role during starvation in fish.

IGF-I, like GH, have metabolic functions so that it can only be expected that nutritional status is one of the factors that regulate GH-IGF-I axis in fish. The typical response of fish to fasting or starvation is an increase in both GH gene expression and GH protein level in the blood, and a decrease in liver IGF-I gene expression and IGF-I protein level in the blood. This opposing picture of GH and IGF-I activity during fasting is explained by the development of GH resistance in the liver which results in the decrease in the number of GH receptors that consequently leads to reduced GH binding and impairment of the GH signaling pathway. The results of the present study is consistent with what is observed in many fish species, except that a significant increase in IGF-I gene expression was observed early in the starvation period, during days 3 and 6 after starvation, which was not observed in many previous studies.

Like IGF-I, IGF-II is also a potent growth factor especially during early development. It stimulates cell proliferation and DNA synthesis in zebrafish embryonic cells (Pozios et al., 2001). Whether it also functions as a metabolic hormone is not yet known although there is preliminary evidence that IGF-II regulates metabolism in trout muscle cells (Codina et al., 2004). Unlike IGF-I, however, where a very clear relationship with GH exists during starvation, nothing is known about the behavior of IGF-II during altered nutritional status in fish. In higher vertebrates, though, there is evidence that IGF-II is regulated by nutritional status and is age dependent as in sheep where it is more sensitive to nutritional status in older but not in younger sheep (Hua et al., 1995). In the present study, the initial response of IGF-II to starvation is an increase in its gene expression which occurred after 3 and 6 days of starvation after which the levels returned back to normal and were no longer different from the fed group.

As expected, the body weight of the fish in the starved group decreased during the 15 day starvation period but the weight reduction was not significant – a mean weight loss of 3.8 g during 15 days. The fed group continued to grow during 15 days experiment gaining a mean body weight gain of 3.7 g. What is interesting is that the starved group showed tendency to catch up growth after re-feeding which in our experiment was only for 6 days. There is some evidence of catch up growth in fish after re-feeding following some period of food restriction (Xie et al., 2001). As shown also in the results, the expression of GH and IGF-I, the major hormones that control growth, were also normalized soon after re-feeding. Considering that the fish will exhibit compensatory growth when re-fed after some period of food restriction, there exists the potential of subjecting the fish to some degree of feed restriction during the culture period. A reduction in the amount of food will be realized during the feed restriction period and yet not affecting the fish’ overall growth. The appropriate duration of feed restriction is critical in this regard. Once established, this promises to be a good approach to reduce feed cost without affecting total production.

Literature cited:

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Felix G. Ayson obtained his Doctor of Science Degree in Zoology with specialization in Fish Physiology and Endocrinology from the University of Tokyo. He was a researcher at the Southeast Asian Fisheries Development Center Aquaculture Department (SEAFDEC AQD) in Iloilo, Philippines since 1989. In August this year, he joined the Food and Agriculture Organization (FAO) of the United Nations as a Chief Technical Adviser.

Evelyn Grace T. de Jesus-Ayson got her Doctor of Science Degree in Zoology with specialization in Fish Physiology and Endocrinology from the University of Tokyo. She is currently the Head of the Research Division of SEAFDEC AQD.

Dr. Akihiro Takemura is an Associate Professor at the Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa, Japan.