We studied the effect of temperature and photoperiod on prolactin (PRL) gene expression in the pituitaries of summer-aclimatized carp (C. carpio). To assess the level of PRL gene transcription, we used a 24mer synthetic oligonucleotide probe derived from the known carp PRL gene sequence. Employing in situ hybridization assays, we observed high expression of PRL mRNA in the rostral pars distalis of summer-acclimatized carp in contrast to the almost negligible PRL transcription which occurs in the winter-acclimatized fish. When experimental combinations of long and short photoperiods with 10°C and 20°C environmental temperatures were studied, only a short photoperiod (8L-16D) in summer-acclimatized carp acclimated to a winter temperature (10°C) markedly depressed PRL gene expression. Our observations indicate that photoperiod constitutes a particularly relevant modulator in the neuroendocrine cascade that activates PRL transcription in the carp.
The basic mechanisms underlying the compensatory response by which eurythermal fish adjust for seasonal changes affecting their habitat, entails reprogramming of the gene expression process both at the transcriptional and translational level (Figueroa et al., 1994; Goldspink, 1995; Krauskopf et al., 1981; Plant et al., 1977; Saez et al., 1982). Carp (Cyprinus carpio) acclimatization involves seasonal modulation of ribosomal RNA transcription, which takes course in different tissues (Vera et al., 1993). In addition, the quantitative expression of liver proteins such as apolipoprotein A-I (Inostroza et al., 1990) and the estrogen induction of vitellogenesis (Hernández et al., 1992) reach the lowest point in cold-acclimatized carp. Likewise, as reported by Goldspink (1995) in muscle of warm-relative to cold-acclimated carp, one myosin heavy chain isoform RNA increases. Because the adaptive response seems to require adjustments of the gene expression process in different tissues, we are working with the hypothesis that seasonal environmental transcription regulation occurs as a result of the transduction of signaling molecules whose synthesis is modulated in connection with the periodic external milieu changes.
Prolactin (PRL), the most versatile pituitary hormone (Sinha, 1995), has been associated with fresh water and salinity adaptation (Bern, 1983; Hirano, 1986; Yada et al., 1992) and with confinement stress response in euryhaline teleosts (Pottinger et al., 1992). It has also been well established that, in mammals, neuroendocrine mechanisms which sense the photoperiod lead to seasonal changes in PRL secretion (Curlewis, 1992). In order to assess if PRL expression was implicated in the acclimatization process, we studied the state of PRL gene expression during the seasonal adjustment of the carp using in situ hybridization assays, and we found high expression of PRL mRNA in the rostral pars distalis (RPD) of summer-acclimatized carp pituitary glands (Figueroa et al., 1994). We observed negligible level of transcription in tissue sections of pituitary glands from the winter-acclimatized fish.
Because seasonal acclimatization of eurythermal fish encompasses molecular responses to changes pertaining to different environmental factors, particularly temperature and photoperiod, it is important to assess if both physical parameters concur in the conformation of the signal that triggers the reprogramming of gene expression. As PRL may be one of the signal molecules coordinating the biochemical adjustment in different tissues, in the present study, we examined, by means of in situ hybridization, the effect of temperature and photoperiod on PRL gene transcription in acclimated carp.
MATERIAL AND METHODS
Male carp (Cyprinus carpio) weighing 1,000–1,500 g were caught in the Calle-Calle River during summer and winter and kept in a fixed 3 × 4m cage submerged 2 m in an effluent of the same river. The temperature of the water in summer and winter was 18–20°C and 8–10°C respectively.
Acclimation of carp
Carp were acclimated in indoor fiber glass tanks (1.0 × 0.5 × 0.7 m) containing 250 l of river water under continuos oxygenation. Water was adjusted to 10°C or 20°C to mimic winter- or summer-environmental temperatures, respectively. Carp maintained in the river were transferred to the fiber glass tanks at the temperature corresponding to their natural environment and adjusted at 1°C per day. Lightbulbs (Spotline, Phillips) were used to emulate daylight. At water surface level a maximum of 5,000 lux was reached. The minimum light intensity at the most distant point was 1,000 lux. Long (16L-8D) and short (8L-16D) photoperiods were used to follow summer and winter conditions, respectively. The fish were kept for at least 21 days under each experimental condition.
Besides summer- and winter-acclimatized control carp, a total of 32 fish were used for this study in two trials. Groups of four carp were subjected to four acclimation conditions (10°C or 20°C combined with either 16L-8D or 8L-16D).
In situ hybridization
The pituitary glands from the control summer- and winter-acclimatized carp, as well as from each of the summer-carp subjected to the four different acclimation conditions, were quickly removed and immersed in 4% (w/v) paraformaldehyde in phosphate saline buffer pH 7.4 (PBS). Sections (12 μm) of the frozen fixed tissues were obtained and placed on slides pre-treated with a solution of 0.01% (w/v) poly-L-Lysine. Samples from each of the set of experiments covering all acclimation conditions were kept at −70°C and processed together. Immediately before hybridization with the oligonucleotide probes, the sections were postfixed with paraformaldehyde, washed in PBS and maintained for 10 min in 2 × standard saline citrate (SSC) (Figueroa et al., 1994).
Synthetic 24mer oligonucleotides probes (sense and antisense), whose sequence included nucleotides G2041-T2064 located in exon V of the carp PRL gene (Chen et al., 1991; Figueroa et al., 1994), were used. The sequences of the oligonucleotides were unique with respect to the carp growth hormone gene (Chiou et al., 1990). The probes were labeled with digoxigenin-11-dUTP at the 3′ end using terminal deoxynucleotidyl transferase and the in situ hybridization was carried out as described elsewhere (Figueroa et al., 1994).
After hybridization, the slides were washed twice with 2 × SSC, once with 1 × SSC for 1 hr at room temperature, followed by a 30 min wash with 0.5 × SSC at 37°C and, lastly, by a 30 min rinse with 0.5 × SSC at room temperature. The immunological detection of the hybridized probes was accomplished as described (Figueroa et al., 1994). Quantification of the label in the pituitary sections was carried out by a modification of the procedure described by Ayson et al., (1994) using an automated image digitizing system (UN-SCAN-IT, Silk Scientific, Inc., USA). Six areas of approximately 0.2 mm2 each were randomly selected in both the rostral pars distalis (RPD) and in the proximal pars distalis (PPD), the later yielding background values. In each of the 12 areas of each pituitary sagital section, the value of pixels representing the density of the label was quantified. The total pixel values representing each of the six areas examined in each microscopic image of the RPD were corrected by substracting the average value rendered by the pixels corresponding to the PPD areas. Statistical analyses were performed using the Student's t-test. P < 0.05 was considered significant.
The in situ hybridization signals for PRL mRNA were always localized in the RPD (Fig. 1). Molecular hybridization did not occur when the digoxigenine-labeled sense oligonucleotide probe derived from the carp PRL gene sequence was used.
As shown in Fig. 1A, high expression of PRL mRNA was observed in summer-acclimatized fish, whereas the winter-adapted carp exhibited a remarkably low hybridization (tissue section not shown, see Fig. 2F). The level of PRL mRNA in the pituitary glands from summer-acclimatized carp did not change upon acclimation at 20°C under long (16L-8D) and short (8L-16D) photoperiods (Fig. 1B, C). A long photoperiod likewise rendered comparable levels of PRL gene expression in 10°C acclimated fish (Fig. 1D). However, upon acclimation of the summer-acclimatized fish at 10°C, a short photoperiod produced a drastic decrease of PRL transcription (Fig. 1E). The quantification and statistical analysis of the level of PRL transcription attained in the summer-acclimatized carp and in each of the four acclimation conditions is shown in Fig. 2. Also, the level of the in situ hybridization signals for PRL mRNA in pituitaries from winter-acclimatized carp (Fig. 2F). Clearly, acclimation of summer-acclimatized fish to cold temperature and a short photoperiod (Fig. 2E) renders a significant difference in PRL transcription, comparable to that attained during the natural seasonal condition of the fish (Fig. 2F).
Both acclimation and acclimatization studies have, in general, favored the consideration of temperature as the direct effector of compensatory responses in fish. Concerning shifts in gene expression, it is clear that reprogramming may be brought about by metabolic effects, by differences in the hormone levels, photoperiod, and even by mechanical activity (Figueroa et al., 1994; Goldspink, 1995).
In euryhaline teleosts, PRL plays a substantial role in maintaining hydromineral balance in freshwater (Bern, 1983; Brown and Brown, 1987; Hirano, 1986; Hirano et al., 1987). Utilizing in situ hybridization, Nishioka et al. (1993) found that either tilapia PRL177 or tilapia PRL188 gene expression varied according to the environmental salinity from which the tilapia (Oreochromis mossambicus) pituitaries were removed. In situ hybridization signal was significantly stronger in freshwater pituitaries than in seawater pituitaries.
In the present study, acclimation of carp, although known to be insufficient to mimic the complexity of acclimatization (Segner and Braunbeck, 1990), provided an unique opportunity to ascertain the effects of temperature and photoperiod on PRL gene transcription. All the acclimation experiments were carried out with male carp to elude hormonal changes linked with the reproductive cycle that takes place throughout seasonal changes.
Using in situ hybridization, we were able to detect a profound photoperiodic effect in summer-acclimatized carp acclimated to the winter temperature. While changes in the photoperiod did not significantly affect summer fish acclimated to their season's temperature, a short photoperiod (8L-16D) remarkably depressed PRL transcription in the rostral pars distalis of the carp acclimated to the winter temperature (Fig. 1E). Acclimation of the summer-acclimatized carp to winter conditions, i.e. low temperature and a short photoperiod, resulted in a PRL gene expression level comparable to the physiological manifestation attained during the cold season (Fig. 2F). While photoperiod by itself at the corresponding summer temperature is insufficient to determine changes in PRL transcription, it certainly plays an important role in the acclimation of summer-acclimatized carp to winter temperatures.
Earlier acclimation studies by McKeown and Peter (1976) showed that in goldfish, Carassius auratus, longer photoperiods and higher temperatures caused pituitary PRL release. Serum PRL changed on a circadian rhythm depending on the length of the photoperiod (McKeown and Peter, 1976). In other fish, such as the trout Onchorhynchus mykiss, plasma PRL levels do not show a pronounced annual cycle (Rand-Weaver et al., 1995). However, in male and female trout, PRL levels were inversely correlated to water temperature (Rand-Weaver et al., 1995).
It is well known that all circadian systems imply a certain degree of cell autonomy and that a photic entrainment pathway is always present as an input (Takahashi, 1993). It is also well established that seasonally breeding eutherian species show the characteristic pattern of high plasma PRL level in spring and summer and a low concentration in the colder seasons, with photoperiod being the major environmental factor controlling this circannual pattern (Curlewis, 1992). Although it is known that in mammals the photoperiod acting via pineal melatonin secretion is involved in the control of the seasonal changes in plasma PRL levels, the mechanism by which melatonin interacts with the lactotrophs remains unclear. In carp, plasma melatonin levels have been assessed for daily cycles and the observed rhythm suggests that melatonin is an important hormone in photoperiodism and circadian rhythm in the fish (Kezuka et al., 1988). Our observations in acclimated carp have been focused on the first measurable event of PRL gene expression and suggest that the photic stimuli constitute a particularly relevant modulator in the neuroendocrine cascade that activates PRL transcription.
We would like to thank Ms. Susan Watson and Prof. Oriana Gonzalez for their critical reading of the manuscript. This work was supported by grants 1940845 from FONDECYT and GE/GLO/90/004 UNIDO/ICGEB.