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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.34 n.1 Santiago  2001

http://dx.doi.org/10.4067/S0716-97602001000100010 

In situ hybridization of somatolactin transcripts in the
pituitary glands from acclimatized carp (Cyprinus carpio)

MAURICIO LÓPEZ,b JAIME FIGUEROA,b GUDRUN KAUSEL,b MARÍA INÉS VERAa
AND MANUEL KRAUSKOPFa *

aFacultad de Ciencias de la Salud, Universidad Nacional Andrés Bello and MIFAB, Santiago, Chile.
bInstituto de Bioquímica, Universidad Austral de Chile, Valdivia, Chile

ABSTRACT

We isolated and cloned a carp somatolactin SL DNA fragment, of which 78% of the nucleotides were identical to the corresponding salmon SL sequence. The results obtained upon Northern blot hybridization of carp pituitary RNA allowed the identification of two transcripts as described for other fish.

When the content of SL transcripts in pituitary sections from summer- and winter- acclimatized carp was quantified by in situ hybridization assays, we found no significant differences between the two seasons. In salmonids, plasma SL reaches higher levels in summer than in winter in synchrony with the water temperature cycle; in the eurythermal carp, however, the complex adaptive responses imposed by seasonal environmental changes do not seem to include the regulation of the somatolactin detected with the probe used at the transcriptional level in pituitary glands.

Key terms: Acclimatization; In situ hybridization; Somatolactin expression; Pituitary; Carp; Fish

INTRODUCTION

Acclimatization and acclimation in eurythermal fish involves the reprogramming of transcription in different tissues (Figueroa et al., 1994; Goldspink 1995; Tiku et al., 1996; Arends et al., 1998; Vera et al., 2000). We have been working with the hypothesis that a molecular signaling system coordinately modulates the compensatory response toward the cyclical habitat changes, i.e. temperature and photoperiod. Furthermore, that the pituitary gland plays a relevant role in the complex process that transduces the periodic external milieu changes into signaling molecules that can be implicated in the control of gene expression. Accordingly, we have found a high level of prolactin (PRL) mRNA in the rostral pars distalis (RPD) of summer-acclimatized carp in comparison to the negligible transcription detected in the winter acclimatized fish (Figueroa et al., 1994) and that photoperiod plays an important role in the neuroendocrine cascade that activates PRL expression (Figueroa et al., 1997). More recently we have shown that the transcription factor Pit-1 that modulates PRL expression as well as the transcription of other anterior pituitary hormones is transcribed at significantly higher levels in summer-acclimatized carp with respect to the winter-adapted fish (Kausel et al., 1999). Furthermore, the expression of the carp prolactin receptor is modulated during the acclimatization process of the fish (San Martin et al., 2000)

Somatolactin (SL), a piscine pituitary hormone belonging to the PRL/GH family, was first characterized in Atlantic cod and flounder (Ono et al., 1990). Anti cod SL serum locates SL in periodic-acid-Schiff (PAS)-positive cells (PIPAS cells) in pars intermedia (PI) of several teleost pituitary glands and in the homologous chromophobic cells in salmonid PI (Rand-Weaver et al., 1991a,b). Prior to SL identification, PIPAS cells were shown to become activated by a variety of external stimuli, such as dark backgrounds (van Eys, 1980), low pH (Wendelaar-Bonga et al., 1986), low calcium, or low osmolarity of the ambient water (Olivereau and Olivereau, 1982), which suggests that the cells may be synthesizing specific molecules in response to environmental changes.

SL production and storage has been shown in SL-immunoreactive cells in rainbow trout (Kaneko et al., 1993). In salmonids, plasma SL fluctuation indicates a role for this hormone in gonadal maturation, smoltification, stress response and seasonal cycle (Rand-Weaver et al., 1993a,b; Rand-Weaver et al., 1995). In red drum (Sciaenops ocellatus), a euryhaline perciform fish, SL seems to play a role in background and low-illumination adaptation (Zhu et al., 1999). Nevertheless, despite the multiple biological activities attributed to SL, its primary function remains unclear.

Carp acclimatization certainly involves the sensing of the physical parameter habitat changes and the corresponding transduction into molecular signals. Searching for environmentally-induced pituitary gland molecules involved in the cascade of events that provides the needed adaptive homeostasis of the fish, we undertook the identification of carp SL gene coding sequences to assess the expression of this protein vis a vis the cyclical seasonal changes that characterize the habitat of this fish. Except for carp, the SL primary coding sequences of several teleost species have already been reported (Ono et al., 1990; Takayama et al., 1991; Iraqi et al., 1993; Pendón et al., 1994; Astola et al., 1996; Cheng et al., 1997; May et al., 1999). In this report, we show that total SL expression appears not to be associated to the seasonal acclimatization of the carp.

MATERIAL AND METHODS

Animals and tissue preparation

Carp fish were caught in summer (8-10°C) and winter (20-22°C) and maintained in a 3 x 4m fixed cage submerged 2m in an affluent of the Calle Calle River two weeks prior to being sacrificed by decapitation. Only male carp were used in this study. Some of the dissected pituitary glands were processed immediately to extract RNA, while others were fixed in 4% paraformaldehyde in phosphate saline buffer pH7.4 (PBS) for 60 min, cryostat sectioned (10 mm) for in situ hybridization (Figueroa et al., 1997) and stored at -70°C to analyze the summer- and winter samples simultaneously.

Isolation and cloning of carp SL DNA fragments

Carp genomic DNA was prepared from nucleated blood cells (Sambrook et al., 1989). Alignment of derived SL amino acid sequences (lumpfish Acc.No:P45640; Atlantic halibut Acc.No.P45641; flounder Acc.No.:P20362; Atlantic cod Acc.No.: P21919; chum salmon Acc.No.:P24405) revealed consensus blocks proceeding from exon IV (amino acids 124 - 130 and 154 - 160). Thus, based on the salmon cDNA sequence, a pair of oligonucleotide primers were designed (sense sSL-1: 5'-CCATTGGTGTACCTGCAAACC-3'; anti-sense sSL-3: 5'-CACCACTCCTTGCTCCA AACT-3') that yielded an amplification product of 112bp using 70ng of carp genomic DNA as template in a standard polymerase chain reaction. The latter was performed with 2.5U Taq DNA polymerase using 30 cycles (93°C for 30 sec, 55°C for 30 sec, 72°C for 10 sec) followed by a 10 min extension step. The unique amplification product was cloned in pGEM-T (Promega) and sequenced with the f-mol Quick Sequencing Kit (Promega) on both strands.

Northern blot analysis

Total pituitary RNA was prepared with Trizol (GibcoBRL) according to the supplier's instructions. Ca. 30µg total RNA were fraccionated on a 1.2% denaturing agarose formaldeyde gel (Sambrook et al., 1989), transferred to Hybond Nylon Membrane (Amersham), hybridized overnight at 42°C with the 112bp carp SL specific fragment labelled during amplification with a32P-dCTP (Sambrook et al., 1989), washed with a final solution of 0.1 x SSC, 0.1% SDS at 65°C for 5min and exposed to Kodak X-Omat film at -70°C for one week.

In situ hybridization

In situ hybridization was performed as described (Figueroa et al., 1997) employing the carp SL antisense specific oligonucleotide, sSL-2 (5'-GGTTTGCAGGTACA CCAATGG-3') and the corresponding complementary sense 21mer oligonucleotide (sSL-1). Labeling of the probes with digoxigenin at the 3'-end and detection of the hybridization signals followed the procedures described (Figueroa et al., 1997).

Digitalized images were captured with a SONY camera directly connected to the microscope (Nikon Eclipse 300). Quantification of the labeled signals was corroborated with two independent automated image-digitizing systems. Using UN-SCAN-IT (Silk Scientific, Inc.) the value of pixels representing the density of the label was quantified in twenty squares (0.5µm2) per section in pituitary sections from various different individuals of summer- and winter-acclimatized fish in the reactive pars intermedia (PI) and corrected for background values in non-stained areas (Kausel et al., 1999). With the system Image Pro Plus 3.0 (Media Cybernetics, USA) the integrated pixel values over the selected reactive area were obtained and expressed as Integrated Optical Density (IOD). Statistical analyses were performed using the Student's t-test. P<0.05 was considered significant.

RESULTS AND DISCUSSION

As shown in Figure 1, the sequence of the cloned 112bp DNA fragment amplified by PCR from carp genomic DNA is 78% identical to the corresponding salmon SL DNA sequence. When the carp SL fragment was used as probe to hybridize total carp pituitary RNA in a Northern blot, two bands of 1.9kb and 0.95kb were detected (Fig. 2). These results are consistent with those reported in red drum, seabream, lumpfish, flounder, halibut and eel, where two SL transcripts were also found (Zhu et al., 1999). In red drum, lumpfish and eel the transcripts appear to be due to the presence of multiple polyadenylation signal sites derived from the same gene (Iraqi et al., 1993; Zhu et al., 1999).


Figure 1: Nucleotide sequence of a partial carp SL gene fragment (accession number AF264044) and alignment with corresponding salmon SL sequence (accession number D10638). The olignucleotides employed to amplify the carp SL DNA fragment are highlighted in bold.


Figure 2: Northern blot analysis of carp pituitary RNA. A) 30µg of total pituitary RNA B) hybridized with the carp SL specific probe.

In situ hybridization with the SL antisense specific 21mer oligonucleotide (sSL-2) confined the labeling of the SL mRNA to cells in the well-developed PI of this teleost (Fig. 3). No labeling was attained with the corresponding sense oligonucleotide (not shown). The sequence analyses, the Northern blot identification of the two bands described for other fish, and the unique location of the signals in the PI, clearly confirmed the specificity of the probe to assess carp SL mRNA.


Figure 3: In situ hybridization in carp pituitary sagittal sections (magnification 20x) with SL specific oligonucleotide. The graphs depict quantification of the SL specific signal in the pars intermedia (PI) of winter (n = 8) and summer (n = 15) acclimatized carp with two different analyzing systems. A) UN-SCAN-IT, B) Image Pro Plus. PPD = proximal pars distalis.

Quantification of the in situ hybridization signals integrating the whole reactive area in the pituitary sections from eight winter- and fifteen summer- acclimatized fish, only revealed an insignificant tendency toward higher SL transcripts in summer carp. Two different digitalizing systems were used. Neither could detect significant differences on the level of SL transcripts in summer- and winter-adapted carp (Fig. 3).

Changes of the SLI and SLII mRNA content in red drum detected by Northern blot analyses seem to reflect light and illumination effects (Zhu et al., 1999). These studies were performed in acclimated fish under varying light conditions while maintaining a constant temperature. Acclimatization is a physiological process that does not precisely mimic the controlled acclimation experiments (Segner and Braunbeck 1990). Additionally, the quantification of red drum SLI and SLII transcripts was accomplished by standardizing with the transcription attained with red drum ß-actin (Zhu et al., 1999) We have recently shown that the seasonal acclimatization of carp results in physiological differential ß-actin transcription (Sarmiento et al., 2000), similarly to what has been observed with the transcription of rRNA (Vera et al., 1997), which puts us be on alert for the reference genes estimated to be constitutive to quantitatively assess gene transcripts in fish subjected to environmental changes.

The in situ hybridization approach has served to determine the remarkable transcriptional modulation of PRL, GH and the transcription factor Pit-1 in pituitary glands from carp undergoing seasonal acclimatization (Figueroa et al., 1994; Figueroa et al., 1997; Kausel et al., 1999), an observation validated by competitive RT-PCR (Kausel et al., 1999). The correlation between the quantitative analyses of the in situ hybridization detection of transcripts and competitive RT-PCR has recently been documented examining ß-actin transcription in different tissues of summer- and winter-adapted carp (Sarmiento et al., 2000).

It is well known that acclimation in the carp is a very complex process that includes compensatory responses in muscle (Goldspink 1995) and cell membranes (Tiku et al., 1996) associated with the regulation of the expression of specific genes. It has also been observed that an essential feature of acclimatization is the generalized nucleolar rearrangement representing profound changes in ribosomal biogenesis (Vera et al., 1997; Vera et al., 2000; Quezada et al., 2000).

Although we acheived the identification of a specific probe for carp SL transcription, we did not find significant changes associated with the acclimatization of the carp, which could, at this control level, respond for a role of SL in the adaptive response. Yet PRL, GH, and SL, appear to be partially controlled by the transcription factor Pit-1 (Kausel et al., 1999) that, as PRL and GH depicts differential expression (Figueroa et al., 1994; Figueroa et al., 1997) during the cyclical seasonal adaptation of the carp. In salmonids, a pronounced seasonal rhythm of plasma SL reaching the highest levels during summer and the lowest in winter is in synchrony with the water temperature cycle (Rand-Weaver et al., 1995). In red drum, the ratio of the two SL mRNAs seems to be influenced by the level of background light stimuli (Zhu et al., 1999). It is known that duplicated genes are common in the tetraploid carp (Ferris and Whitt, 1977) and that there are two POMC genes that appear to be differentially expressed during temperature stress (Arends et al., 1998). Therefore, although our results did not show that SL gene expression was seasonally modulated, the in situ hybridization with a probe representing a highly conserved coding carp SL sequence cannot rule out the differential expression of two or more mRNA.

ACKNOWLEDGEMENTS

This work was supported by Grant 1970651 from FONDECYT, and DI-UNAB 60-A/99. The Millenium Institute of Fundamental and Applied Biology (MIFAB) is financed in part by the Ministerio de Planificación y Cooperación (Chile)

Corresponding author: Dr. Manuel Krauskopf, Universidad Nacional Andres Bello, Avenida República 237, Piso 2, Santiago, Chile. Fax: (562 ) 698-0414, E-mail: mkrausk@abello.unab.cl

Received: December 29, 2000. Accepted: March 6, 2001.

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