Gudrun Kausel

Environmental acclimatization of the carp modulates the transcription of β-actin

A cascade of mechanisms involving changes in gene expression are substantial to shape the adaptive responses that a eurythermal fish requires upon environmental changes in its habitat. We have previously shown that the cyclical reprogramming of rRNA transcription is a remarkable feature in carp under seasonal acclimatization. Using in situ hybridization and competitive RT‐PCR we found significant differences in β‐actin transcripts, generally accepted to be coded by a typical housekeeping gene, in tissues from summer‐ and winter‐acclimatized carp. The physiological differential β‐actin transcription herein reported places us on the alert for the reference genes estimated to be constitutive to quantitatively assess gene transcripts. J. Cell. Biochem. 80:223–228, 2000.

Transcription factor Pit-1 expression is modulated upon seasonal acclimatization of eurythermal ectotherms: Identification of two Pit-1 genes in the carp

A second Pit‐1 gene in carp (Cyprinus carpio), including the complete structural gene and 1.1 kb of promoter region, was identified and completely sequenced. The exon‐intron structure was determined, and reverse transcription‐polymerase chain reaction (RT‐PCR) experiments suggest that only one Pit‐1 splice variant is present in carp pituitary. The effect of seasonal acclimatization on the extent of Pit‐1 gene expression was studied in summer‐ and winter‐acclimatized carp. Quantitative RT‐PCR analysis revealed a clear increase of Pit‐1 mRNA in the pituitaries from summer‐acclimatized carp compared with the winter‐adapted fish. In situ hybridization of pituitary gland sections with riboprobes representing the complete 5′‐transactivating region of carp Pit‐1 depicted a significantly higher Pit‐1 mRNA level in the rostral pars distalis of the summer‐acclimatized fish where prolactin is expressed in a manner that resembles the seasonal increase observed in the proximal pars distalis and the pars intermedia. The cell‐ and temporal‐specific transcription of Pit‐1 supports its role in the molecular mechanisms that underly the acclimatization process undergone by eurythermal fish as a result of the physical effects of seasonal changes on their habitat. J. Cell. Biochem. 75:598–609, 1999.

Molecular cloning of Salmo salar Toll‐like receptors (TLR1, TLR22, TLR5M and TLR5S) and expression analysis in SHK‐1 cells during Piscirickettsia salmonis infection

In fish, the innate immune system is the primary response against infection. Toll-like receptors (TLRs) recognize pathogens through pathogen-associated molecular patterns (PAMPs), and some target molecules of TLRs are homologous between fish and mammals. Piscirickettsia salmonis is one of the main pathogens affecting the salmon industry in Chile. Better knowledge of mechanisms underlying its invasive capacity and recognition of target cells is crucial for vaccine development. Therefore, Salmo salar L. TLR1, TLR22, membrane TLR5M and soluble TLR5S sequences were cloned, and expression kinetics were analysed by RT-qPCR in salmon head kidney cells (SHK-1) infected with three different P. salmonis preparations: alive, formaldehyde treated, extract. Clearly, all analysed TLRs were expressed and transcription level changes were revealed at 2 hpi, 12 or 16 hpi and 24 hpi depending on P. salmonis infection scheme. Increased IL1-beta expression confirmed TLR pathway response. Furthermore, significant expression modulations of several members of the TLR pathway in this in vitro model suggest that P. salmonis extract rather than formaldehyde-inactivated bacteria might strengthen the salmon immune system.

Seasonal modulation of growth hormone mRNA and protein levels in carp pituitary: evidence for two expressed genes

Adaptation of eurythermal fish to naturally varying environmental conditions involves modulation of expressions of various factors in the hypothalamo-hypophyseal axis. Here we used three complementary approaches to assess the seasonal variation of growth hormone (GH) protein and mRNA levels in pituitary glands of acclimatized carp fish. First, a polyclonal antibody raised against an oligopeptide derived from the carp GH sequence was used for immunohistochemistry; second, oligonucleotides specific for GH transcripts were used for in situ hybridization. Specific immunodetection of GH coincides with visualization of GH mRNA in the proximal pars distalis, the specific location of somatotroph cells in carp pituitary gland. Finally, competitive RT-PCR analyses confirmed that GH expression exhibits seasonal cyclical reprogramming with higher levels in summer- than in winter-adapted fish. The expression pattern suggests an important role for GH in the molecular mechanisms underlying the acclimatization process. In parallel, amplification of sequences from the fourth intron and adjacent sites from exons IV and V demonstrates the existence of a new GH gene previously undescribed. The detection of transcripts corresponding to each gene suggests that both GH gene copies are active in the duplicated carp genome and that they are similarly affected by seasonal adaptation.

Cloning of the prepro C-RFa gene and brain localization of the active peptide in Salmo salar

In all vertebrates, the synthesis and release of prolactin (Prl) from pituitary lactotroph cells is tightly controlled by hypothalamic factors. We have cloned and characterized a hypothalamic cDNA from Atlantic salmon (Salmo salar) encoding C-RFa, a peptide structurally related to mammalian Prl-releasing peptide (PrRP). The deduced preprohormone precursor is composed of 155 amino acid residues presenting a 87.1% similarity to chum salmon C-RFa and a 100% similarity to all fish C-RFa in the bioactive precursor motifs. C-RFa-immunoreactive perikarya and fibres were located in the brain of S. salar, especially in the hypothalamus, olfactory tract, optic tectum and cerebellum. In contrast, immunolabelled fibres were not observed in the pituitary stalk or in the hypophysis. However, interestingly, we detected immunolabelled cells in the rostral pars distalis of the pituitary in the basolateral region in which Prl is synthesized. These results were confirmed by obtaining a strong signal by using reverse transcription/polymerase chain reaction (RT-PCR) on mRNA from both hypothalamus and pituitary. These data show, for the first time, by immunohistochemistry and RT-PCR, that C-RFa is produced in pituitary cells. Finally, based on these results, a possible function for C-RFa as a locally produced PrRP in this teleost is discussed.

Epigenetic processes in a tetraploid mammal

Polyploidy has played a most important role in speciation and evolution of plants and animals. It is thought that low frequency of polyploidy in mammals is due to a dosage imbalance that would interfere with proper development in mammalian polyploids. The first tetraploid mammal, Tympanoctomys barrerae (Octodontidae), appears to be an exception to this rule. In this study we investigated X chromosome inactivation (XCI) and genomic imprinting in T. barrerae, two epigenetic processes usually involved in dosage control in mammalian genomes. The imprinting status of the Peg1 gene was determined by Peg1 allelic expression studies. The inactive X chromosome was identified on interphase nuclei by immunofluorescence using specific antisera raised against Met3H3K27 and macroH2A1. Quantitative PCR was used to compare the Peg1/Dmd ratio in T. barrerae and in its most closely related diploid species, Octomys mimax. Our data demonstrate that parental-specific silencing of at least one gene and normal X chromosomal dosage mechanism are conserved in the tetraploid genome. We hypothesize a concerted action of genetic and epigenetic mechanisms during the process of functional diploidization of this tetraploid genome.

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

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.

Effect of seasonal acclimatization on the expression of the carp transcription factor Pit-1

We isolated a clone comprising four exons of the carp Pit‐1 gene. Using synthetic oligonucleotide probes derived from the carp Pit‐1 sequence Pit‐1 expression was assessed by in situ hybridization in pituitary sections from summer‐ and winter‐acclimatized carp. Semiquantitative analyses of the hybridization signals revealed a significant higher Pit‐1 expression in the proximal pars distalis (PPD) and pars intermedia (PI) of the pituitary glands from summer‐acclimatized carp, compared to the winter‐acclimatized fish. In both adaptive states, relative to the PPD and PI, only a basal Pit‐1 expression was detected in the rostral pars distalis. Thus, during seasonal acclimatization of an eurythermal fish, Pit‐1 seems to be involved in the mechanisms that underlie the compensatory response.

Glandular kallikreins in the teleost Cyprinus carpio: tissue distribution, possible involvement in prolactin processing, and effect of 17 β-estradiol in vivo

We examined glandular kallikrein (GK), a putative prolactin processing protease, in the teleost Cyprinus carpio. When employing an anti-Centropristis striata GK antibody proteins of 39 kDa in muscle, 52 kDa in gill, 52 kDa in kidney, and two proteins of 46 and 72 kDa in pituitary gland were detected. Immunoreactive kallikreins were recognized in intermuscle cell tissue, epithelial gill cells, apical region of tubular cells, and prolactin producing lactotrophs in pituitary gland, suggesting a osmoregulatory role for this enzyme. We found three prolactin (PRL) variants using anti-tilapia PRL antibodies, in pituitary gland 23 and 16 kDa, and in plasma 23 and 22 kDa forms. Clearly co-localization of GK and PRL in lactotrophs could be demonstrated. In winter-acclimatized male carp, where the pituitary PRL level is low, 17beta-estradiol treatment increased PRL but not GK immunoreactivity. In contrast to GK and PRL co-regulation by estrogen in mammalian pituitary gland, no similar effect on immunoreactive PRL and GK was observed in the ichtyc pituitary. No changes in GK immunostaining occurred in gill or muscle tissue in response to estrogen treatment. These results, taken with the observation of significantly increased GK immunoreactivity in the apical region of kidney tubular cells in estrogen treated male carp, indicate that the regulation of GK expression in pituitary and kidney could be different in fish with respect to mammals.

Environmental reprogramming of the expression of protein kinase CK2β subunit in fish

The dramatic segregation of the nucleolar components in winter-acclimatized carp is the most striking cellular-phenotypical feature observed during the seasonal adaptation of this fish toward the circannual changes in its habitat. Our studies also show that the carp habitat temperature and photoperiod winter conditions provoke a remarkable reduction of both rRNA transcription and the processing of their precursors. To gain knowledge on the mechanisms involved in the regulation of nucleolar activity during the seasonal adaptation process, we studied the behavior of some genes, specifically snoRNA U3 and protein kinase CK2.Consistent with the reduction in the synthesis and processing of pre-rRNA observed during the cold season, the level of CK2β expression decreases in winter when compared to that attained in summer. Similarly, in winter, liver and kidney cells contain lower levels of CK2β subunit protein compared to summer. CK2 is associated with or modifies different factors and enzymes involved in the nucleolar activity; therefore, its higher or lower content could be part of the molecular mechanisms underlying the nucleolar seasonal changes that occur during the compensatory acclimatization process.