Preprint volume - SIBM

Pre-print Volume –Posters
Topic 4: THE ELASMOBRANCHS
C. GAMBARDELLA 1,2 , L. GALLUS 2 , S. FASULO 1 , G. TAGLIAFIERRO 2 ,
M. VACCHI 3 , S. FERRANDO 2
1 Dip. di Biologia Animale ed Ecologia Marina, Università di Messina,
Salita Sperone, 31 – 98166 Messina, Italia.
2 LIBiOM, Dip. di Biologia, Università di Genova, Italia.
chiara.gambardella@unige.it
3 ISPRA, c/o National Antarctic Museum, Università di Genova, Italia.
FIRST REPORT ON LEPTIN IN A CARTILAGINOUS FISH
PRIMA EVIDENZA DELLA LEPTINA IN UN PESCE CARTILAGINEO
Abstract - Leptin is a hormone involved in food intake. It is conserved through evolution, but no studies that
have focused on its presence in cartilaginous fish are available to date. Here we report the presence of
leptin-like immunoreactivity in the gut of the cartilaginous fish Scyliorhinus canicula (Linnaeus,1758) using
Western Blot and immunohistochemical analyses. A 16 kDa band, corresponding to leptin, was detected in
the homogenate of the stomach, while no immunoreactivity was observed in the intestine.
Immunohistochemistry of the gastric mucosa revealed leptin-like staining localized to mucous-secreting
cells and endocrine cells. This is the first report on the presence of a leptin-like peptide in a cartilaginous
fish.
Key-words: fish, histochemistry, stomach, hormones.
Introduction - Leptin is a 16 kDa circulating hormone produced by the ob gene
identified in mammalian adipose tissue (Zhang et al., 1994) that plays a key role in the
regulation of food intake and energy balance (Schwartz et al., 2000). The leptin gene
has been sequenced in mammals, birds, amphibians and bony fish (Zhang et al., 1994;
Doyon et al., 2001; Kurokawa et al., 2005) but no information on leptin is available in
cartilaginous fish. Leptin expression has been reported in adipose tissue, liver and gut
of many vertebrates (Johnson et al., 2000; Muruzàbal et al., 2002; Kurokawa et al.,
2005; Gambardella et al., in press). Among fish, most studies on leptin have been
restricted to bony fish, therefore we attempted to verify the presence of leptin in the
small-spotted catshark (Scyliorhinus canicula) as a representative cartilaginous fish,
since it has often been used as a model species in the study of vertebrate evolution.
Materials and methods - Five adult specimens of S. canicula were collected in the
Ligurian Sea, anesthetized, killed and then dissected to collect the gut. For
immunohistochemistry, the stomach and the intestine were fixed in 4%
paraformaldehyde in a 0.1 mol/L phosphate-buffered solution (pH 7.4) at 4 °C,
embedded, and cut into 5-μm thick sections. Immunohistochemical labeling was
performed using rabbit polyclonal antiserum against leptin (LEP) (1:200; Santa Cruz
Biotechnology Inc.). Reactions were visualized by immunofluorescence and by
immunoperoxidase, using DAB (di-amino-benzidin, Sigma). Negative controls were
performed by neutralising LEP primary antiserum with its antigen (1:200; Santa Cruz
Biotechnology Inc.). For Western Blot analyses stomach and intestine homogenates
were prepared according to the Laemmli method (Laemmli, 1970) on a 10%-23%
gradient SDS-polyacrylamide gel.
Results - Leptin-like immunoreactivity (ir) was only observed in the gastric mucosa.
Leptin-like ir cells showing two different morphological aspects were located in the
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