Page 2 - Brain_morphology_2008

G. Palmieri et al.

To increase the possibility of success in bluefin Immunohistochemistry
tuna domestication, a better understanding of the De-waxed and re-hydrated tissue sections were
reproductive biology of this fish is required, with immersed in 3% H2O2 for 10 min to suppress
special focus on the brain-pituitary-gonad axis. endogenous peroxidase activity and then rinsed with
The aim of this paper was i) to provide data on distilled water and phosphate buffered saline (PBS,
the gross morphology of bluefin tuna brain; ii) to 0.01M, pH 7.4) containing 0.15 M NaCl, 1%
describe the main diencephalic nuclei involved in bovine serum albumin (BSA) and 0.5% Triton X-
reproductive function; iii) to immunolocalize, in 100. Non-specific binding sites for immunoglobu-
both the forebrain and midbrain, the neurons lins were blocked by incubation for 30 min with 5%
responsible for GnRH synthesis. normal goat serum (NHS) in PBS containing 1%
BSA. Sections were then incubated for 15-24 h at
4°C in a moist chamber with nine different dilutions
Materials and Methods (1:100, 1:500, 1:1000, 1:1500, 1:2000, 1:2500,
1:3000, 1:4000, 1:5000) of polyclonal primary
Sampling antisera against salmon(s)GnRH, chicken(c)GnRH
Both brain and pituitary were collected from 18 and sea bream(sb)GnRH, used to determine the
sexually mature bluefin tuna killed during the peri- optimal working dilution. Anti sGnRH (lot n. 1667)
od of natural spawning of the species (June-July, and anti cGnRH (lot n. 675) were kindly supplied
2005 and 2006) at several locations in the by R. P. Millar; anti cGnRH (lot n. 6) and anti
Mediterranean Sea.The total body mass and brain sbGnRH (lot n. 691) were kindly provided by M.
mass of each specimen were measured to the near- Amano.
est kg and to 0.01 g, respectively. All antisera were diluted in 5% BSA/0.01 M
Samples were fixed in 10% buffered formalin for PBS/0.5% Triton X-100, pH 7.4. The resulting
24 h, dehydrated through graded alcohols, cleared suitable working dilutions (no background) for each
in xylene and embedded in paraffin wax.Twelve- or antiserum are given in Table 1. The slices were
five-µm thick sections were cut for morphology or rinsed in Triton-PBS for 15 min and incubated with
immunohistochemistry investigations, respectively. biotinylated secondary antibody for 30 min to be
washed in PBS and re-incubated in avidin-biotin-
Encephalization quotient and morphology peroxidase complex (ABC) (Vector, Burlingame,
The encephalization quotient (QE) was calculat- CA). Peroxidase activity was visualised by 10-min.
ed as the ratio of actual brain size to expected brain incubation with Vector DAB Peroxidase Substrate
size using the formula: Kit (Vector, Burlingame, CA), which produces a
brown precipitate. Finally, the sections were coun-
QE = EA EE -1 terstained with hematoxylin, dehydrated and
mounted.
where EA = actual brain mass and EE = expected Negative controls were performed by: (1)
brain mass. replacement of primary antibody with NGS, and
The EE for bluefin tuna was calculated by the (2) omission of primary antibody.
allometric equation for the brain mass to body
mass relationship (Log10 brain mass = 0.5301
Log10 body mass 0.8735 ) reported by Lisney and Collin Table 1. Anti-GnRH sera immunoreactivity of the bluefin tuna
(2006). diencephalons.
Morphological investigations were performed by Antisera against Lot No. Dilution PO-PV LT
haematoxylin-eosin or Nissl stainings and % (S.D.) % (S.D.)
Bielschowsky silver technique modified by Servier
salmonGnRH 1667* 1:1500 25 (8) 43 (9)
and Munger (1965).Neuron size was determined on chickenGnRH-II 675* 1:2500 47 (12) -
histological slides by a Quantimet 500W (Leica, chickenGnRH-II 6** 1:3000 43 (6) -
Cambridge, U.K.) image analyser by measuring the seabreamGnRH 691** 1:5000 - -
minor and major axis of at least 100 cell bodies for PO-PV, nucleus preopticus-periventricularis; LT, lateralis tuberis nucleus; S.D., standard
deviation; -, no immunostaining; *, provided by R.P . Millar (MRC, Edinburgh, UK);
each cell type.Cell body size is given as mean ± S.E. **, provided by M. Amano (Kitasato University, Japan).

20