Abstract
Reproduction of fish is regulated by a complex interaction between environmental signals
and various neurohormones including gonadotropin-inhibitory hormone (GnIH) secreted
from particular neurons in the brain. GnIH is a multifunctional hypophysiotropic
neurohormone which was first identified in the quail brain as an inhibitor of luteinizing
hormone (LH) secretion. There have been few studies on the neuroanatomical and
functional analysis of GnIH orthologs in teleosts, which have provided contradictory results.
Therefore, the first aim of the present study was to determine precise localization of GnIH
and GnIH receptor (GnIH-R)-immunoreactive cells in grass puffer (gp) brain.
Immunohistochamical method was used where specific antisera were generated against
gpGnIH-2 and GnIH-R peptides. GnIH-ir neurons were located in two different regions,
prominently in nucleus preoptic magnocellularis (NPOm) and a small population in
semicircular torus (TS) with their wide innervation throughout the brain. GnIHR-ir cells
were detected consistently with GnIH-ir fibers in lateral and ventral part of the dorsal
telencephalon, lateral part of anterior preoptic nucleus, anterior part of the posterior
preoptic nucleus and the medial preglomerular nucleus. Second attempt was to clarify
neuroendocrine mechanisms underlying the effect of changes in water temperature on
reproduction in fish. Hence, the changes in expression of GnIH and GnIH-R genes were
examined in the brain and pituitary along with growth hormone (gh) and prolactin (prl)
genes in the pituitary of male grass puffer exposed to low temperature (14°C), normal
temperature (21°C, as initial control) and high temperature (28°C) conditions for 7 days.
Levels of gnih and gnihr mRNAs were significantly decreased in both low and high
temperature conditions compared to normal temperature in the brain and pituitary.
Similarly, glı mRNA levels were significantly decreased in both low and high temperature
conditions. The prl mRNAs were drastically decreased at low temperature but showed no
significant changes at high temperature. Third effort was made to elucidate the lunar-age
dependent oscillations in the expression of GnIH and GnIH-R genes where brain and
pituitary samples collected at ZT9 (Zeitgeber Time) and ZT18 at intervals of five days in a
lunar month. The gnih and gnihr mRNA levels were significantly higher during the new
moon period when compared to the full moon period both at ZT9 and ZT18 in the brain as
well as in the pituitary with interestingly two peaks in the pituitary at ZT18 in new moon
and first quarter period. Finally, gpGnIH-1 peptide was administered with different doses
(0.01, 0.1, 1.0 µg/g BW) to reveal the functional relationship with the brain and pituitary
hormones. The gpGnIH-1 (0.01 µg) stimulated significantly both gnrh2 and gnrh3 but not
gnrh1 gene expression. Kisspeptin (Kiss2) and Kisspeptin receptor (Kiss2-r) genes expression
were also increased significantly with the same level (0.01 µg) of gpGnIH-1 application. At
the pituitary level, fshß, lhf, gh and prl but gpa genes expression were significantly increased
with low (0.01 µg) and high (1.0 µg) dose of gpGnIH-1 administration. Taken altogether, the
above results suggest that secreting from particular neurons in the brain, GnIH/GnIH-R
system may potentially regulate grass puffer reproduction exerting their actions directly on
the pituitary hormones or indirectly interacting with GnRH and Kisspeptin neurons where
temperature and lunar-age might play as external signals in the neuroendocrine regulatory
mechanisms of grass puffer reproduction.