Role of IGF-1 and IGF-1R/PI3K/AKT/mTOR signalling pathway with respect to the effects of 11-KT on GnRH3 neurons in the female tilapia brain
According to the present results obtained with the TN-containing brain slices of mature females (Fig. 2), the in vitro treatment with 11-KT increased GnRH3 neurons, indicating that 11-KT can increase the number of GnRH3 neurons without mediation via other organs, such as the gonad and liver. Since there are no AR binding sites in the promoter regions of GnRH3 gene in tilapia, we considered that IGF-1 might mediate the action of 11-KT on GnRH3 neurons. Our hypothesis is strongly supported by the following four findings: 1) Treatment with IGF-1 increased the number of GnRH3 neurons (Fig. 3). 2) The expression of IGF-1 mRNA increased shortly after 11-KT treatment (Fig. 4). 3) The potent IGF-1R inhibitor, BMS-754807, strongly suppressed the 11-KT-induced increase of GnRH3 neurons (Fig. 5). 4) Immunohistochemical staining with anti-GnRH3 and anti-IGF-1R antibodies revealed the co-localization of IGF-1R and GnRH3 (Fig. 6). These results support the possibility that the effect of 11-KT on GnRH3 neurons is mediated by IGF-1 produced in the brain and IGF-1R on GnRH3 neurons. Furthermore, studies using inhibitors (Fig. 5, 7, and 8) clearly indicate the contribution of the IGF-1R downstream signalling pathway, PI3K/AKT/mTOR, to the effect of 11-KT on GnRH3 neurons. Therefore, our findings indicate that the effect of 11-KT on the number of GnRH3 neurons is mediated by the IGF-1 and IGF-1R/PI3K/AKT/mTOR signalling pathways.
Although there were no binding sites for ARs in GnRH3 promoters, the expression of AR mRNA was reported near, but not in, the TN of female medaka . Ogawa and Parhar showed by single-cell gene profiling that, although Arb mRNA as well as Tr, Gr, and Er mRNAs were expressed in GnRH3 neurons, the expression level of Arb mRNA was so low that nested-PCR was necessary to observe it. Combining these results with ours, it can be proposed that the effect of 11-KT on GnRH3 neurons is mediated by other molecules such as IGF-1 and its receptor, although it cannot be completely ruled out that 11-KT affects GnRH3 neurons directly.
The role of IGF-1 and IGF-1R/PI3K/AKT/mTOR signalling pathway in the regulatory process of GnRH neurons reported in other animals
IGF-1 is known to possess powerful neuroprotective effects in promoting neuronal survival, neuronal differentiation, neurite elongation, neurogenesis, and neurite regeneration in vivo and in vitro. It is a regulator of reproductive neuroendocrine function that affects the development and functions of GnRH neurons in the vertebrate brain, although this has been reported mainly in mammals. [31,32] GnRH perikarya express IGF-1R before puberty in mice, suggesting a potential direct anatomical locus where IGF-1 can control reproductive development and function.[31,33] Furthermore, the action of IGF-1 on IGF-1R in GnRH neurons regulates the onset of puberty in rodents.[34,35]
In fish like Mozambique tilapia, while IGF-1 and IGF-2 are mainly produced in the liver, their transcripts are expressed in various tissues including the brain. The absolute amounts of Igf-1 and Igf-2 mRNAs were also determined in many regions, such as the liver and brain of Nile tilapia. In yellow catfish, the expression of Igf-1 mRNA in the hypothalamus was higher in adult males than in adult females. Additionally, the effect of androgen (MT) treatment affected its expression in a sex-dependent manner; MT treatment in females increased the expression of Igf-1 mRNA.
The activation of IGF-1R leads to the stimulation of the following two major downstream signalling pathways: (i) the PI3K-AKT-mTOR pathway and (ii) Ras-Raf-MAP pathway. Regarding the relationship between GnRH and the intracellular signalling pathway following IGF-1R, Shao et al. investigated the expression of genes and proteins related to the IGF-1 pathway in the rat hypothalamus after combined treatments with di-(2-ethylhexyl) phthalate (DEHP), a common environmental endocrine disruptor. Their study indicated that DEHP might activate the hypothalamic GnRH neurons prematurely through the IGF-1 and IGF-1R/PI3K/AKT/mTOR signalling pathway to promote GnRH release. It was recently reported that administering IGF-1 significantly increased the firing rate and frequency of spontaneous postsynaptic currents in rodents, and that IGF-1R and PI3K are involved in this process. These findings indicate that IGF-1 affects the activity of GnRH neurons through IGF-1R and the PI3K/AKT/mTOR pathways in rodents.
In teleosts, there are only a few studies on IGF-1R downstream signalling pathways in GnRH neurons. Onuma et al. revealed that IGF-1 affects the development of GnRH2 and GnRH3 through IGF-1R in zebrafish embryos. They also examined whether PI3K inhibitors and/or ERK inhibitors disturbed the distribution of GnRH3 neurons in embryos. Treatment of embryos with a PI3K inhibitor (LY294002 or wortmannin) caused abnormal GnRH3 distribution similar to that in IGF signalling-deficient embryos. Contrastingly, U0126 had no effects. These results indicate that the influence of IGF-1 on the development and migration of GnRH2 and GnRH3 neurons was mediated through IGF-1R and the subsequent PI3K signalling pathway. According to findings regarding GnRH neurons in mammals and teleosts, it is highly likely that IGF-1 and the IGF-1R/PI3K/AKT/mTOR pathway play an important regulatory role in GnRH3 neurons in Mozambique tilapia.
In our previous studies, when 11-KT induced male-specific reproductive behaviours in mature female tilapia, GnRH3 neurons were increased in the TN region.[14,24] Moreover, newly proliferated GnRH3 neurons have been found in the TN of 11-KT-treated mature females.  Considering these findings with the present results, we conclude that IGF-1 and the IGF-1R/PI3K/AKT/mTOR signalling pathway are likely involved in the 11-KT-induced sex reversal of the female brain in terms of GnRH3 neurons.
Reduced involvement of ERK and JAK in the 11-KT-induced increase of GnRH3 neurons
IGF-1R signalling can also activate the Ras/Raf/Mek/ERK pathway, which is broadly involved in the regulation of neuron proliferation, differentiation, and apoptosis.[40–42] The co-activation of the ERK and AKT signalling pathways has been reported to promote the survival of GnRH neurons or Gn10 GnRH cells.[43,44] Additionally, positive regulation of Gnrh mRNA levels induced by kisspeptin was mediated through the activation of ERK and AKT signalling pathways in mammalian GT1-7 (a clonal cell line of mature, differentiated GnRH neurons) and GN11 (a clonal cell line with GnRH expression) cells. JAK2 is necessary for the neuroendocrine control of female reproduction and development of GnRH neurons.[46,47] In vitro, GnRH neurons and GnRH cell lines respond to a variety of ligands, including cytokines, which activate the JAK/signal transducers and activators of transcription intracellular signalling pathway.[48,49] However, in this study, the ERK and JAK inhibitors did not significantly suppress the 11-KT-induced increase of GnRH3 neurons, indicating a reduced contribution of ERK and JAK in mediating the effect of 11-KT on GnRH3 neurons compared to the PI3K/AKT/mTOR pathway.
We focused on GnRH3 neurons, which have been reported to control male-specific reproductive behaviour in tilapia, and found a possible role of IGF-1 in mediating the effect of androgen on GnRH3 neurons. IGF-1 is a very important molecule commonly used in controlling reproduction by regulating GnRH neurons in mammals, as well as in controlling somatic development in vertebrates. Therefore, our findings propose an attractive idea that IGF-1 and its downstream signalling pathway play an important role during the sex reversal of the brain in tilapia.