Single-Cell Gene Profiling Reveals Social Status-Dependent Modulation of Nuclear Hormone Receptors in GnRH Neurons in a Male Cichlid Fish

Abstract

Gonadotropin-releasing hormone (GnRH) is essential for the initiation and maintenance of reproductive functions in vertebrates. To date, three distinct paralogue lineages, GnRH1, GnRH2, and GnRH3, have been identified with different functions and regulatory mechanisms. Among them, hypothalamic GnRH1 neurons are classically known as the hypophysiotropic form that is regulated by estrogen feedback. However, the mechanism of action underlying the estrogen-dependent regulation of GnRH1 has been debated, mainly due to the coexpression of low levels of estrogen receptor (ER) genes. In addition, the role of sex steroids in the modulation of GnRH2 and GnRH3 neurons has not been fully elucidated. Using single-cell real-time PCR, we revealed the expression of genes for estrogen, androgen, glucocorticoid, thyroid, and xenobiotic receptors in GnRH1, GnRH2, and GnRH3 neurons in the male Nile tilapia Oreochromis niloticus. We further quantified expression levels of estrogen receptor genes (ERα, ERβ, and ERγ) in three GnRH neuron types in male tilapia of two different social statuses (dominant and subordinate) at the single cell level. In dominant males, GnRH1 mRNA levels were positively proportional to ERγ mRNA levels, while in subordinate males, GnRH2 mRNA levels were positively proportional to ERβ mRNA levels. These results indicate that variations in the expression of nuclear receptors (and possibly steroid sensitivities) among individual GnRH cells may facilitate different physiological processes, such as the promotion of reproductive activities through GnRH1 neurons, and the inhibition of feeding and sexual behaviors through GnRH2 neurons.

Involvement of IGF-1R-PI3K-AKT-mTOR Pathway in Increased Number of GnRH3 Neurons During Androgen-induced Sex Reversal of the Brain in Female Tilapia

“…Ogawa and Parhar showed by single-cell gene pro ling 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. [29] 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.…”

Direct and Indirect Effects of Sex Steroids on Gonadotrope Cell Plasticity in the Teleost Fish Pituitary

“…However, a few studies have reported the presence of Esrs and Ars in teleost Gnrh neurons, suggesting the possibility of a direct sex steroid feedback. The erα paralog was found to be expressed in Gnrh3 neurons in medaka ( 195 ) and in Gnrh1, 2, and 3 neurons in Nile tilapia ( 242 ), while both arα and arβ were found in Gnrh1 neurons in the cichlid Astatotilapia burtoni ( 179 ). Thus, it is possible that sex steroids might regulate Gnrh activity and proliferation directly in teleosts, although as described below there is more evidence of indirect pathways via effects on Gnrh regulatory factors (e.g., kisspeptin and dopamine), as seen in mammals.…”

Morphological Evidence for Functional Crosstalk Between Multiple GnRH Systems in the Male Tilapia, Oreochromis niloticus

“…Single cell harvesting of GnRH-GAP-immunoreactive cells and total RNA extraction was performed as described previously with slight modifications ( 38 , 39 , 42 , 44 ). Briefly, the brain sections were immunoreacted with GAP antiserum for overnight followed by incubation with secondary antibodies (AlexaFluor 488- or 594-labeled anti-rabbit IgG) as described above in RNase-free buffer.…”