From mammals to fish, reproduction is driven by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) temporally secreted from the pituitary gland. Teleost fish are an excellent model for addressing the unique regulation and function of each gonadotropin cell since, unlike mammals, they synthesize and secrete LH and FSH from distinct cells. Only very distant vertebrate classes (such as fish and birds) demonstrate the mono-hormonal strategy, suggesting a potential convergent evolution. Cell-specific transcriptome analysis of double-labeled transgenic tilapia expressing GFP and RFP in LH or FSH cells, respectively, yielded genes specifically enriched in each cell type, revealing differences in hormone regulation, receptor expression, cell signaling, and electrical properties. Each cell type expresses a unique GPCR signature that reveals the direct regulation of metabolic and homeostatic hormones. Comparing these novel transcriptomes to that of rat gonadotrophs revealed conserved genes that might specifically contribute to each gonadotropin activity in mammals, suggesting conserved mechanisms controlling the differential regulation of gonadotropins in vertebrates.

In brief: The hypothalamus–pituitary–gonad axis integrates environmental and internal signals to tune reproductive functions. We hypothesized that in fish, Gnrh predominantly stimulates luteinizing hormone (LH) secretion, while Cck selectively regulates follicle-stimulating hormone (FSH), thereby uncovering a novel role for Cck as a metabolic gatekeeper that links reproductive activity with nutritional status. In fish, the hypothalamus–pituitary–gonad axis governs reproduction in response to environmental and internal signals. FSH regulates gonadal growth, while LH controls maturation. Gonadotropin-releasing hormone (Gnrh) plays a key role in this process, while the role of the satiety hormone cholecystokinin (Cck) is under investigation. We hypothesized that Gnrh and Cck differentially regulate tilapia LH and FSH, reflecting distinct reproductive and metabolic roles. Gnrh receptor (r) 1 was localized to FSH cells and detected only in juvenile fish, while GnRrhr3 was found in LH cells across both juvenile and mature stages. GnRH stimulation significantly increased LH release, while FSH levels showed only a moderate rise. Structural analysis revealed that Gnrhr3 exhibits more stable and rapid ligand binding and activates protein kinase A and protein kinase C (PKC) pathways, whereas Gnrhr1 signals exclusively through the PKC/Ca ²⁺ pathway. Elevated FSH levels and gnrhr 1 expression were observed during early vitellogenesis, coinciding with increased cck-rba expression in FSH cells. Cck-positive neurons originating in the brain were observed to terminate in the adenohypophysis, specifically near FSH-secreting cells. In addition, c ck-rba was exclusively expressed in FSH cells, and tilapia Cck injection significantly elevated plasma FSH levels while reducing food intake, highlighting the role of Cck in linking reproduction to nutritional status. Together, these findings indicate that Gnrh predominantly regulates LH secretion via Gnrhr3, while Cck, through Cck-rba, specifically controls FSH, linking reproduction to nutritional status. Cck acts as a metabolic gatekeeper, aligning gonadal development with energy availability and ensuring reproduction when energy is sufficient.

The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshβ, mdLhβ, mdFshβα, and mdLhβα by Pichia pastoris, generating specific antibodies against their respective β subunits, and their use within the development of ELISAs. The advantages of this protocol include: •The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%).•The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity.•Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time.

Life histories of oviparous species dictate high metabolic investment in the process of gonadal development leading to ovulation. In vertebrates, these two distinct processes are controlled by the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH), respectively. While it was suggested that a common secretagogue, gonadotropin-releasing hormone (GnRH), oversees both functions, the generation of loss-of-function fish challenged this view. Here, we reveal that the satiety hormone cholecystokinin (CCK) is the primary regulator of this axis in zebrafish. We found that FSH cells express a CCK receptor, and our findings demonstrate that mutating this receptor results in a severe hindrance to ovarian development. Additionally, it causes a complete shutdown of both gonadotropins secretion. Using in-vivo and ex-vivo calcium imaging of gonadotrophs, we show that GnRH predominantly activates LH cells, whereas FSH cells respond to CCK stimulation, designating CCK as the bona fide FSH secretagogue. These findings indicate that the control of gametogenesis in fish was placed under different neural circuits, that are gated by CCK.

Introduction: As in all vertebrates, reproduction in fish is regulated by gonadotrophin-releasing hormone (GnRH) control on gonadotrophic hormones (GtHs) activity. However, the neuroendocrine factors that promote GnRH and GtH activity are unknown. In Nile tilapia (Oreochromis niloticus), sexual activity and reproduction ability depend on social rank; only dominant males and females reproduce. Here, this characteristic of dominant fish allows us to compare brain and pituitary gene expression in animals that do and do not reproduce, aiming to reveal mechanisms that regulate reproduction. Methods: An extensive transcriptome analysis was performed, combining two sets of transcriptomes: a novel whole-brain and pituitary transcriptome of established dominant and subordinate males, together with a cell-specific transcriptome of luteinizing hormone (LH) and follicle-stimulating hormone cells. Pituitary incubation assay validated the direct effect of steroid application on chosen genes and GtH secretion. Results: In most dominant fish, as determined behaviorally, the gonadosomatic index was higher than in subordinate fish, and the leading upregulated pituitary genes were those coding for GtHs. In the brain, various neuropeptide genes, including isotocin, cholecystokinin, and MCH, were upregulated; these may be related to reproductive status through effects on behavior and feeding. In a STRING network analysis combining the two transcriptome sets, brain aromatase, highly expressed in LH cells, is the most central gene with the highest number of connections. In the pituitary incubation assay, testosterone and estradiol increased the secretion of LH and specific gene transcription. Conclusions: The close correlation between behavioral dominance and reproductive capacity in tilapia allows unraveling novel genes that may regulate the hypothalamic-pituitary-gonadal axis, highlighting aromatase as the main factor affecting the brain and pituitary in maintaining a sexually active organism.

From mammals to fish, gametogenesis and sexual maturation are driven by LH and FSH, the two gonadotropic hormones temporally secreted from the pituitary. Teleost fish are an excellent model for addressing the unique regulation and function of each gonadotropin hormone since, unlike mammals; they synthesize and secrete LH and FSH from distinct cells. By performing cell specific transcriptome analysis of double-labelled transgenic Nile tilapia (Oreochromis niloticus) expressing GFP and RFP in LH or FSH cells, respectively, we identified genes specifically enriched in each cell type. Though GnRH is considered the main neuropeptide regulating LH and FSH, we found that each LH and FSH cell express unique GPCR signature that reveals the direct regulation of additional metabolic and homeostatic hormones (like cholecystokinin, somatostatin and glutamate). Moreover, some of those GPCRs were conserved also in gonadotrophs of mammals (like PACAP receptor, Adropin receptor and GABBA receptor). Next, we had exploited the unique behavior of Nile tilapia where a behavioral hierarchy is created between males, to compare the gene expression in the pituitary and brain of dominant (reproducing) males to a subordinate (non-reproducing) males. By combining the two transcriptome sets we had identified novel players in the hypothalamic regulation of the HPG axis, and revealed how brain aromatase (cyp19a1b), that is enriched specifically in LH cells, is the key factor in regulating the activity of LH and FSH cells in dominant reproducing fish. Thereby, unraveling novel mechanisms in the differential regulation of LH and FSH. The research was funded by the Israel Science Foundation (ISF) no. 1540/17.