Leptin Is an Important Endocrine Player That Directly Activates Gonadotropic Cells in Teleost Fish, Chub Mackerel

Leptin, secreted by adipocytes, directly influences the onset of puberty in mammals. Our previous study showed that leptin stimulation could promote the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from pituitary cells in primary culture and ovarian development in chub mackerel. This study aimed to elucidate the detailed mechanism of leptin-induced effects on gonadotropin hormone-producing cells. We produced recombinant leptin using silkworm pupae and investigated the effects of leptin on FSH and LH secretion and gene expression in the primary culture of pituitary cells from chub mackerel. The presence or absence of co-expression of lepr mRNA, FSH and LH b-subunit mRNA in gonadotropic cells was examined by double-labeled in situ hybridization. The addition of leptin significantly increased the secretion and gene expression of FSH and LH from male and female pituitary cells in primary culture. In contrast, gonadotropin-releasing hormone 1 affected neither FSH secretion in cells from females nor fshb and lhb expression in cells from males and females. The expression of lepr was observed in FSH- and LH-producing cells of both males and females. The results indicate that leptin directly regulates gonadotropin synthesis and secretion and plays an important role in the induction of puberty in teleost fish.

Transcriptomic heterogeneity of Sox2-expressing pituitary cells

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells (HPC), nonhormonal folliculostellate cells (FSC) and pituicytes, vascular pericytes and endothelial cells, and putative Sox2-expressing stem cells. Here, we used scRNAseq analysis of adult female rat pituitary cells to study the heterogeneity of pituitary cells with a focus on evaluating the transcriptomic profile of the Sox2-expressing population. Samples containing whole pituitary and separated anterior and posterior lobe cells allowed the identification of all expected pituitary resident cell types and lobe-specific subpopulations of vascular cells. Sox2 was expressed uniformly in all FSC, pituicytes, and a fraction of HPC. FSC comprised two subclusters; FSC1 contained more cells but expressed less genetic diversity compared to FSC2. The latter contained proliferative cells, expressed genes consistent with stem cell niche formation, including tight junctions, and shared genes with HPC. The FSC2 transcriptome profile was also consistent with the activity of pathways regulating cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. The expression of other stem cell marker genes was common for FSC and pituicytes (Sox9, Cd9, Hes1, Vim, S100b) or cell type-specific (FSC: Prop1, Prrx1, Pitx1, Pitx2, Lhx3; pituicytes: Fgf10, Tbx3, Lhx2, Nkx2-1, Rax). FSC and pituicytes also expressed other astroglial marker genes, some common and other distinct, consistent with their identities as astroglial cells of the pituitary. These data suggest functional heterogeneity of FSC, with a larger fraction representing classical FSC, and a smaller fraction containing active stem-like cells and HPC-committed progenitors.

Carbon Black Nanoparticles Selectively Alter Follicle-Stimulating Hormone Expression in vitro and in vivo in Female Mice

Toxic effects of nanoparticles on female reproductive health have been documented but the underlying mechanisms still need to be clarified. Here, we investigated the effect of carbon black nanoparticles (CB NPs) on the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are key regulators of gonadal gametogenesis and steroidogenesis. To that purpose, we subjected adult female mice to a weekly non-surgical intratracheal administration of CB NPs at an occupationally relevant dose over 4 weeks. We also analyzed the effects of CB NPs in vitro, using both primary cultures of pituitary cells and the LβT2 gonadotrope cell line. We report here that exposure to CB NPs does not disrupt estrous cyclicity but increases both circulating FSH levels and pituitary FSH β-subunit gene (Fshb) expression in female mice without altering circulating LH levels. Similarly, treatment of anterior pituitary or gonadotrope LβT2 cells with increasing concentrations of CB NPs dose-dependently up-regulates FSH but not LH gene expression or release. Moreover, CB NPs enhance the stimulatory effect of GnRH on Fshb expression in LβT2 cells without interfering with LH regulation. We provide evidence that CB NPs are internalized by LβT2 cells and rapidly activate the cAMP/PKA pathway. We further show that pharmacological inhibition of PKA significantly attenuates the stimulatory effect of CB NPs on Fshb expression. Altogether, our study demonstrates that exposure to CB NPs alters FSH but not LH expression and may thus lead to gonadotropin imbalance.

Leptin: A Metabolic Signal for the Differentiation of Pituitary Cells

Pituitary cell function is impacted by metabolic states and therefore must receive signals that inform them about nutritional status or adiposity. A primary signal from adipocytes is leptin, which recent studies have shown regulates most pituitary cell types. Subsets of all pituitary cell types express leptin receptors and leptin has been shown to exert transcriptional control through classical JAK/STAT pathways. Recent studies show that leptin also signals through post-transcriptional pathways that involve the translational regulatory protein Musashi. Mechanistically, post-transcriptional control would permit rapid cellular regulation of critical pre-existing pituitary transcripts as energy states change. The chapter will review evidence for transcriptional and/or post-transcriptional regulation of leptin targets (including Gnrhr, activin, Fshb, Gh, Ghrhr, and Pou11f1) and the consequences of the loss of leptin signaling to gonadotrope and somatotrope functions.

Novel Pituitary Actions of TAC4 Gene Products in Teleost

Tachykinin 4 (TAC4) is the latest member of the tachykinin family involved in several physiological functions in mammals. However, little information is available about TAC4 in teleost. In the present study, we firstly isolated TAC4 and six neurokinin receptors (NKRs) from grass carp brain and pituitary. Sequence analysis showed that grass carp TAC4 could encode two mature peptides (namely hemokinin 1 (HK1) and hemokinin 2 (HK2)), in which HK2 retained the typical FXGLM motif in C-terminal of tachyinin, while HK1 contained a mutant VFGLM motif. The ligand-receptor selectivity showed that HK2 could activate all 6 NKRs but with the highest activity for the neurokinin receptor 2 (NK2R). Interestingly, HK1 displayed a very weak activation for each NKR isoform. In grass carp pituitary cells, HK2 could induce prolactin (PRL), somatolactin α (SLα), urotensin 1 (UTS1), neuromedin-B 1 (NMB1), cocaine- and amphetamine-regulated transcript 2 (CART2) mRNA expression mediated by NK2R and neurokinin receptor 3 (NK3R) via activation cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3)/protein kinase C (PKC) and calcium2+ (Ca2+)/calmodulin (CaM)/calmodulin kinase-II (CaMK II) cascades. However, the corresponding stimulatory effects triggered by HK1 were found to be notably weaker. Furthermore, based on the structural base for HK1, our data suggested that a phenylalanine (F) to valine (V) substitution in the signature motif of HK1 might have contributed to its weak agonistic actions on NKRs and pituitary genes regulation.

The Hippo Pathway Effectors YAP and TAZ Regulate LH Release by Pituitary Gonadotrope Cells in Mice

The Hippo transcriptional coactivators YAP and TAZ exert critical roles in morphogenesis, organ size determination and tumorigenesis in many tissues. Although Hippo kinase cascade activity was recently reported in the anterior pituitary gland in mice, the role of the Hippo effectors in regulating gonadotropin production remains unknown. The objective of this study was therefore to characterize the roles of YAP and TAZ in gonadotropin synthesis and secretion. Using a conditional gene targeting approach (cKO), we found that gonadotrope-specific inactivation of Yap and Taz resulted in increased circulating levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in adult male mice, along with increased testosterone levels and testis weight. Female cKO mice had increased circulating LH (but not FSH) levels, which were associated with a hyperfertility phenotype characterized by higher ovulation rates and larger litter sizes. Unexpectedly, the loss of YAP/TAZ did not appear to affect the expression of gonadotropin subunit genes, yet both basal and GnRH-induced LH secretion were increased in cultured pituitary cells from cKO mice. Likewise, pharmacologic inhibition of YAP binding to the TEAD family of transcription factors increased both basal and GnRH-induced LH secretion in LβT2 gonadotrope-like cells in vitro without affecting Lhb expression. Conversely, mRNA levels of ChgA and SgII, which encode key secretory granule cargo proteins, were decreased following pharmacologic inhibition of YAP/TAZ, suggesting a mechanism whereby YAP/TAZ regulate the LH secretion machinery in gonadotrope cells. Together, these findings represent the first evidence that Hippo signaling may play a role in regulating pituitary LH secretion.

Reproductive Regulation of PrRPs in Teleost: The Link Between Feeding and Reproduction

Prolactin-releasing peptide (PrRP), a sort of vital hypothalamic neuropeptide, has been found to exert an enormous function on the food intake of mammals. However, little is known about the functional role of PrRP in teleost. In the present study, two PrRP isoforms and four PrRP receptors were isolated from grass carp. Ligand-receptor selectivity displayed that PrRP1 preferentially binds with PrRP-R1a and PrRP-R1b, while PrRP-R2a and PrRP-R2b were special receptors for PrRP2. Tissue distribution indicated that both PrRPs and PrRP-Rs were highly expressed in the hypothalamus-pituitary-gonad axis and intestine, suggesting a latent function on food intake and reproduction. Using grass carp as a model, we found that food intake could significantly induce hypothalamus PrRP mRNA expression, which suggested that PrRP should be also an anorexigenic peptide in teleost. Interestingly, intraperitoneal (IP) injection of PrRPs could significantly induce serum luteinizing hormone (LH) secretion and pituitary LHβ and GtHα mRNA expression in grass carp. Moreover, using primary culture grass carp pituitary cells as a model, we further found that PrRPs could directly induce pituitary LH secretion and synthesis mediated by AC/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II pathways. Finally, estrogen treatment of prepubertal fish elicited increases in PrRPs and PrPR receptors expression in primary cultured grass carp hypothalamus cells, which further confirmed that the PrRP/PrRPR system may participate in the neuroendocrine control of fish reproduction. These results, taken together, suggest that PrRPs might act as a coupling factor in feeding metabolism and reproductive activities in teleost.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish ( Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Pituitary Somatotroph Adenoma-derived Exosomes: Characterization of Nonhormonal Actions

Context The identification and biological actions of pituitary-derived exosomes remain elusive. Objective This work aimed to validate production of exosomes derived from human and rat pituitary and elucidate their actions. Methods Isolated extracellular vesicles (EVs) were analyzed by Nanoparticle Tracking Analysis (NTA) and expressed exosomal markers detected by Western blot, using nonpituitary fibroblast FR and myoblast H9C2 cells as controls. Exosome inhibitor GW4869 was employed to detect attenuated EV release. Exosomal RNA contents were characterized by RNA sequencing. In vitro and in vivo hepatocyte signaling alterations responding to GH1-derived exosomes (GH1-exo) were delineated by mRNA sequencing. GH1-exo actions on protein synthesis, cAMP (3′,5′-cyclic adenosine 5′-monophosphate) response, cell motility, and metastases were assessed. Results NTA, exosomal marker detection, and GW4869 attenuated EV release, confirming the exosomal identity of pituitary EVs. Hydrocortisone increased exosome secretion in GH1 and GH3 cells, suggesting a stress-associated response. Exosomal RNA contents showed profiles distinct for pituitary cells, and rat primary hepatocytes exposed to GH1-exo exhibited transcriptomic alterations distinct from those elicited by growth hormone or prolactin. Intravenous GH1-exo injection into rats attenuated hepatic Eif2ak2 and Atf4 mRNA expression, both involved in cAMP responses and amino acid biosynthesis. GH1-exo suppressed protein synthesis and forskolin-induced cAMP levels in hepatocytes. GH1-exo–treated HCT116 cells showed dysregulated p53 and mitogen-activated protein kinase (MAPK) pathways and attenuated motility of malignant HCT116 cells, and decreased tumor metastases in nude mice harboring splenic HCT116 implants. Conclusion Our findings elucidate biological actions of somatotroph-derived exosomes and implicate exosomes as nonhormonal pituitary-derived messengers.

Effective Perturbations of the Amplitude, Gating, and Hysteresis of IK(DR) Caused by PT-2385, an HIF-2α Inhibitor

PT-2385 is currently regarded as a potent and selective inhibitor of hypoxia-inducible factor-2α (HIF-2α), with potential antineoplastic activity. However, the membrane ion channels changed by this compound are obscure, although it is reasonable to assume that the compound might act on surface membrane before entering the cell´s interior. In this study, we intended to explore whether it and related compounds make any adjustments to the plasmalemmal ionic currents of pituitary tumor (GH3) cells and human 13-06-MG glioma cells. Cell exposure to PT-2385 suppressed the peak or late amplitude of delayed-rectifier K+ current (IK(DR)) in a time- and concentration-dependent manner, with IC50 values of 8.1 or 2.2 µM, respectively, while the KD value in PT-2385-induced shortening in the slow component of IK(DR) inactivation was estimated to be 2.9 µM. The PT-2385-mediated block of IK(DR) in GH3 cells was little-affected by the further application of diazoxide, cilostazol, or sorafenib. Increasing PT-2385 concentrations shifted the steady-state inactivation curve of IK(DR) towards a more hyperpolarized potential, with no change in the gating charge of the current, and also prolonged the time-dependent recovery of the IK(DR) block. The hysteretic strength of IK(DR) elicited by upright or inverted isosceles-triangular ramp voltage was decreased during exposure to PT-2385; meanwhile, the activation energy involved in the gating of IK(DR) elicitation was noticeably raised in its presence. Alternatively, the presence of PT-2385 in human 13-06-MG glioma cells effectively decreased the amplitude of IK(DR). Considering all of the experimental results together, the effects of PT-2385 on ionic currents demonstrated herein could be non-canonical and tend to be upstream of the inhibition of HIF-2α. This action therefore probably contributes to down-streaming mechanisms through the changes that it or other structurally resemblant compounds lead to in the perturbations of the functional activities of pituitary cells or neoplastic astrocytes, in the case that in vivo observations occur.