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To use magnetic resonance fingerprinting (MRF)–derived T1 and T2 values to differentiate gonadotroph from non-gonadotroph pituitary macroadenomas based on the 2017 World Health Organization classification of pituitary adenomas. A total of 57 patients with suspected pituitary macroadenomas were enrolled for analyses in this study between May 2018 and January 2020. Conventional magnetic resonance imaging (MRI) and MRF were performed in all patients before surgery using a 3-T MRI scanner. MRF-derived T1 and T2 values were compared between the gonadotroph and non-gonadotroph pituitary macroadenomas using a Mann-Whitney U test. The Knosp classification was used to evaluate cavernous sinus invasion by the adenomas. Receiver operating characteristic analyses were used to determine the diagnostic performance of T1 and T2 values. Quantitative T1 and T2 values yielded from MRF of gonadotroph pituitary macroadenomas were significantly higher than those of the non-gonadotroph pituitary macroadenomas (p < 0.001 and = 0.002, respectively). The AUC for the T2 value (0.888) was significantly greater than that for the T1 value (0.742) (p = 0.034). The AUC for combined T1 and T2 values was 0.885. Non-gonadotroph pituitary macroadenomas were more likely to invade the cavernous sinus than gonadotroph pituitary macroadenomas (55% vs 26%, p = 0.026). MRF may help to preoperatively differentiate between gonadotroph and non-gonadotroph pituitary macroadenomas and may be useful in guiding the treatment of these adenomas. • Somatostatin receptor type 3 is the most abundant receptor subtype in gonadotroph pituitary adenomas. • Magnetic resonance fingerprinting may help to preoperatively differentiate between gonadotroph and non-gonadotroph pituitary macroadenomas. • Magnetic resonance fingerprinting shows potential for guiding the treatment of pituitary macroadenomas.

Background Pituitary neuroendocrine tumors (PitNETs) are the second most common intracranial tumor. We lacked a comprehensive understanding of the pathogenesis and heterogeneity of these tumors. Methods We performed high-precision single-cell RNA sequencing for 2679 individual cells obtained from 23 surgically resected samples of the major subtypes of PitNETs from 21 patients. We also performed single-cell multi-omics sequencing for 238 cells from 5 patients. Results Unsupervised clustering analysis distinguished all tumor subtypes, which was in accordance with the classification based on immunohistochemistry and provided additional information. We identified 3 normal endocrine cell types: somatotrophs, lactotrophs, and gonadotrophs. Comparisons of tumor and matched normal cells showed that differentially expressed genes of gonadotroph tumors were predominantly downregulated, while those of somatotroph and lactotroph tumors were mainly upregulated. We identified novel tumor-related genes, such as AMIGO2, ZFP36, BTG1, and DLG5. Tumors expressing multiple hormone genes showed little transcriptomic heterogeneity. Furthermore, single-cell multi-omics analysis demonstrated that the tumor had a relatively uniform pattern of genome with slight heterogeneity in copy number variations. Conclusions Our single-cell transcriptome and single-cell multi-omics analyses provide novel insights into the characteristics and heterogeneity of these complex neoplasms for the identification of biomarkers and therapeutic targets.

Dexamethasone-induced Ras-related protein 1 (Rasd1) is a member of the Ras superfamily of monomeric G proteins that have a regulatory function in signal transduction. Rasd1, also known as Dexras1 or AGS1, is rapidly induced by dexamethasone (Dex). While prior data indicates that Rasd1 is highly expressed in the pituitary and that the gene may function in regulation of corticotroph activity, its exact cellular localization in this tissue has not been delineated. Nor has it been determined which endocrine pituitary cell type(s) are responsive to Dex-induced expression of Rasd1. We hypothesized that Rasd1 is primarily localized in corticotrophs and furthermore, that its expression in these cells would be upregulated in response to exogenous Dex administration. Rasd1 expression in each pituitary cell type both under basal conditions and 1-hour post Dex treatment were examined in adult male mice. While a proportion of all endocrine pituitary cell types expressed Rasd1, a majority of corticotrophs and thyrotrophs expressed Rasd1 under basal condition. In vehicle treated animals, approximately 50-60% of corticotrophs and thyrotrophs cells expressed Rasd1 while the gene was detected in only 15-30% of lactotrophs, somatotrophs, and gonadotrophs. In Dex treated animals, Rasd1 expression was significantly increased in corticotrophs, somatotrophs, lactotrophs, and gonadotrophs but not thyrotrophs. In Dex treated animals, Rasd1 was detected in 80-95% of gonadotrophs and corticotrophs. In contrast, Dex treatment increased Rasd1 expression to a lesser extent (55-60%) in somatotrophs and lactotrophs. Corticotrophs of the pars intermedia, which lack glucocorticoid receptors, failed to display increased Rasd1 expression in Dex treated animals. Rasd1 is highly expressed in corticotrophs under basal conditions and is further increased after Dex treatment, further supporting its role in glucocorticoid negative feedback. In addition, the presence and Dex-induced expression of Rasd1 in endocrine pituitary cell types, other than corticotrophs, may implicate Rasd1 in novel pituitary functions.

Anti-Müllerian hormone (AMH) is primarily produced by ovarian granulosa cells and contributes to follicle development. AMH is also produced in other tissues, including the brain and pituitary; however, its roles in these tissues are not well understood. In this study, we examined the effect of AMH on pituitary gonadotrophs. We detected AMH and AMH receptor type 2 expression in LβT2 cells. In these cells, the expression of FSHβ- but not α- and LHβ-subunits increased significantly as the concentration of AMH increased. LβT2 cells expressed Kiss-1 and Kiss-1R. AMH stimulation resulted in decreases in both Kiss-1 and Kiss-1R. The siRNA-mediated knockdown of Kiss-1 in LβT2 cells did not alter the basal expression levels of α-, LHβ-, and FSHβ-subunits. In LβT2 cells overexpressing Kiss-1R, exogenous kisspeptin stimulation significantly increased the expression of all three gonadotropin subunits. However, kisspeptin-induced increases in these subunits were almost completely eliminated in the presence of AMH. In contrast, GnRH-induced increases in the three gonadotropin subunits were not modulated by AMH. Our observations suggested that AMH acts on pituitary gonadotrophs and induces FSHβ-subunit expression with concomitant decreases in Kiss-1 and Kiss-1R gene expression. Kisspeptin, but not GnRH-induced gonadotropin subunit expression, was inhibited by AMH, suggesting that it functions in association with the kisspeptin/Kiss-1R system in gonadotrophs.

Functional relationship between nuclear receptor subfamily 4 group A member 3 (Nr4a3) and annexin A5 (Anxa5), which are two gonadotropin-releasing hormone (GnRH)-inducible genes, has been established while evaluating pituitary gonadotropes in relation to follicle-stimulating hormone beta (Fshb) expression. However, the physiological variations that arise due to the differential expression of these genes in the pituitary gland during rat estrous cycle remain unknown. This study aimed to evaluate the Nr4a3 and Anxa5 mRNA expression during the estrous cycle in rats in comparison with the expression of the gonadotropin subunit genes, luteinizing hormone beta (Lhb) and Fshb. Nr4a3 mRNA expression showed a single peak at 1400 h of proestrus during the 4-d estrous cycle. Anxa5 mRNA level was elevated along with increased Fshb mRNA expression after the decline of Nr4a3 mRNA until 2300 h. Lhb mRNA expression levels were not significantly changed during the estrous cycle. Notably, addition of a GnRH antagonist at 1100 h completely eradicated luteinizing hormone secretion at 1400 h and 1700 h of proestrus, and significantly reduced the Nr4a3 mRNA expression level at both the time points. These results suggest that GnRH is, at least partly, responsible for the increase in pituitary Nr4a3, and that the interaction between NR4A3 and ANXA5 is required to regulate Fshb expression during the preovulatory gonadotropin surge.

Reproductive functions may be affected by internal and external factors that are integrated in the central nervous system (CNS). Stressful stimuli induce the neuroendocrine response of the hypothalamic-pituitary-adrenal axis, as well as the synthesis of the neurosteroid allopregnanolone (AL) in the brain. This study tested the hypothesis that centrally administered AL could affect the expression of certain genes involved in reproductive functions at the hypothalamus and pituitary levels, as well as pulsatile gonadotropin secretion in sheep under both natural and stressful conditions. Luteal-phase sheep (n = 24) were subjected to a three-day (day 12–14 of the estrous cycle) series of control or AL (4 × 15 μg/60 μL/30 min, at 30 min intervals) infusions into the third ventricle. Acute stressful stimuli (isolation from other sheep and partial movement restriction) were used in the third day of infusion. Stressful stimuli reduced kisspeptin-1 mRNA levels in both the mediobasal hypothalamus (MBH) and the preoptic area (POA), while pro-dynorphin (PDYN) mRNA level only in the MBH. AL alone decreased the abundances of these transcripts in both structures. Stress increased the expression of gonadotropin-releasing hormone (GnRH) mRNA in the MBH and POA, luteinizing hormone (LH) β subunit (LHβ) mRNA in the anterior pituitary (AP) and pulsatile LH secretion. In contrast, mRNA level of follicle stimulating hormone (FSH) β subunit (FSHβ) was decreased in the AP, with no effect of stress on pulsatile FSH secretion. In stressed sheep, AL counteracted the increase in GnRH mRNA expression only in the POA, but it decreased the level of this transcript in both hypothalamic tissues when infused alone. AL prevented the stress-induced increase in LHβ mRNA expression in the AP and pulsatile LH secretion, as well as inhibited almost all aspects of FSH secretion when administered alone. The suppressive effect of AL on GnRH receptor mRNA expression was also observed in both MBH and AP. We concluded that acute stress and AL exerted multidirectional effects on hypothalamic centers that regulate reproductive functions and secretory activity of AP gonadotrophs in sheep. However, we indicated the dominant inhibitory effect of AL under natural and stressful conditions.

The expression of the gonadotrophin-releasing hormone receptor expression on pituitary gonadotrophs in humans is well characterized. In nervous system they have also been found in hippocampi and cerebral cortex. However, gonadotrophin-releasing hormone receptor expression in human spinal cord has not been reported. This study was to analyze the gonadotrophin-releasing hormone receptor expression in human spinal cord by immunohistochemistry, mRNAs by reverse transcriptase polymerase chain reaction, cDNA cloning and Western blot. The results show immunoreactive material to gonadotrophin-releasing hormone receptor in motoneurons of the spinal cord. Further, the study revealed that spinal cord expressed the gonadotrophin-releasing hormone receptor mRNA. The amplicon sequence corresponds to 100% of identity to GenBank. In Western blot, a band of 37 kDa were found in extracts of spinal cord and placenta as a control. In conclusion, human spinal cord expresses gonadotrophin-releasing hormone receptor analyzed through immunohistochemistry, the expression of its mRNA, cloning its cDNA and Western blot analysis. The presence of gonadotrophin-releasing hormone receptor in the spinal cord suggests the possibility of an extrapituitary functional role independent of reproductive system.

To more clearly understand the equine gonadotrope response to kisspeptin and gonadotropin releasing hormone (GnRH), peripheral LH and FSH were quantified in diestrous mares after treatment with either equine kisspeptide (eKp-10, 0.5 mg iv), GnRH (25 μg iv), or a combination thereof every 4 h for 3 days. The following observations were made: 1) a diminished LH and FSH response to eKp-10 and GnRH was observed by Day 3, but was not different by treatment, 2) a decrease in basal LH concentration was observed from Day 1 to Day 3 for the eKp-10, but not the GnRH treated mares, 3) there was no change in basal FSH with either treatment. Additionally, pre-treatment with GnRH antagonist (antide 1.0 mg iv) eliminated any measurable change in LH after eKp-10 (1.0 mg iv) treatment. Both GnRH and kisspeptin are Gαq/11 coupled receptors, therefore quantifying the rise in intracellular calcium following treatment with cognate ligand allows simultaneous assessment of receptor activation. Direct stimulation of equine primary pituitary cells with GnRH and/or eKp-10 demonstrates three distinct populations of pituitary cells: one population responded to both eKp-10 and GnRH, a second, independent population, responded to only eKp-10, and a third population responded only to GnRH. These populations were confirmed using co-immunofluorescence of hemipituitaries from mares in diestrus. Although the rise in peripheral LH concentration elicited by eKp-10 is dependent on GnRH, this work suggests that kisspeptin also has a specific and direct effect on the equine gonadotrope, independent of GnRH.

The anterior pituitary gland plays a central role in regulating various physiological processes, including body growth, reproduction, metabolism and stress response. Here, we perform single-cell RNA-sequencing (scRNA-seq) of 4113 individual cells from human fetal pituitaries. We characterize divergent developmental trajectories with distinct transitional intermediate states in five hormone-producing cell lineages. Corticotropes exhibit an early intermediate state prior to full differentiation. Three cell types of the PIT-1 lineage (somatotropes, lactotropes and thyrotropes) segregate from a common progenitor coexpressing lineage-specific transcription factors of different sublineages. Gonadotropes experience two multistep developmental trajectories. Furthermore, we identify a fetal gonadotrope cell subtype expressing the primate-specific hormone chorionic gonadotropin. We also characterize the cellular heterogeneity of pituitary stem cells and identify a hybrid epithelial/mesenchymal state and an early-to-late state transition. Here, our results provide insights into the transcriptional landscape of human pituitary development, defining distinct cell substates and subtypes and illustrating transcription factor dynamics during cell fate commitment., Editor’s summary_NCOMMS-19-41732B The anterior pituitary gland controls body growth and reproduction but how early development is dynamically regulated is unclear. Here, the authors use scRNA-seq of human fetal pituitaries to identify different developmental routes and state transitions of five hormone-producing cell lineages, and a hybrid epithelial/mesenchymal state of pituitary stem cells.

Through their receptors at each level of hypothalamo-pituitary-gonadal axis glucocorticoid excess, either endogenous or administered or stress-induced, could affect steroid production in the testis and thus male fertility. The main ways by which glucocorticoids act are as follows: 1) Affecting gonadoliberin and LH synthesis and release through glucocorticoid receptors in hypothalamic neurons and pituitary gonadotropes. 2) By so far not clearly evidenced reduction of the number of LH receptors on the membrane of Leydig cells. 3) By affecting expression and function of steroidogenic enzymes in the testis. 4) By regulation of in situ access of glucocorticoid to its target cells in the testis. 5) By promotion Leydig cell apoptosis. The review provides a survey of physiological and molecular mechanisms staying behind these effects. It does not deal with the clinical effects of glucocorticoid treatment which would substantially exceed the scope of the pater.

With the introduction of the WHO 2017 classification of endocrine neoplasms, the use of the pituitary transcription factors PIT-1, Tpit and SF-1 has become the standard of care. However, immunohistochemistry for these transcription factors is not available in all institutions, and their reliability has been questioned. We read with interest the findings of Mete et al. that GATA-3 expression was detected in some pituitary neuroendocrine tumours (PitNET). We therefore sort to validate this in our large cohort of PitNETs. We searched the database of Royal North Shore Hospital for PitNETs between 1998 and 2012, constructed a tissue microarray and reclassified these entities based on their expression for PIT-1, Tpit and SF-1. We then scored the expression of GATA-3 immunohistochemistry on a scale of 0-2, where 0 was no staining, 1 was patchy or weak staining and 2 was strong and diffuse staining. 265 of 346 tumours were able to be classified into a specific tumour subtype, and 263 tumours had tissue available for GATA-3 immunohistochemistry. 89% of gonadotrophs and 93% of triple-negative tumours with expression for luteinising hormone and follicle-stimulating hormone were positive for GATA-3. In the triple-negative group, GATA-3 was positive in 1 mammosomatotroph and 80% of tumours with thyroid-stimulating hormone expression. In the triple-negative hormone-negative group, 21 of 33 tumours were positive (64%). The results demonstrate that GATA-3 is a useful marker to supplement the existing pituitary transcription factors, albeit slightly less sensitive and specific than previously reported. GATA-3 may be employed in addition to the current array of immunohistochemical transcription factors, especially in the resource poor setting. However, given its potential cross-reactivity with other entities of the Sella, positive staining should be interpreted with caution and in the morphological and clinical context.

Unlike gonadotropin-releasing hormone (GnRH) analogues characterized by amino acid replacement in decapeptide primary structure, Cu-GnRH molecule preserves the native sequence but contains a Cu2+ ion stably bound to the nitrogen atoms including that of the imidazole ring of His2. Cu-GnRH can operate via cAMP/PKA signalling in anterior pituitary cells, suggesting that it may affect selected gonadotropic network gene transcription in vivo. We analysed pituitary mRNA expression of Egr-1, Nr5a1, and Lhb based on their role in luteinizing hormone (LH) synthesis; and Nos1, Adcyap1, and Prkaca due to their dependence on cAMP/PKA activity. In two independent experiments, ovariectomized rats received intracerebroventricular pulsatile (one pulse/h or two pulses/h over 5 h) microinjections of 2 nM Cu-GnRH; 2 nM antide (GnRH antagonist) + 2 nM Cu-GnRH; 100 nM PACAP6–38 (PACAP receptor antagonist) + 2 nM Cu-GnRH. Relative expression of selected mRNAs was determined by qRT-PCR. LH serum concentration was examined according to RIA. All examined genes responded to Cu-GnRH stimulation with increased transcriptional activity in a manner dependent on pulse frequency pattern. Increased expression of Nr5a1, Lhb, Nos1, Adcyap1, and Prkaca mRNA was observed solely in rats receiving the complex with frequency of two pulses/h over 5 h. Egr-1 transcription was up-regulated for both applied Cu-GnRH pulsatile patterns. The stimulatory effect of Cu-GnRH on gene transcription was dependent on both GnRH receptor and PAC-1 activation. In conclusion, obtained results indicate that Cu-GnRH complex is a GnRH analogue able to induce both IP3/PKC and cAMP/PKA-dependent gonadotrope network gene transcription in vivo.

The progesterone receptor (PR, encoded by Pgr) plays essential roles in reproduction. Female mice lacking the PR are infertile, due to the loss of the protein’s functions in the brain, ovary, and uterus. PR is also expressed in pituitary gonadotrope cells, but its specific role therein has not been assessed in vivo. We therefore generated gonadotrope-specific Pgr conditional knockout mice (cKO) using the Cre-LoxP system. Overall, both female and male cKO mice appeared phenotypically normal. cKO females displayed regular estrous cycles (vaginal cytology) and normal fertility (litter size and frequency). Reproductive organ weights were comparable between wild-type and cKO mice of both sexes, as were production and secretion of the gonadotropins, LH and FSH, with one exception. On the afternoon of proestrus, the amplitude of the LH surge was blunted in cKO females relative to controls. Contrary to predictions of earlier models, this did not appear to derive from impaired GnRH self-priming. Collectively, these data indicate that PR function in gonadotropes may be limited to regulation of LH surge amplitude in female mice via a currently unknown mechanism.

Negative energy balance in domestic animals suppresses their reproductive function. These animals commonly use long-chain fatty acids (LCFAs) from adipocytes as an energy source under states of malnutrition. The G-protein coupled receptor, GPR120, is a specific receptor for LCFAs, but its role in reproductive function remains unknown in domestic animals. The purpose of this study was to examine whether GPR120 is involved in the reproductive system of cattle. GPR120 mRNA expression was evaluated in brain, pituitary, and ovarian tissue samples by RT-PCR. GPR120 gene expression was detected with high intensity only in the anterior pituitary sample, and GPR120-immunoreactive cells were found in the anterior pituitary gland. Double immunohistochemistry of GPR120 in the anterior pituitary hormone-producing cells, such as gonadotropes, thyrotropes, lactotropes, somatotropes, and corticotropes, was performed to clarify the distribution of GPR120 in the anterior pituitary gland of ovariectomized heifers. Luteinizing hormone β subunit (LHβ)- and follicle-stimulating hormone β subunit (FSHβ)-immunoreactive cells demonstrated GPR120 immunoreactivity at 80.7% and 85.9%, respectively. Thyrotropes, lactotropes, somatotropes, and corticotropes coexpressed GPR120 at 21.1%, 5.4%, 13.6%, and 14.5%, respectively. In conclusion, the present study suggests that GPR120 in the anterior pituitary gland might mediate LCFA signaling to regulate gonadotrope functions, such as hormone secretion or production, in cattle.

High-fat diet (HFD) is associated with the regulation of reproductive functions. This study aimed to investigate the effects of short-term HFD on the mRNA expression levels of follicle-stimulating hormone β subunit (FSHβ), luteinizing hormone β subunit (LHβ), gonadotropin-releasing hormone receptor, and long-chain fatty acid receptor, GPR120, in the matured male mouse pituitary gland. Adult male mice were fed either control chow or HFD for 1, 2, 5, 10, 30 and 150 days. Fshb and Gpr120 mRNA expression levels in the pituitary glands were significantly increased during 2 to 30 days of HFD feeding. Gnrh-r mRNA in the 30 days HFD fed group and body weight in the 30 and 150 days HFD fed groups were higher than control. However, there were no significant differences in plasma non-esterified fatty acids or glucose levels during the 150 days of HFD feeding. These results suggest that male mice feeding a short-term HFD induces FSHβ synthesis and GPR120 expression in their pituitary gonadotropes.

Activins selectively stimulate follicle-stimulating hormone (FSH) secretion by pituitary gonadotrope cells. More recently, other members of the TGFbeta superfamily, the bone morphogenetic proteins (BMPs), were reported to regulate FSH synthesis. Activins and BMPs independently and synergistically stimulate transcription of the FSHbeta subunit (Fshb) gene in immortalized gonadotrope-like cells. Both ligands can signal via the activin receptor type IIA (ACVR2A) to regulate FSH synthesis in vitro. In vivo, global Acvr2a knockout mice exhibit a 60% reduction in circulating FSH relative to wild-type animals, suggesting that activins, BMPs, or related ligands might signal through additional type II receptors to regulate FSH in vivo. Although the leading candidates are ACVR2B and the BMP type II receptor (BMPR2), only the latter mediates activin or BMP2 induction of Fshb transcription in vitro. Here, we generated mice carrying a loss of function mutation in Bmpr2 specifically in gonadotropes. Puberty onset, estrous cyclicity, and reproductive organ weights were similar between control and conditional knockout females. Serum FSH and luteinizing hormone (LH) and pituitary expression of Fshb and the LHbeta subunit (Lhb) were similarly unaffected by the gene deletion in both sexes. These results suggest that BMPR2 might not play a necessary role in FSH synthesis or secretion in vivo or that another type II receptor, such as ACVR2A, can fully compensate for its absence. These data also further contribute to the emerging concept that BMPs may not be physiological regulators of FSH in vivo.

Roles of Clock genes and the bone morphogenetic protein (BMP) system in the regulation of gonadotropin secretion by gonadotropin-releasing hormone (GnRH) were investigated using mouse gonadotropin L beta T2 cells. It was found that luteinizing hormone (LH)beta mRNA expression level in L beta T2 cells changed gradually over time, with LH beta expression being suppressed in the early phase up to 12 h and then elevated in the late phase 24 h after GnRH stimulation. In addition, the mRNA expression levels of Clock genes, including Bmal1, Clock, Per2, and Cry1, also showed temporal changes mimicking the pattern of LH beta expression in the presence and absence of GnRH. Notably, the expression levels of Bmal1 and Clock showed strong positive correlations with LH beta mRNA expression levels. Moreover, a functional link of the ERK signaling of mitogen-activated protein kinases (MAPKs) in the suppression of LH beta mRNA expression, as well as Bmal1 and Clock mRNA expression by GnRH at the early phase, was revealed. Inhibition of Bmal1 and Clock expression using siRNA was involved in the reduction in LH beta mRNA levels in the late phase 24 h after GnRH stimulation. Furthermore, in the presence of BMP-6 and -7, late-phase Bmal1 and LH beta mRNA expression after GnRH stimulation was significantly attenuated. Collectively, the results indicated that LH expression in gonadotrope cells exhibits Bmal1/Clock-dependent fluctuations under the influence of GnRH and that the fluctuations are regulated by ERK and BMPs in the early and late stages, respectively, in a phase-dependent manner after GnRH stimulation.

Gonadotropin-releasing hormone (GnRH) stimulation of annexin A1 (ANXA1) and A5 (ANXA5) mRNA expression was analyzed in LβT2 gonadotrope cells. Quantitative polymerase chain reaction results showed that a GnRH analog (GnRHa) stimulated the expression of both ANXA1 and A5 mRNA with a peak at 12 h of incubation; however, ANXA1 mRNA was extremely stimulated (60 folds). Immunocytochemical analysis confirmed these findings. A GnRH antagonist inhibited the effect of GnRHa. ANXA1 and A5 mRNA levels were significantly increased by protein kinase C (PKC) activator (12-O-Tetradecanoylphorbol-13-acetate; TPA), but not by dibutyryl cAMP. GnRHa-stimulated induction of ANXA1 and A5 mRNA was inhibited by PKC (GF109203) and MEK inhibitors (PD98059). TPA increased ANXA1 and A5 mRNA expression in a dose-dependent manner (1 nM to 10 μM), while the extent of the increase was much greater in ANXA1. After stimulation with 10 nM or 1 μM TPA, ANXA1 and A5 mRNA levels were increased at 6 h. ANXA1 mRNA levels were higher in the 1 μM TPA than in the 10 nM TPA treatment, whereas 1 μM TPA did not show further stimulation of ANXA5 mRNA compared to 10 nM TPA. These results clearly show that ANXA1 mRNA expression is stimulated by GnRH through PKC like ANXA5, and the response of ANXA1 is much larger than that of ANXA5. A close relationship between these annexins and a significant role for ANXA1 in GnRH action at gonadotropes is suggested.

GnRH1 and GnRH2 receptors (provisonal nomenclature [39], also called Type I and Type II GnRH receptor, respectively [85]) have been cloned from numerous species, most of which express two or three types of GnRH receptor [85, 84, 114]. GnRH I (p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) is a hypothalamic decapeptide also known as luteinizing hormone-releasing hormone, gonadoliberin, luliberin, gonadorelin or simply as GnRH. It is a member of a family of similar peptides found in many species [85, 84, 114] including GnRH II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2 (which is also known as chicken GnRH-II). Receptors for three forms of GnRH exist in some species but only GnRH I and GnRH II and their cognate receptors have been found in mammals [85, 84, 114]. GnRH1 receptors are expressed by pituitary gonadotrophs, where they mediate the effects of GnRH on gonadotropin hormone synthesis and secretion that underpin central control of mammalian reproduction. GnRH analogues are used in assisted reproduction and to treat steroid hormone-dependent conditions [58]. Notably, agonists cause desensitization of GnRH-stimulated gonadotropin secretion and the consequent reduction in circulating sex steroids is exploited to treat hormone-dependent cancers of the breast, ovary and prostate [58]. GnRH1 receptors are selectively activated by GnRH I and all lack the COOH-terminal tails found in other GPCRs. GnRH2 receptors do have COOH-terminal tails and (where tested) are selective for GnRH II over GnRH I. GnRH2 receptors are expressed by some primates but not by humans [88]. Phylogenetic classifications divide GnRH receptors into three [85] or five groups [129] and highlight examples of gene loss through evolution, with humans retaining only one ancient gene. The structure of the GnRH1 receptor in complex with elagolix has been elucidated [132].

In vertebrates, the anterior pituitary plays a crucial role in regulating several essential physiological processes via the secretion of at least seven peptide hormones by different endocrine cell types. Comparative and comprehensive knowledge of the spatial distribution of those endocrine cell types is required to better understand their physiological functions. Using medaka as a model and several combinations of multi-color fluorescence in situ hybridization, we present the first 3D atlas revealing the gland-wide distribution of seven endocrine cell populations: lactotropes, thyrotropes, Lh and Fsh gonadotropes, somatotropes, and pomca-expressing cells (corticotropes and melanotropes) in the anterior pituitary of a teleost fish. By combining in situ hybridization and immunofluorescence techniques, we deciphered the location of corticotropes and melanotropes within the pomca-expressing cell population. The 3D localization approach reveals sexual dimorphism of tshba-, pomca-, and lhb-expressing cells in the adult medaka pituitary. Finally, we show the existence of bi-hormonal cells co-expressing lhb-fshb, fshb-tshba and lhb-sl using single-cell transcriptomics analysis and in situ hybridization. This study offers a solid basis for future comparative studies of the teleost pituitary and its functional plasticity.

Protein tyrosine phosphatase non-receptor type 5 (PTPN5), also called striatal-enriched protein tyrosine phosphatase (STEP), is highly expressed in neurons of the basal ganglia, hippocampus, cortex, and related structures, also in the pituitary. Gonadotropins are the key regulator of the reproduction in mammals. In this study, PTPN5 is detected to express in murine pituitary in a developmental manner. Moreover, the expression of PTPN5 in the pituitary is heavily reduced after ovary removal. Follicle-stimulating hormone (FSH) secretion in gonadotropes is regulated by PTPN5 via binding GnRH to GnRH-R. Two parallel signaling pathways, Gs-protein kinase A (PKA)-PTPN5 and Gq-phospholipases C (PLC)-p38 MAPK-PTPN5, cooperatively regulate GnRH-induced FSH secretion. We also show that influx of Ca2+ activates the Ca2+ -dependent phosphatase calcineurin, leading to the phosphorylation and activation of PTPN5. The intracellular release of Ca2+ is reduced via TC2153. In conclusion, blocking or knocking out of PTPN5 reduces the release of FSH in whole pituitary. Mechanically, PTPN5 regulates gonadotropes' function through regulating intracellular calcium homeostasis.

Follicle-stimulating hormone (FSH) is a dimeric glycoprotein secreted by the anterior pituitary gonadotrope that is necessary for reproductive function in mammals. FSH primarily regulates granulosa cells and follicular growth in females, and Sertoli cell function in males. Since its identification in the 1930s and sequencing in the 1970s, significant progress has been made in elucidating its regulation and downstream function. Recent advances provide deeper insight into FSH synthesis, and effects in the gonads suggest potential roles in extragonadal tissues and examine pharmacological approaches and clinical applications in infertility treatment that now affect 18% of couples. These advances were discussed in detail in a number of reviews published in the last 2 years in Endocrinology. In this brief commentary, we summarize these reviews and point to the outstanding questions that should be answered in the near future to bridge a gap in our understanding of this hormone.

The world of long non-coding RNAs (lncRNAs) has opened up massive new prospects in understanding the regulation of gene expression. Not only are there seemingly almost infinite numbers of lncRNAs in the mammalian cell, but they have highly diverse mechanisms of action. In the nucleus, some are chromatin-associated, transcribed from transcriptional enhancers (eRNAs) and/or direct changes in the epigenetic landscape with profound effects on gene expression. The pituitary gonadotrope is responsible for activation of reproduction through production and secretion of appropriate levels of the gonadotropic hormones. As such, it exemplifies a cell whose function is defined through changes in developmental and temporal patterns of gene expression, including those that are hormonally induced. Roles for diverse distal regulatory elements and eRNAs in gonadotrope biology have only just begun to emerge. Here, we will present an overview of the different kinds of lncRNAs that alter gene expression, and what is known about their roles in regulating some of the key gonadotrope genes. We will also review various screens that have detected differentially expressed pituitary lncRNAs associated with changes in reproductive state and those whose expression is found to play a role in gonadotrope-derived nonfunctioning pituitary adenomas. We hope to shed light on this exciting new field, emphasize the open questions, and encourage research to illuminate the roles of lncRNAs in various endocrine systems.

Within pituitary gonadotropes, the gonadotropin-releasing hormone receptor (GnRHR) receives hypothalamic input from GnRH neurons that is critical for reproduction. Previous studies have suggested that androgens may regulate GnRHR, although the mechanisms remain unknown. In this study, we demonstrated that androgens positively regulate Gnrhr mRNA in mice. We then investigated the effects of androgens and androgen receptor (AR) on Gnrhr promoter activity in immortalized mouse LβT2 cells, which represent mature gonadotropes. We found that AR positively regulates the Gnrhr proximal promoter, and that this effect requires a hormone response element (HRE) half site at -159/-153 relative to the transcription start site. We also identified nonconsensus, full-length HREs at -499/-484 and -159/-144, which are both positively regulated by androgens on a heterologous promoter. Furthermore, AR associates with the Gnrhr promoter in ChIP. Altogether, we report that GnRHR is positively regulated by androgens through recruitment of AR to the Gnrhr proximal promoter.

Extracellular acidification regulates endocrine cell functions. Ovarian cancer G protein-coupled receptor 1 (OGR1), also known as GPR68, is a proton-sensing G protein-coupled receptor and is activated by extracellular acidification, resulting in the activation of multiple intracellular signaling pathways. In the present study, we found that OGR1 was expressed in some gonadotropic cells in rat anterior pituitary and in LβΤ2 cells, which are used as a model of gonadotropic cells. When we reduced extracellular pH, a transient intracellular Ca2+ increase was detected in LβT2 cells. The Ca2+ increase was inhibited by a Gq/11 inhibitor and Cu2+, which is known as an OGR1 antagonist. We also found that extracellular acidification enhanced GnRH-induced Gaussia luciferase secretion from LβT2 cells. These results suggest that OGR1 may play a role in the regulation of gonadotropic cell function such as its hormone secretion.

Kisspeptin is a hypothalamic neuropeptide, which acts directly on gonadotropin-releasing hormone (GnRH)-secreting neurons via its cognate receptor (GPR54 or Kiss-R) to stimulate GnRH secretion in mammals. In non-mammalian vertebrates, there are multiple kisspeptins (Kiss1 and Kiss2) and Kiss-R types. Recent gene knockout studies have demonstrated that fish kisspeptin systems are not essential in the regulation of reproduction. Studying the detailed distribution of kisspeptin receptor in the brain and pituitary is important for understanding the multiple action sites and potential functions of the kisspeptin system. In the present study, we generated a specific antibody against zebrafish Kiss2-R (=Kiss1Ra/GPR54-1/Kiss-R2/KissR3) and examined its distribution in the brain and pituitary. Kiss2-R-immunoreactive cell bodies are widely distributed in the brain including in the dorsal telencephalon, preoptic area, hypothalamus, optic tectum, and in the hindbrain regions. Double-labeling showed that not all but a subset of preoptic GnRH3 neurons expresses Kiss2-R, while Kiss2-R is expressed in most of the olfactory GnRH3 neurons. In the posterior preoptic region, Kiss2-R immunoreactivity was seen in vasotocin cells. In the pituitary, Kiss2-R immunoreactivity was seen in corticotropes, but not in gonadotropes. The results in this study suggest that Kiss2 and Kiss2-R signaling directly serve non-reproductive functions and indirectly subserve reproductive functions in teleosts.

Gonadotropes represent approximately 5–15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches.

Visfatin, also called the pre-B cell colony enhancing factor, is produced mainly by the visceral fat and in the target cells activates the 3-phosphoinositide pathway, the cascade of mitogen-activated protein kinases and a number of the other effector systems. In the recent years, evidence has been obtained that visfatin regulates the functional activity of the hypothalamic-pituitary-gonad axis. Visfatin controls the synthesis and secretion of both gonadoliberin by the hypothalamic neurons and gonadotropins by the pituitary gonadotrophs, and also affects the steroidogenesis and folliculogenesis, acting on the reproductive tissues. One of the mechanisms of the steroidogenic action of visfatin is the potentiation of the stimulating effect of insulin-like growth factor-1 on the synthesis of sex steroid hormones. Since the production of visfatin and the activity of its signaling system vary significantly in the conditions of diabetes mellitus and metabolic syndrome, this directly affects its regulation of the reproductive functions in these metabolic disorders. Visfatin plays an important role in maintaining the functional activity of the ovaries and the normal quality of oocytes during aging. The review is devoted to the problem of the structural and functional organization of visfatin and its signaling system, the role of visfatin in the regulation of the female and male reproductive systems, and the mechanisms of visfatin action on the hypothalamic-pituitary-gonad axis.

Decapeptide gonadoliberin (GnRH) is the most important regulator of the hypothalamic-pituitary-gonadal (HPG) axis that controls the synthesis and secretion of the luteinizing and follicle-stimulating hormones by gonadotrophs in the adenohypophysis. GnRH is produced by the specialized hypothalamic neurons using the site-specific proteolysis of the precursor protein and is secreted into the portal pituitary system, where it binds to the specific receptors. These receptors belong to the family of G protein-coupled receptors, and they are located on the surface of gonadotrophs and mediate the regulatory effects of GnRH on the gonadotropins production. The result of GnRH binding to them is the activation of phospholipase C and the calcium-dependent pathways, the stimulation of different forms of mitogen-activated protein kinases, as well as the activation of the enzyme adenylyl cyclase and the triggering of cAMP-dependent signaling pathways in the gonadotrophs. The gonadotropins, kisspeptin, sex steroid hormones, insulin, melatonin and a number of transcription factors have an important role in the regulation of GnRH1 gene expression, which encodes the GnRH precursor, as well as the synthesis and secretion of GnRH. The functional activity of GnRH-producing neurons depends on their migration to the hypothalamic region at the early stages of ontogenesis, which is controlled by anosmin, ephrins, and lactosamine-rich surface glycoconjugate. Dysregulation of the migration of GnRH-producing neurons and the impaired production and secretion of GnRH, lead to hypogonadotropic hypogonadism and other dysfunctions of the reproductive system. This review is devoted to the current state of the problem of regulating the synthesis and secretion of GnRH, the mechanisms of migration of hypothalamic GnRH-producing neurons at the early stages of brain development, the functional activity of the GnRH-producing neurons in the adult hypothalamus and the molecular mechanisms of GnRH action on the pituitary gonadotrophs. New experimental data are analyzed, which significantly change the current understanding of the functioning of GnRH-producing neurons and the secretion of GnRH, which is very important for the development of effective approaches for correcting the functions of the HPG axis.

Cyclic adenosine monophosphate (cAMP) plays a pivotal role in gonadotrope responses in the pituitary. Gonadotropin-releasing hormone (GnRH) mediated synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are regulated by both the Gs/cAMP and Gq/Ca2+ signaling pathways. Pituitary adenylate cyclase-activating polypeptide (PACAP) also regulates GnRH responsiveness in gonadotropes through the PACAP receptor, which activates the Gs/cAMP signaling pathway. Therefore, measuring intracellular cAMP levels is important for elucidating the molecular mechanisms of FSH and LH synthesis and secretion in gonadotropes. The GloSensor cAMP assay is useful for detecting cAMP levels in intact, living cells. In this study, we found that increased GloSensor luminescence intensity did not correlate with cAMP accumulation in LβT2 cells under low pH conditions. This result indicates that cell type and condition must be considered when using GloSensor cAMP.

Bone morphogenetic proteins (BMPs) are pleiotropic ligands in the TGF-β superfamily. In the early to mid-2000s, several BMPs, including BMP2, were shown to regulate FSH synthesis alone and in synergy with activins in immortalized gonadotrope-like cell lines and primary pituitary cultures. Activins are also TGF-β family members, which were identified and named based on their abilities to stimulate FSH production selectively. Mechanistic analyses suggested that BMP2 promoted expression of the FSHβ subunit gene (Fshb) via at least two nonmutually exclusive mechanisms. First, BMP2 stimulated the production of the inhibitor of DNA-binding proteins 1, 2, and 3 (Id1, Id2, and Id3), which potentiated the stimulatory actions of homolog of Drosophila mothers against decapentaplegic 3 (SMAD3) on the Fshb promoter. SMAD3 is an intracellular signaling protein that canonically mediates the actions of activins and is an essential regulator of Fshb production in vitro and in vivo. Second, BMP2 was shown to activate SMAD3-dependent signaling via its canonical type IA receptor, BMPR1A (also known as ALK3). This was a surprising result, as ALK3 conventionally activates distinct SMAD proteins. Although these initial results were compelling, they were challenged by contemporaneous and subsequent observations. For example, inhibitors of BMP signaling did not specifically impair FSH production in cultured pituitary cells. Of perhaps greater significance, mice lacking ALK3 in gonadotrope cells produced FSH normally. Therefore, the physiological role of BMPs in FSH synthesis in vivo is presently uncertain.

Fertility is dependent on follicle-stimulating hormone (FSH), a product of gonadotrope cells of the anterior pituitary gland. Hypothalamic gonadotropin-releasing hormone (GnRH) and intra-pituitary activins are regarded as the primary drivers of FSH synthesis and secretion. Both stimulate expression of the FSH beta subunit gene (Fshb), although the underlying mechanisms of GnRH action are poorly described relative to those of the activins. There is currently no consensus on how GnRH regulatesFshbtranscription, as results vary across species and betweenin vivoandin vitroapproaches. One of the more fully developed models suggests that the murineFshbpromoter is tonically repressed by histone deacetylases (HDACs) and that GnRH relieves this repression, at least in immortalized murine gonadotrope-like cells (LβT2 and αT3-1). In contrast, we observed that the class I/II HDAC inhibitor trichostatin A (TSA) robustly inhibited basal, activin A-, and GnRH-inducedFshbmRNA expression in LβT2 cells and in primary murine pituitary cultures. Similar results were obtained with the class I specific HDAC inhibitor, entinostat, whereas two class II-specific inhibitors, MC1568 and TMP269, had no effects onFshbexpression. Collectively, these data suggest that class I HDACs are positive, not negative, regulators ofFshbexpressionin vitroand that, contrary to earlier reports, GnRH may not stimulateFshbby inhibiting HDAC-mediated repression of the gene.

The hypothalamic decapeptide, GnRH, is the gatekeeper of mammalian reproductive development and function. Activation of specific, high-affinity cell surface receptors (GnRH receptors) on gonadotropes by GnRH triggers signal transduction cascades to stimulate the coordinated synthesis and secretion of the pituitary gonadotropins FSH and LH. These hormones direct gonadal steroidogenesis and gametogenesis, making their tightly regulated production and secretion essential for normal sexual maturation and reproductive health. FSH and LH are glycoprotein heterodimers comprised of a common α-subunit and a unique β-subunit (FSHβ and LHβ, respectively), which determines the biological specificity of the gonadotropins. The unique β-subunit is the rate-limiting step for the production of the mature gonadotropins. Therefore, FSH synthesis is regulated at the transcriptional level by Fshb gene expression. The overarching goal of this review is to expand our understanding of the mechanisms and pathways underlying the carefully orchestrated control of FSH synthesis and secretion by GnRH, focusing on the transcriptional regulation of the Fshb gene. Identification of these regulatory mechanisms is not only fundamental to our understanding of normal reproductive function but will also provide a context for the elucidation of the pathophysiology of reproductive disorders and infertility to lead to potential new therapeutic approaches.

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene result in central hypothyroidism, often associated with macroorchidism. Testicular enlargement in these patients might be caused by increases in follicle-stimulating hormone (FSH) levels, as IGSF1 has been proposed to function as an inhibin B receptor or as an inhibitor of activin type I receptor (ALK4) activity in pituitary gonadotrope cells. If true, loss of IGSF1 should lead to reduced inhibin B action or disinhibition of activin signaling, thereby increasing FSH synthesis. Here, we show that FSH levels and sperm counts are normal in male Igsf1 knockout mice, although testis size is mildly increased. Sperm parameters are also normal in men with IGSF1 deficiency, although their FSH levels may trend higher and their testes are enlarged. Inhibin B retains the ability to suppress FSH synthesis in pituitaries of Igsf1-knockout mice and IGSF1 does not interact with ALK4 or alter activin A/ALK4 stimulation of FSHβ (Fshb/FSHB) subunit transcription or expression. In light of these results, it is unlikely that macroorchidism in IGSF1 deficiency derives from alterations in spermatogenesis or inhibin/activin regulation of FSH.

Restricted food intake, either from lack of food sources or endogenous fasting, during reproductive periods is a widespread phenomenon across the animal kingdom. Considering previous studies show the canonical upstream regulator of reproduction in vertebrates, the hypothalamic Gonadotropin-releasing hormone (Gnrh), is inhibited in some fasting animals, we sought to understand the neuroendocrine control of reproduction in fasted states. Here, we explore the roles of the midbrain neuropeptide, Gnrh2, in inducing reproduction via its pituitary prevalence, gonadotropin synthesis, gametogenesis, and reproductive outputs in the zebrafish model undergoing different feeding regimes. We discovered a fasting-induced four-fold increase in length and abundance of Gnrh2 neuronal projections to the pituitary and in close proximity to gonadotropes, whereas the hypothalamic Gnrh3 neurons are reduced by six-fold in length. Subsequently, we analyzed the functional roles of Gnrh2 by comparing reproductive parameters of a Gnrh2-depleted model, gnrh2−/−, to wild-type zebrafish undergoing different feeding conditions. We found that Gnrh2 depletion in fasted states compromises spawning success, with associated decreases in gonadotropin production, oogenesis, fecundity, and male courting behavior. Gnrh2 neurons do not compensate in other circumstances by which Gnrh3 is depleted, such as in gnrh3−/− zebrafish, implying that Gnrh2 acts to induce reproduction specifically in fasted zebrafish.

The proper expression of gonadotropin-releasing hormone receptors (GnRHRs) by pituitary gonadotropes is critical for maintaining maximum reproductive capacity. GnRH receptor expression must be tightly regulated in order to maintain the normal pattern of expression through the estrous cycle in rodents, which is believed to be important for interpreting the finely tuned pulses of GnRH from the hypothalamus. Much work has shown that Gnrhr expression is heavily regulated at the level of transcription. However, researchers have also discovered that Gnrhr is regulated post-transcriptionally. This review will discuss how RNA-binding proteins and microRNAs may play critical roles in the regulation of GnRHR expression. We will also discuss how these post-transcriptional regulators may themselves be affected by metabolic cues, specifically with regards to the adipokine leptin. All together, we present evidence that Gnrhr is regulated post-transcriptionally, and that this concept must be further explored in order to fully understand the complex nature of this receptor.

Ethanolamine plasmalogens (EPls), unique alkenylacyl-glycerophospholipids, are the only known ligands of G-protein-coupled receptor 61—a novel receptor co-localised with gonadotropin-releasing hormone receptors on anterior pituitary gonadotrophs. Brain EPl decreases with age. Commercial EPl—extracted from the cattle brain (unidentified age)—can independently stimulate FSH secretion from gonadotrophs. We hypothesised that there exists an age-related difference in the quality, quantity, and ability of bovine brain EPls to stimulate bovine gonadotrophs. We compared the brains of young (about 26 month old heifers) and old (about 90 month old cows) Japanese Black bovines, including EPls obtained from both groups. Additionally, mRNA expressions of the EPl biosynthesis enzymes, glyceronephosphate O-acyltransferase, alkylglycerone phosphate synthase, and fatty acyl-CoA reductase 1 (FAR1) were evaluated in young and old hypothalami. The old-brain EPl did not stimulate FSH secretion from gonadotrophs, unlike the young-brain EPl. Molecular species of EPl were compared using two-dimensional liquid chromatography-mass spectrometry. We identified 20 EPl molecular species of which three and three exhibited lower (P P

The adenohypophysis contains five secretory cell types (somatotrophs, lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs), each secreting a different hormone, and controlled by different hypothalamic releasing hormones (HRHs). Exocytic secretion is regulated by cytosolic Ca2+ signals ([Ca2+]C), which can be generated either by Ca2+ entry through the plasma membrane and/or by Ca2+ release from the endoplasmic reticulum (ER). In addition, Ca2+ entry signals can eventually be amplified by ER release via calcium-induced calcium release (CICR). We have investigated the contribution of ER Ca2+ release to the action of physiological agonists in pituitary gland. Changes of [Ca2+] in the ER ([Ca2+]ER) were measured with the genetically encoded low-affinity Ca2+ sensor GAP3 targeted to the ER. We used a transgenic mouse strain that expressed erGAP3 driven by a ubiquitous promoter. Virtually all the pituitary cells were positive for the sensor. In order to mimick the physiological environment, intact pituitary glands or acute slices from the transgenic mouse were used to image [Ca2+]ER. [Ca2+]C was measured simultaneously with Rhod-2. Luteinizing hormone-releasing hormone (LHRH) or thyrotropin releasing hormone (TRH), two agonists known to elicit intracellular Ca2+ mobilization, provoked robust decreases of [Ca2+]ER and concomitant rises of [Ca2+]C. A smaller fraction of cells responded to thyrotropin releasing hormone (TRH). By contrast, depolarization with high K+ triggered a rise of [Ca2+]C without a decrease of [Ca2+]ER, indicating that the calcium-induced calcium-release (CICR) via ryanodine receptor amplification mechanism is not present in these cells. Our results show the potential of transgenic ER Ca2+ indicators as novel tools to explore intraorganellar Ca2+ dynamics in pituitary gland in situ.

Gonadotropes cells located in the anterior pituitary gland are critical for reproductive fitness. A rapid surge in the serum concentration of luteinizing hormone (LH) secreted by anterior pituitary gonadotropes is essential for stimulating ovulation and is thus required for a successful pregnancy. To meet the requirements to mount the LH surge, gonadotrope cells display plasticity at the cellular, molecular and morphological level. First, gonadotrope cells heighten their sensitivity to an increasing frequency of hypothalamic GnRH pulses by dynamically elevating the expression of the GnRH receptor (GnRHR). Following ligand binding, GnRH initiates highly organized intracellular signaling cascades that ultimately promote the synthesis of LH and the trafficking of LH vesicles to the cell periphery. Lastly, gonadotrope cells display morphological plasticity, where there is directed mobilization of cytoskeletal processes towards vascular elements to facilitate rapid LH secretion into peripheral circulation. This mini review discusses the functional and organizational plasticity in gonadotrope cells including changes in sensitivity to GnRH, composition of the GnRHR signaling platform within the plasma membrane, and changes in cellular morphology. Ultimately, multimodal plasticity changes elicited by gonadotropes are critical for the generation of the LH surge, which is required for ovulation.

Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide that is secreted in pulses and acts via G-protein coupled receptors on pituitary gonadotropes to drive the synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropin hormones are packaged into secretory vesicles for release. GnRH receptors (GnRHR) are primarily Gq-coupled and mediate a rapid (within seconds-minutes) increase in vesicle fusion with the plasma membrane, whereas in longer time frames GnRH stimulates gonadotropin synthesis and thereby influences the hormone content of these vesicles. Together, these effects underpin the central control of reproduction by GnRH. Here, we review GnRHR and their mechanism of action, focusing on atypical structural and functional characteristics, and also on the combination of wet-lab and computational approaches to explore pulse frequency decoding and information transfer via GnRHR.

Accumulating evidence indicates that some pituitary cell types are organized in complex networks in both mammals and fish. In this study, we have further investigated the previously described cellular extensions formed by the medaka (Oryzias latipes) luteinizing hormone gonadotropes (Lh cells). Extensions, several cell diameters long, with varicosity-like swellings, were common both in vitro and in vivo. Some extensions approached other Lh cells, while others were in close contact with blood vessels in vivo. Gnrh further stimulated extension development in vitro. Two types of extensions with different characteristics could be distinguished, and were classified as major or minor according to size, origin and cytoskeleton protein dependance. The varicosity-like swellings appeared on the major extensions and were dependent on both microtubules and actin filaments. Immunofluorescence revealed that Lhβ protein was mainly located in these swellings and at the extremity of the extensions. We then investigated whether these extensions contribute to network formation and clustering, by following their development in primary cultures. During the first two days in culture, the Lh cells grew long extensions that with time physically attached to other cells. Successively, tight cell clusters formed as cell somas that were connected via extensions migrated towards each other, while shortening their extensions. Laser photolysis of caged Ca2+ showed that Ca2+ signals originating in the soma propagated from the soma along the major extensions, being particularly visible in each swelling. Moreover, the Ca2+ signal could be transferred between densely clustered cells (sharing soma-soma border), but was not transferred via extensions to the connected cell. In summary, Lh gonadotropes in medaka display a complex cellular structure of hormone-containing extensions that are sensitive to Gnrh, and may be used for clustering and possibly hormone release, but do not seem to contribute to communication between cells themselves.

Reproduction is a complex process that is controlled centrally via a network of hypothalamic neurons to modulate the pulsatile release of gonadotropin-releasing hormone (GnRH) and subsequently pituitary gonadotropins. The gonadotropins, luteinizing hormone, and follicle-stimulating hormone, drive gametogenesis and hormone production from the gonads. The hypothalamic-pituitary exchange is controlled by gonadal steroids through negative and positive feedback mechanisms via steroid receptors. Due to the expression of these receptors, GnRH neurons, the hypothalamic neurons that control them, and pituitary gonadotropes are sensitive to exogenous compounds that interact with steroid and nuclear receptors or alter hormone production and metabolism. The compounds, called endocrine-disrupting compounds (EDCs), are ubiquitous and persistent in human environments and could bioaccumulate in the body. EDCs include plasticizers (like bisphenol A), dioxin, polychlorinated biphenyls (PCBs), organochlorine pesticides, flame retardants, and perfluorinated alkyl substances (PFAS). Numerous studies have reported that perinatal, juvenile, or adult exposure to these EDCs, primarily in rats, disrupt the hypothalamic control of pituitary gonadotropin production leading to disruption of gonadal steroid production and estrous cyclicity. The purpose of this review is to evaluate these studies primarily focusing on GnRH and kisspeptin neurons and anterior pituitary gonadotropins and to discuss the need for deeper investigations into the hypothalamic-pituitary-gonadal axis.

The discovery of novel neurohormones is important for the advancement of neuroendocrinology. In early 1970s, gonadotropin-releasing hormone (GnRH), a hypothalamic neuropeptide that promotes gonadotropin release, was identified to be an endogenous neurohormone in mammals. In 2000, thirty years later, another hypothalamic neuropeptide, gonadotropin-inhibitory hormone (GnIH), that inhibits gonadotropin release, was found in quail. GnIH acts via GPR147 and inhibits gonadotropin release and synthesis and reproductive function in birds through actions on GnRH neurons in the hypothalamus and pituitary gonadotrophs. Later, GnIH was found in other vertebrates including humans. GnIH studies have advanced the progress of reproductive neuroendocrinology. Furthermore, recent GnIH studies have indicated that abnormal changes in GnIH expression may cause pubertal disorder and reproductive dysfunction. Here, we describe GnIH discovery and its impact on the progress of reproductive neuroendocrinology. This review also highlights advancement and perspective of GnIH studies on drug development for pubertal disorder and reproductive dysfunction. (149/150).

Reproduction is regulated by the gonadotropins, LH and FSH, which are synthesized and secreted by pituitary gonadotrophs in response to hypothalamic GnRH in a pulse frequency dependent manner. The gonadotroph decodes GnRH pulsatility via the GnRH receptor (GnRHR), which increases in expression and cell surface density before estrus and is responsible for downstream signaling cascades that differentially favor gonadotropin expression. The gonadotroph Gnrhr promoter contains a tripartite enhancer, including an AP-1 element that is necessary for full GnRH induction of Gnrhr expression in vitro. We previously generated an AP-1 knock-in (KI) mouse model with a single point mutation (C-269T) in the Gnrhr promoter AP-1 binding motif that resulted in an abnormal reproductive phenotype in female mice. Compared to wildtype (WT) littermates, female KI mice had a significant delay in first estrus, disrupted estrous cyclicity, fewer corpora lutea, and smaller litters. Males had no apparent reproductive phenotype. Basal serum gonadotropin levels were similar between WT and KI mice, but gonadectomy induced a significantly lower rise in serum LH levels of KI mice relative to WT mice, concomitant with significantly lower pituitary Gnrhr, Lhb, and Fshb mRNA levels in both sexes. We have now extended the characterization of these mice by measuring LH pulsatility and assessing GnRH induction of LH in vivo and in vitro. The frequency and amplitude of LH pulses over three hours were similar in ovariectomized WT and KI mice; however, KI mice had significantly reduced LH secretion, as measured by area under the curve. Similarly, GnRH treatment induced a diminished LH response in intact KI compared to WT males. In vitro cultures of hemi-pituitaries from gonadectomized WT and KI males were exposed to 0.01 nM GnRH and LH secretion into culture media was measured by ELISA at 0, 0.5, 1, 2, and 4 hours. There was no difference in basal LH secretion between WT and KI pituitaries but GnRH induction of LH was significantly lower in cultures from AP-1 mutant mice, indicating a direct impairment of GnRH action at the level of the pituitary. Taken together, these data indicate that the gonadotroph Gnrhr AP-1 promoter motif is critical for normal reproductive function. Prevention of AP-1 binding to the Gnrhr proximal promoter element decreases GnRH-induced Gnrhr, Lhb, and Fshb levels, impairs GnRH-stimulated LH secretion, and disrupts pubertal development and reproductive cyclicity in female mice.

Follicle-stimulating hormone (FSH) regulates gonadal function and fertility. Measurement of FSH in bodily fluids and tissues is possible with radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs). Recently, several novel assays were developed to measure pituitary hormones including growth hormone, prolactin, and luteinizing hormone in mice from small sample volumes. Here, we describe a novel and sensitive ELISA that enables the accurate measurement of FSH in serum, plasma, and whole blood from female and male mice. The assay can also be used to measure FSH in murine pituitary lysates and cell culture media. In summary, the new methodology described here will enable investigators to measure FSH from a variety of biological samples in mice accurately, at low cost, and in their own laboratories.

The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone, signal the gonads to regulate male and female fertility. FSH is critical for female fertility as it regulates oocyte maturation, ovulation, and hormone synthesis. Multiple genome-wide association studies (GWAS) link a 130 Kb locus at 11p14.1, which encompasses the FSH beta-subunit (FSHB) gene, with fertility-related traits that include polycystic ovary syndrome, age of natural menopause, and dizygotic twinning. The most statistically significant single nucleotide polymorphism from several GWAS studies (rs11031006) resides within a highly conserved 450 bp region 26 Kb upstream of the human FSHB gene. Given that sequence conservation suggests an important biological function, we hypothesized that the region could regulate FSHB transcription. In luciferase assays, the conserved region enhanced FSHB transcription and gel shifts identified a binding site for Steroidogenic factor 1 (SF1) contributing to its function. Analysis of mouse pituitary single-cell ATAC-seq demonstrated open chromatin at the conserved region exclusive to a gonadotrope cell-type cluster. Additionally, enhancer-associated histone markers were identified by immunoprecipitation of chromatin from mouse whole pituitary and an immortalized mouse gonadotrope-derived LβT2 cell line at the conserved region. Furthermore, we found that the rs11031006 minor allele upregulated FSHB transcription via increased SF1 binding to the enhancer. All together, these results identify a novel upstream regulator of FSHB transcription and indicate that rs11031006 can modulate FSH levels.

While extracellular vesicles play a role in intercellular communication, it is not known how their release is regulated. We show here that hypothalamic gonadotropin-releasing hormone (GnRH) stimulates extracellular vesicle (EV) formation associated with annexin A5 (ANXA5) from pituitary gonadotropes. The results show that 1) membrane blebs containing ANXA5 are produced after GnRH agonist (GnRHa) stimulation of gonadotropes and that this is observedin situas a loss of distinction at cell-cell boundaries, 2) EV containing ANXA5 are increased by GnRHa, 3) a gonadotrope-derived EV fraction stimulates LH release from other gonadotropes, and 4) an increase in ANXA5-loaded EV occurs in the plasma of ovariectomized rats. Finally, we also showed that 5) GnRHa stimulation of blebbing and EV-ANXA5 were suppressed by a protein kinase A inhibitor. These present results demonstrate a novel autocrine/paracrine mechanism mediated through the production of EV containing ANXA5. A hormonal regulation mechanism of cell-cell communication by means of EV is suggested.