Peak-agnostic high-resolution cis-regulatory circuitry mapping using single cell multiome data.

Single same cell RNAseq/ATACseq multiome data provide unparalleled potential to develop high resolution maps of the cell-type specific transcriptional regulatory circuitry underlying gene expression. We present CREMA, a framework that recovers the full cis-regulatory circuitry by modeling gene expression and chromatin activity in individual cells without peak-calling or cell type labeling constraints. We demonstrate that CREMA overcomes the limitations of existing methods that fail to identify about half of functional regulatory elements which are outside the called chromatin 'peaks'. These circuit sites outside called peaks are shown to be important cell type specific functional regulatory loci, sufficient to distinguish individual cell types. Analysis of mouse pituitary data identifies a Gata2-circuit for the gonadotrope-enriched disease-associated Pcsk1 gene, which is experimentally validated by reduced gonadotrope expression in a gonadotrope conditional Gata2-knockout model. We present a web accessible human immune cell regulatory circuit resource, and provide CREMA as an R package.

Seasonality of Reproduction in a Subtropical Free-Living Finch Amandava amandava: Plasticity of Adenohypophyseal Gonadotropes, Lactotropes, and Thyrotropes.

INTRODUCTION: This study sought to decipher the mechanism of transitions between life-history stages in a seasonally reproducing subtropical finch, Amandava amandava delineating the plasticity of the gonadotropes (LH cells), lactotropes (PRL cells), and thyrotropes (TSH cells) in the pituitary gland including the pars tuberalis, with regard to the in situ expression, morphological characteristics, and alteration in the plasma levels of hormones. METHODS: Immunohistochemistry of LH, PRL, TSH cells, morphometry and densitometry of expressed hormones (Image J software analysis), and ELISA for plasma hormonal levels were performed. RESULTS: LH, PRL, and TSH cells showed remarkable plasticity during the annual seasonal reproductive cycle. In the PT, all the 3 cell types were detected during the breeding phase, with additional detection of the TSH immunoreactivity during the pre-breeding and the PRL immunoreactivity during post-breeding phases. Pars distalis (PD) expressions and the plasma levels of the LH and TSH were at the peak during the breeding phase, but the PRL peak was during the post-breeding phase. In addition to PRL in the neurohypophysis and in the median eminence, hypothalamic PRL, and TSH were also elucidated. CONCLUSIONS: This study suggests activation of the gonadal axis by the PT TSH which might transduce seasonal cues, but not specifically photoperiod, in the birds of the tropics/subtropics. Post-breeding phase sustained high plasma TSH and peak plasma PRL might coordinate the transition to the non-breeding phase including the trigger of parental care as the later hormone assigned with. Hypothalamic TSH and PRL might influence events of seasonality through central modulation.

Role of microRNAs in pituitary gonadotrope cells.

The gonadotrope cells within the pituitary control vital processes of reproduction by producing follicle stimulating hormone (FSH) and luteinizing hormone (LH). Both external stimuli and internal regulatory factors contribute to the regulation of gonadotrope development and function. In recent years, growing evidences indicate that microRNAs (miRNAs), which regulate gene expression post-transcriptionally, play critical roles in multiple processes of gonadotrope development and function, including the syntheses of α or ß subunits of FSH and LH, the secretion of LH, the regulation of GnRH signaling, and the maintenance of gonadotrope cell kinetics. Here, we review recent advances of miRNAs' expression, functions and mechanisms approached by using miRNA knockout mouse models, in silico analysis and the in vitro cultures of primary pituitary cells and gonadotrope-derived cell lines. By summarizing and discussing different roles of miRNAs in gonadotropes, this minireview helps to gain insights into the complex molecular network in gonadotropes and reproduction.

The ecdysone-induced protein 93 is a key factor regulating gonadotrophic cycles in the adult female mosquito Aedes aegypti.

Repeated blood feedings are required for adult female mosquitoes to maintain their gonadotrophic cycles, enabling them to be important pathogen carriers of human diseases. Elucidating the molecular mechanism underlying developmental switches between these mosquito gonadotrophic cycles will provide valuable insight into mosquito reproduction and could aid in the identification of targets to disrupt these cycles, thereby reducing disease transmission. We report here that the transcription factor ecdysone-induced protein 93 (E93), previously implicated in insect metamorphic transitions, plays a key role in determining the gonadotrophic cyclicity in adult females of the major arboviral vector Aedes aegypti Expression of the E93 gene in mosquitoes is down-regulated by juvenile hormone (JH) and up-regulated by 20-hydroxyecdysone (20E). We find that E93 controls Hormone Receptor 3 (HR3), the transcription factor linked to the termination of reproductive cycles. Moreover, knockdown of E93 expression via RNAi impaired fat body autophagy, suggesting that E93 governs autophagy-induced termination of vitellogenesis. E93 RNAi silencing prior to the first gonadotrophic cycle affected normal progression of the second cycle. Finally, transcriptomic analysis showed a considerable E93-dependent decline in the expression of genes involved in translation and metabolism at the end of a reproductive cycle. In conclusion, our data demonstrate that E93 acts as a crucial factor in regulating reproductive cycle switches in adult female mosquitoes.

Single-Cell Transcriptional Profile Construction of Rat Pituitary Glands before and after Sexual Maturation and Identification of Novel Marker Spp1 in Gonadotropes.

The mammalian pituitary gland drives highly conserved physiological processes such as somatic cell growth, pubertal transformation, fertility, and metabolism by secreting a variety of hormones. Recently, single-cell transcriptomics techniques have been used in pituitary gland research. However, more studies have focused on adult pituitary gland tissues from different species or different sexes, and no research has yet resolved cellular differences in pituitary gland tissue before and after sexual maturation. Here, we identified a total of 15 cell clusters and constructed single-cell transcriptional profiles of rats before and after sexual maturation. Furthermore, focusing on the gonadotrope cluster, 106 genes were found to be differentially expressed before and after sexual maturation. It was verified that Spp1, which is specifically expressed in gonadotrope cells, could serve as a novel marker for this cell cluster and has a promotional effect on the synthesis and secretion of follicle-stimulating hormone. The results provide a new resource for further resolving the regulatory mechanism of pituitary gland development and pituitary hormone synthesis and secretion.

Transcription factor GATA2 may potentiate follicle-stimulating hormone production in mice via induction of the BMP antagonist gremlin in gonadotrope cells.

Mammalian reproduction depends on the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone, which are secreted by pituitary gonadotrope cells. The zinc-finger transcription factor GATA2 was previously implicated in FSH production in male mice; however, its mechanisms of action and role in females were not determined. To directly address GATA2 function in gonadotropes, we generated and analyzed gonadotrope-specific Gata2 KO mice using the Cre-lox system. We found that while conditional KO (cKO) males exhibited ∼50% reductions in serum FSH levels and pituitary FSHß subunit (Fshb) expression relative to controls, FSH production was apparently normal in cKO females. In addition, RNA-seq analysis of purified gonadotropes from control and cKO males revealed a profound decrease in expression of gremlin (Grem1), a bone morphogenetic protein (BMP) antagonist. We show Grem1 was expressed in gonadotropes, but not other cell lineages, in the adult male mouse pituitary. Furthermore, Gata2, Grem1, and Fshb mRNA levels were significantly higher in the pituitaries of WT males relative to females but decreased in males treated with estradiol and increased following ovariectomy in control but not cKO females. Finally, we found that recombinant gremlin stimulated Fshb expression in pituitary cultures from WT mice. Collectively, the data suggest that GATA2 promotes Grem1 expression in gonadotropes and that the gremlin protein potentiates FSH production. The mechanisms of gremlin action have not yet been established but may involve attenuation of BMP binding to activin type II receptors in gonadotropes, facilitating induction of Fshb transcription by activins or related ligands.

Clinical characteristics of functioning gonadotroph adenoma in women presenting with ovarian hyperstimulation: Audit of UK pituitary centres.

OBJECTIVE: Functioning gonadotroph adenomas (FGAs) are rare pituitary tumours stimulating ovarian function with potential life-threatening consequences in women. However, a lack of aggregated clinical experience of FGAs impairs management in affected women. The aim of this study is to present the clinical course of FGA-induced ovarian hyperstimulation syndrome (OHSS) cases as identified by some of the largest UK pituitary endocrine tertiary centres with a view to increasing awareness and improving diagnosis and management of women with FGA. DESIGN: A retrospective observational study; audit of eight UK regional pituitary centres for cases of FGAs. SETTING: Specialist neuroendocrine centres in the United Kingdom. PATIENTS AND MEASUREMENTS: Women diagnosed with FGA-induced OHSS. Description of their clinical course. RESULTS: Seven cases of FGA were identified in women, all causing OHSS. Mean age was 33.4 years at diagnosis. Abdominal pain, irregular periods, headache, and visual disturbances were reported at presentation by 100%, 71%, 57% and 43% of women, respectively. Three of seven women underwent ovarian surgery before FGA diagnosis. Six women underwent transsphenoidal surgery (TSS) with incomplete tumour resection in five of those, but all showed improvement or resolution in symptoms and biochemistry postoperatively. CONCLUSION: FGA is a rare cause of spontaneous OHSS. TSS improves clinical and biochemical features of ovarian hyperstimulation in FGAs. Improved awareness of FGA will prevent inappropriate emergency ovarian surgery.

Concurrent IgG4-related hypophysitis and clinically nonfunctioning gonadotroph pituitary neuroendocrine tumor.

BACKGROUND: Some patients develop immunoglobulin G4 (IgG4)-related hypophysitis associated with systemic diseases. More than 30 cases of IgG4-related hypophysitis have been reported. However, biopsy has rarely been performed in these patients, and none have had an associated pituitary neuroendocrine tumor (PitNET). We present a case of concurrent IgG4-related hypophysitis and PitNET. CASE PRESENTATION: A 56-year-old Japanese man arrived at the hospital with visual impairment, bitemporal hemianopia, and right abducens nerve palsy. Magnetic resonance imaging revealed pituitary body and stalk swelling as well as a small poorly enhanced right anterior lobe mass. Laboratory and loading test results suggested hypopituitarism. Because IgG4 level was elevated, a systemic examination was performed; multiple nodules were found in both lung fields. The diagnosis was based on an endoscopic transnasal biopsy of the pituitary gland. A histopathological examination revealed a marked infiltration of plasma cells into the pituitary gland, which was strongly positive for IgG4. The histological features of the resected tumor were consistent with those of gonadotroph PitNET, which was immunohistochemically positive for follicle-stimulating hormone-ß and steroidogenic factor-1, and no plasma cell infiltration was observed. Based on the histopathological examination results, steroid therapy was initiated, which reduced pituitary gland size and serum IgG4 levels. DISCUSSION AND CONCLUSIONS: This is the first reported case of IgG4-related hypophysitis with PitNET. Although no pathological findings indicating a relationship between the two conditions were found, we were able to preoperatively differentiate multiple lesions via detailed diagnostic imaging.

TGFBR3L is associated with gonadotropin production in non-functioning gonadotroph pituitary neuroendocrine tumours.

PURPOSE: Transforming growth factor-beta receptor 3-like (TGFBR3L) is a pituitary enriched membrane protein selectively detected in gonadotroph cells. TGFBR3L is named after transforming growth factor-beta receptor 3 (TGFBR3), an inhibin A co-receptor in mice, due to sequence identity to the C-terminal region. We aimed to characterize TGFBR3L detection in a well-characterized, prospectively collected cohort of non-functioning pituitary neuroendocrine tumours (NF-PitNETs) and correlate it to clinical data. METHODS: 144 patients operated for clinically NF-PitNETs were included. Clinical, radiological and biochemical data were recorded. Immunohistochemical (IHC) staining for FSHß and LHß was scored using the immunoreactive score (IRS), TGFBR3L and TGFBR3 were scored by the percentage of positive stained cells. RESULTS: TGFBR3L staining was selectively present in 52% of gonadotroph tumours. TGFBR3L was associated to IRS of LHß (median 2 [IQR 0-3] in TGFBR3L negative and median 6 [IQR 3-9] in TGFBR3L positive tumours, p < 0.001), but not to the IRS of FSHß (p = 0.32). The presence of TGFBR3L was negatively associated with plasma gonadotropin concentrations in males (P-FSH median 5.5 IU/L [IQR 2.9-9.6] and median 3.0 [IQR 1.8-5.6] in TGFBR3L negative and positive tumours respectively, p = 0.008) and P-LH (median 2.8 IU/L [IQR 1.9-3.7] and median 1.8 [IQR 1.1-3.0] in TGFBR3L negative and positive tumours respectively, p = 0.03). TGFBR3 stained positive in 22% (n = 25) of gonadotroph tumours with no correlation to TGFBR3L. CONCLUSION: TGFBR3L was selectively detected in half (52%) of gonadotroph NF-PitNETs. The association to LHß staining and plasma gonadotropins suggests that TGFBR3L may be involved in hormone production in gonadotroph NF-PitNETs.

Functioning gonadotroph adenomas in premenopausal women: clinical and molecular characterization and review of the literature.

PURPOSE: To summary the clinical features of premenopausal women with functioning gonadotroph adenomas (FGAs) and preliminarily explore their molecular characterization. METHODS: 12 premenopausal females with FGAs in our center were retrospectively analyzed. Previously reported cases were also summarized. The patients were clinically divided into FSH- or LH-predominant types according to their preoperative serum FSH/LH ratio. The expressions of related genes in the tumor tissues of female FGAs, non-functioning gonadotroph adenomas (NFGAs), and silent corticotropin adenomas were evaluated by RT-qPCR. RESULTS: Of all the 12 patients with FGAs from our center, 11 (91.7%) were diagnosed as FSH-predominant type, and they all had menstrual disorders, including 9 with spontaneous ovarian hyperstimulation syndrome (sOHSS). Their hormonal profiles showed non-suppressed FSH (12.45 ± 7.34 IU/L) with hyperestrogenemia [median estradiol level 1353.0 pg/mL (636.0, 3535.0)]. The other patient (8.3%) with LH-predominant type mainly manifested with infertility and sustained elevated serum LH without FSH or estradiol increasing. 65 premenopausal FGAs patients were systematic reviewed. 60 patients (92.3%) were FSH-predominant type, including 86.7% presented with menstrual disorders, 16.7% reported infertility, and 98.2% (55/56) showed sOHSS. No sOHSS or hyperestrogenemia were found in the 5 patients (7.7%) with LH-predominant type. Pituitary imaging data revealed macroadenomas and microadenomas accounted for 89.2% and 10.8%, respectively. Of 63 patients (96.9%) who underwent pituitary adenoma resection, 77.8% had complete tumor resection and no recurrence at the last follow-up. The relative expressions of KISS1 mRNA were significantly higher in FGA group than in NFGA group (p = 0.018), and significantly positively correlated with the preoperative serum estradiol levels (p = 0.004). CONCLUSIONS: Different clinical features were observed in premenopausal women with FGAs of FSH- or LH-predominant types. The elevated KISS1 expression in tumor tissues might involve in the secretion function of FGAs.

The expression of Annexin A1 and A5 mRNA by gonadotropin-releasing hormone in LßT2 gonadotrope cells.

Gonadotropin-releasing hormone (GnRH) stimulation of annexin A1 (ANXA1) and A5 (ANXA5) mRNA expression was analyzed in LßT2 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.

Pituitary apoplexy secondary to metastatic breast carcinoma into a gonadotroph cell adenoma of the pituitary.

A 65 year-old lady with metastatic breast cancer presented with pituitary apoplexy. Following surgery, histopathology confirmed metastatic breast carcinoma into a gonadotroph cell adenoma of the pituitary. Tumours that metastasise to a normal pituitary gland are unusual. More so, such neoplasm-to-neoplasm metastasis is extremely rare. This is, as far as we are aware, the first description of a metastasis into a gonadotroph cell pituitary adenoma presenting as apoplexy.

Intronic Enhancer Is Essential for Nr5a1 Expression in the Pituitary Gonadotrope and for Postnatal Development of Male Reproductive Organs in a Mouse Model.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) is expressed in the pituitary gonadotrope and regulates their differentiation. Although several regulatory regions were implicated in Nr5a1 gene expression in the pituitary gland, none of these regions have been verified using mouse models. Furthermore, the molecular functions of NR5A1 in the pituitary gonadotrope have not been fully elucidated. In the present study, we generated mice lacking the pituitary enhancer located in the 6th intron of the Nr5a1 gene. These mice showed pituitary gland-specific disappearance of NR5A1, confirming the functional importance of the enhancer. Enhancer-deleted male mice demonstrated no defects at fetal stages. Meanwhile, androgen production decreased markedly in adult, and postnatal development of reproductive organs, such as the seminal vesicle, prostate, and penis was severely impaired. We further performed transcriptomic analyses of the whole pituitary gland of the enhancer-deleted mice and controls, as well as gonadotropes isolated from Ad4BP-BAC-EGFP mice. These analyses identified several genes showing gonadotrope-specific, NR5A1-dependent expressions, such as Spp1, Tgfbr3l, Grem1, and Nr0b2. These factors are thought to function downstream of NR5A1 and play important roles in reproductive organ development through regulation of pituitary gonadotrope functions.

Functional pituitary gonadotroph adenoma in male patients: Case report.

Pituitary gonadotroph adenomas are common but very rarely do they secrete biologically active luteinizing hormone (LH) and follicle-stimulating hormone (FSH). There have been case studies reporting high sex hormones (testosterone/estrogen) in the presence of high or normal LH and FSH. Here we report two cases (with their consent) who presented with visual disturbance and headache at a tertiary care hospital (Aga Khan university hospital) Karachi, Pakistan. Brain imaging revealed a pituitary macroadenoma. Further workup was consistent with pituitary gonadotroph adenoma with high FSH (case 1) and normal LH/FSH (case 2) and elevated serum testosterone in both cases. Transsphenoidal resection was performed and the tissue sample histopathology confirmed pituitary adenoma. Postoperatively, improvement in hormonal profile was observed along with a resolution of visual disturbances and headaches. Thus, functional gonadotroph adenoma should be considered in the presence of elevated testosterone/estrogen and normal or elevated follicle-stimulating hormone (FSH)/ luteinizing hormone (LH). Early diagnosis leads to a better outcome.

Updating the Landscape for Functioning Gonadotroph Tumors.

Functioning gonadotroph adenomas (FGAs) are rare tumors, as the overwhelming majority of gonadotroph tumors are clinically silent. Literature is based on case reports and small case series. Gonadotroph tumors are poorly differentiated and produce and secrete hormones inefficiently, but in exceptional cases, they cause clinical syndromes due to hypersecretion of intact gonadotropins. The clinical spectrum of endocrine dysfunction includes an exaggerated response of ovaries characterized as ovarian hyperstimulation syndrome (OHSS) in premenopausal females and adolescent girls, testicular enlargement in males, and isosexual precocious puberty in children. Transsphenoidal surgery and removal of tumor reduces hormonal hypersecretion, improves endocrine dysfunction, and provides tissue for further analysis. Medical therapies (somatostatin analogues, dopamine agonists, GnRH agonists/antagonists) are partially or totally ineffective in many cases, especially with respect to antitumor effect. This review aims to update recent literature on these rare functioning tumors and highlight their therapeutic management.

Magnetic resonance fingerprinting for preoperative differentiation between gonadotroph and non-gonadotroph pituitary macroadenomas.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: MRF may help to preoperatively differentiate between gonadotroph and non-gonadotroph pituitary macroadenomas and may be useful in guiding the treatment of these adenomas. KEY POINTS: • 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.

Clinical profiles of silent corticotroph adenomas compared with silent gonadotroph adenomas after adopting the 2017 WHO pituitary classification system.

PURPOSE: Silent corticotroph adenomas (SCAs) can be redefined according to the 2017 World Health Organization pituitary classification system with the introduction of T­PIT, a transcription factor. We studied the clinical features of these redefined SCAs. METHODS: We compared 112 patients with SCAs and 198 patients with silent gonadotroph adenomas (SGAs) who underwent surgery from January 2019 to May 2020. RESULTS: The prevalence of SCAs increased from 21.3 to 30.2% under the new classification rules. T-PIT-positive, adrenocorticotropic hormone-negative SCAs and T-PIT-positive, adrenocorticotropic hormone-positive SCAs exhibited similar clinical features. SCAs exhibited significant female preponderance (90.2% vs. 29.8%, P < 0.0001); more frequent invasion (36.6% vs. 7.6%, P < 0.0001), especially multiple-site invasion (P < 0.0001); and marked cystic changes on imaging compared with SGAs (54.5% vs. 19.2%, P < 0.0001). SCAs had a softer tumor consistency (89.2% vs. 61.1%, P < 0.0001). Gross total resection was achieved in 66.1% of SCAs and 66.2% of SGAs (P > 0.9999). The overall recurrence/progression rates of SCAs and SGAs were 9.8% and 6.6% at 14.1 and 13.5 months of follow-up, respectively (P = 0.3765). The proportion of patients with more than two recurrences requiring multiple surgeries and radiation was similar between SCAs and SGAs (7.1% vs. 3.0%, P = 0.1514). However, multiple recurrences of SCAs affected younger patients than SGAs (39.0 vs. 53.5 years, P = 0.0433). CONCLUSIONS: The prevalence of SCAs increased with the introduction of T-PIT. SCAs and SGAs exhibited comparable size and recurrence/progression rates, but SCAs showed increased invasion and more marked cystic change. Aggressive SCAs tended to affect younger patients. Close long-term monitoring for SCA recurrence/progression is required.

Effect of anti-Müllerian hormone on the regulation of pituitary gonadotropin subunit expression: roles of kisspeptin and its receptors in gonadotroph LßT2 cells.

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 LßT2 cells. In these cells, the expression of FSHß- but not α- and LHß-subunits increased significantly as the concentration of AMH increased. LßT2 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 LßT2 cells did not alter the basal expression levels of α-, LHß-, and FSHß-subunits. In LßT2 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 FSHß-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.

Biphasic Roles of Clock Genes and Bone Morphogenetic Proteins in Gonadotropin Expression by Mouse Gonadotrope Cells.

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 LßT2 cells. It was found that luteinizing hormone (LH)ß mRNA expression level in LßT2 cells changed gradually over time, with LHß 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 LHß expression in the presence and absence of GnRH. Notably, the expression levels of Bmal1 and Clock showed strong positive correlations with LHß mRNA expression levels. Moreover, a functional link of the ERK signaling of mitogen-activated protein kinases (MAPKs) in the suppression of LHß 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 LHß mRNA levels in the late phase 24 h after GnRH stimulation. Furthermore, in the presence of BMP-6 and -7, late-phase Bmal1 and LHß 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.

Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

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.