Tet3 CXXC domain and dioxygenase activity cooperatively regulate key genes for Xenopus eye and neural development.

Ten-Eleven Translocation (Tet) family of dioxygenases dynamically regulates DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functions and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further demonstrate that the Tet3 CXXC domain is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are both crucial for Tet3's biological function. Together, these findings define Tet3 as a transcription regulator and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development.

Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma.

DNA methylation at the 5 position of cytosine (5-mC) is a key epigenetic mark that is critical for various biological and pathological processes. 5-mC can be converted to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) family of DNA hydroxylases. Here, we report that "loss of 5-hmC" is an epigenetic hallmark of melanoma, with diagnostic and prognostic implications. Genome-wide mapping of 5-hmC reveals loss of the 5-hmC landscape in the melanoma epigenome. We show that downregulation of isocitrate dehydrogenase 2 (IDH2) and TET family enzymes is likely one of the mechanisms underlying 5-hmC loss in melanoma. Rebuilding the 5-hmC landscape in melanoma cells by reintroducing active TET2 or IDH2 suppresses melanoma growth and increases tumor-free survival in animal models. Thus, our study reveals a critical function of 5-hmC in melanoma development and directly links the IDH and TET activity-dependent epigenetic pathway to 5-hmC-mediated suppression of melanoma progression, suggesting a new strategy for epigenetic cancer therapy.

Trafficking-defective mutant PROKR2 cycles between endoplasmic reticulum and Golgi to attenuate endoplasmic reticulum stress.

G protein-coupled receptors (GPCRs) play crucial roles in numerous physiological and pathological processes. Mutations in GPCRs that result in loss of function or alterations in signaling can lead to inherited or acquired diseases. Herein, studying prokineticin receptor 2 (PROKR2), we initially identify distinct interactomes for wild-type (WT) versus a mutant (P290S) PROKR2 that causes hypogonadotropic hypogonadism. We then find that both the WT and mutant PROKR2 are targeted for endoplasmic reticulum (ER)-associated degradation, but the mutant is degraded to a greater extent. Further analysis revealed that both forms can also leave the ER to reach the Golgi. However, whereas most of the WT is further transported to the cell surface, most of the mutant is retrieved to the ER. Thus, the post-ER itinerary plays an important role in distinguishing the ultimate fate of the WT versus the mutant. We have further discovered that this post-ER itinerary reduces ER stress induced by the mutant PROKR2. Moreover, we extend the core findings to another model GPCR. Our findings advance the understanding of disease pathogenesis induced by a mutation at a key residue that is conserved across many GPCRs and thus contributes to a fundamental understanding of the diverse mechanisms used by cellular quality control to accommodate misfolded proteins.

Long-Term Changes in the Size of Pituitary Microadenomas.

BACKGROUND: The estimated prevalence of pituitary lesions is 10% to 38.5% in radiologic studies. However, how frequently these incidental lesions should be monitored by serial pituitary magnetic resonance imaging (MRI) remains unclear. OBJECTIVE: To evaluate changes in pituitary microadenomas over time. DESIGN: Retrospective, longitudinal cohort study. SETTING: Mass General Brigham, Boston, Massachusetts. PATIENTS: Evidence of pituitary microadenoma from MRI. MEASUREMENTS: Dimensions of pituitary microadenomas. RESULTS: During the study period (from 2003 to 2021), 414 patients with pituitary microadenomas were identified. Of the 177 patients who had more than 1 MRI, 78 had no change in the size of the microadenoma over time, 49 had an increase in size, 34 had a decrease in size, and 16 had both an increase and decrease in size. By linear mixed model analysis, the estimated slope was 0.016 mm/y (95% CI, -0.037 to 0.069). In the subgroup analysis, pituitary adenomas with a baseline size of 4 mm or less tended to increase in size. The estimated slope was 0.09 mm/y (CI, 0.020 to 0.161). In contrast, in the subgroup with baseline tumor size greater than 4 mm, the size tended to decrease. The estimated slope was -0.063 mm/y (CI, -0.141 to 0.015). LIMITATION: Retrospective cohort, some patients were lost to follow-up for unknown reasons, and data were limited to local large institutions. CONCLUSION: During the study period, approximately two thirds of the microadenomas remained unchanged or decreased in size. The growth, if any, was slow. These findings suggest that less frequent pituitary MRI surveillance for patients with incidental pituitary microadenomas may be safe. PRIMARY FUNDING SOURCE: None.

Alcohol intake and risk of pituitary adenoma.

BACKGROUND: The association between alcohol intake and incidence of pituitary adenoma has not been reported previously. We examined this association in three large, prospective cohort studies. METHODS: Using data from the Nurses' Health Study, Nurses' Health Study II, and Health Professionals Follow-Up Study, we computed multivariable-adjusted hazard ratios (MVHR) and 95% confidence intervals (CI) for pituitary adenoma by levels of alcohol intake using Cox proportional hazards regression. RESULTS: We identified 292 incident cases of pituitary adenoma (225 among women, 67 among men) among 235,973 participants with 6,548,732 person-years of follow-up. Compared with intake of ≤ 0.5 g/day, cumulative average alcohol intake in all categories was associated with reduced risk of pituitary adenoma (MVHR = 0.60, 95% CI 0.43-0.83 for 0.5-≤ 2 g/day, MVHR = 0.57, 95% CI 0.41-0.79 for > 2.0-≤ 8.0, MVHR = 0.70, 95% CI 0.47-1.04 for > 8.0-≤ 15.0, and MVHR = 0.51, 95% CI 0.32-0.83 for > 15.0 g/day). Significant inverse findings were present in women and were similar but non-significant in men. For specific alcoholic beverages, inverse associations were statistically significant for total wine (MVHR = 0.58, 95% CI 0.43-0.79 comparing 0.5-≤ 2 to ≤ 0.5 g/day), red wine (MVHR = 0.65, 95% CI 0.46-0.92 comparing 0.5-≤ 2 to ≤ 0.5 g/day), and white wine (MVHR = 0.72, 95% CI 0.53-0.97 comparing 0.5-≤ 2 to ≤ 0.5 g/day). Results were consistent using baseline intake, recent intake, and with an 8-year lag. CONCLUSION: In three prospective cohorts, compared to almost no consumption, alcohol consumption was associated with reduced risk of pituitary adenoma. Sensitivity analyses suggest that these results are unlikely to be the result of reverse causation or diagnostic bias.

Hypothalamic miR-30 regulates puberty onset via repression of the puberty-suppressing factor, Mkrn3.

Mkrn3, the maternally imprinted gene encoding the makorin RING-finger protein-3, has recently emerged as putative pubertal repressor, as evidenced by central precocity caused by MKRN3 mutations in humans; yet, the molecular underpinnings of this key regulatory action remain largely unexplored. We report herein that the microRNA, miR-30, with three binding sites in a highly conserved region of its 3' UTR, operates as repressor of Mkrn3 to control pubertal onset. Hypothalamic miR-30b expression increased, while Mkrn3 mRNA and protein content decreased, during rat postnatal maturation. Neonatal estrogen exposure, causing pubertal alterations, enhanced hypothalamic Mkrn3 and suppressed miR-30b expression in female rats. Functional in vitro analyses demonstrated a strong repressive action of miR-30b on Mkrn3 3' UTR. Moreover, central infusion during the juvenile period of target site blockers, tailored to prevent miR-30 binding to Mkrn3 3' UTR, reversed the prepubertal down-regulation of hypothalamic Mkrn3 protein and delayed female puberty. Collectively, our data unveil a novel hypothalamic miRNA pathway, involving miR-30, with a prominent role in the control of puberty via Mkrn3 repression. These findings expand our current understanding of the molecular basis of puberty and its disease states.

Quality of life after long-term biochemical control of acromegaly.

PURPOSE: To assess long-term quality of life (QoL) in patients with sustained biochemical control of acromegaly, comparing those receiving vs not receiving pharmacotherapy (primary analysis); to assess change in QoL over time (secondary analysis). METHODS: Cross-sectional study, with a secondary longitudinal component, of 58 patients with biochemically controlled acromegaly. All had participated in studies assessing QoL years previously, after having undergone surgery ± radiotherapy. One cohort received medical therapy [MED (n = 33)]; the other did not [NO-MED (n = 25)]. QoL was assessed by the 36-Item-Short-Form Health Survey (SF-36), Acromegaly Quality of Life Questionnaire (AcroQoL), Gastrointestinal Quality of Life Index (GIQLI), Symptom Questionnaire, and QoL-Assessment of Growth Hormone Deficiency in Adults (QoL-AGHDA). RESULTS: Mean (± SD) duration of biochemical control was 15.0 ± 6.4 years for MED and 20.4 ± 8.2 years for NO-MED (p = 0.007). 58% of subjects scored < 25% of normal on ≥ 1 SF-36 domain and 32% scored < 25% of normal on ≥ 4 of 8 domains. Comparing MED vs NO-MED and controlling for duration of biochemical control, there were no significant differences in QoL by SF-36, AcroQOL, GIQLI, Symptom Questionnaire, or QoL-AGHDA. Growth hormone deficiency (GHD) but not radiotherapy predicted poorer QoL. In MED, QoL improved over time in three AcroQoL domains and two GIQLI domains. In NO-MED, QoL worsened in two SF-36 domains and two Symptom Questionnaire domains; QoL-AGHDA scores also worsened in subjects with GHD. CONCLUSION: A history of acromegaly and development of GHD, but not pharmacologic or radiotherapy, are detrimental to QoL, which remains poor over the long-term despite biochemical control.

Functional Rescue of Inactivating Mutations of the Human Neurokinin 3 Receptor Using Pharmacological Chaperones.

G protein-coupled receptors (GPCRs) facilitate the majority of signal transductions across cell membranes in humans, with numerous diseases attributed to inactivating GPCR mutations. Many of these mutations result in misfolding during nascent receptor synthesis in the endoplasmic reticulum (ER), resulting in intracellular retention and degradation. Pharmacological chaperones (PCs) are cell-permeant small molecules that can interact with misfolded receptors in the ER and stabilise/rescue their folding to promote ER exit and trafficking to the cell membrane. The neurokinin 3 receptor (NK3R) plays a pivotal role in the hypothalamic-pituitary-gonadal reproductive axis. We sought to determine whether NK3R missense mutations result in a loss of cell surface receptor expression and, if so, whether a cell-permeant small molecule NK3R antagonist could be repurposed as a PC to restore function to these mutants. Quantitation of cell surface expression levels of seven mutant NK3Rs identified in hypogonadal patients indicated that five had severely impaired cell surface expression. A small molecule NK3R antagonist, M8, increased cell surface expression in four of these five and resulted in post-translational receptor processing in a manner analogous to the wild type. Importantly, there was a significant improvement in receptor activation in response to neurokinin B (NKB) for all four receptors following their rescue with M8. This demonstrates that M8 may have potential for therapeutic development in the treatment of hypogonadal patients harbouring NK3R mutations. The repurposing of existing small molecule GPCR modulators as PCs represents a novel and therapeutically viable option for the treatment of disorders attributed to mutations in GPCRs that cause intracellular retention.

Central precocious puberty: Recent advances in understanding the aetiology and in the clinical approach.

Central precocious puberty (CPP) results from early activation of the hypothalamic-pituitary-gonadal (HPG) axis. The current state of knowledge of the complex neural network acting at the level of the hypothalamus and the GnRH neuron to control puberty onset has expanded, particularly in the context of molecular interactions. Along with these advances, the knowledge of pubertal physiology and pathophysiology has also increased. This review focuses on regulatory abnormalities occurring at the hypothalamic level of the HPG axis to cause CPP. The clinical approach to diagnosis of puberty and pubertal disorders is also reviewed, with a particular focus on aetiologies of CPP. The recent identification of mutations in MKRN3 and DLK1 in familial as well sporadic forms of CPP has changed the state of the art of the approach to patients with CPP. Genetic advances have also had important repercussions beyond consideration of puberty alone. Syndromic disorders and central nervous system lesions associated with CPP are also discussed. If untreated, these conditions may lead to adverse physical, psychosocial and medical outcomes.

Effects of growth hormone receptor antagonism and somatostatin analog administration on quality of life in acromegaly.

OBJECTIVE: Acromegaly is associated with impaired quality of life (QoL). We investigated the effects of biochemical control of acromegaly by growth hormone receptor antagonism vs somatostatin analog therapy on QoL. DESIGN: Cross-sectional. PATIENTS: 116 subjects: n = 55 receiving a somatostatin analog (SSA group); n = 29 receiving pegvisomant (PEG group); n = 32 active acromegaly on no medical therapy (ACTIVE group). MEASUREMENTS: Acromegaly QoL Questionnaire (AcroQoL), Rand 36-Item Short Form Survey (SF-36) and Gastrointestinal QoL Index (GIQLI); fasting glucose, insulin and IGF-1 levels (LC/MS, Quest Diagnostics). RESULTS: There were no group differences in mean age, BMI or sex [(whole cohort mean ± SD) age 52 ± 14 years, BMI 30 ± 6 kg/m2 , and male sex 38%]. Mean IGF-1 Z-scores were higher in ACTIVE (3.9 ± 1.0) vs SSA and PEG, which did not differ from one another (0.5 ± 0.7 and 0.5 ± 0.7, P < .0001 vs ACTIVE). Eighty-three per cent of PEG previously received somatostatin analogs, which had been discontinued due to lack of efficacy (52%) or side effects (41%). There were no differences in the four QoL primary end-points (AcroQoL Global Score, SF-36 Physical Component Summary Score, SF-36 Mental Health Summary Score and GIQLI Global Score) between SSA and PEG. Higher HbA1c, BMI and IGF-1 Z-scores were associated with poorer QoL in several domains. CONCLUSION: Our data support a comparable QoL in patients receiving pegvisomant vs somatostatin analogs, despite the fact that the vast majority receiving pegvisomant did not respond to or were not able to tolerate somatostatin analogs.

The oestrous cycle and skeletal muscle atrophy: Investigations in rodent models of muscle loss.

NEW FINDINGS: What is the central question of this study? Is the oestrous cycle affected during disuse atrophies and, if so, how are oestrous cycle changes related to musculoskeletal outcomes? What is the main finding and its importance? Rodent oestrous cycles were altered during disuse atrophy, which was correlated with musculoskeletal outcomes. However, the oestrous cycle did not appear to be changed by Lewis lung carcinoma, which resulted in no differences in muscle size in comparison to healthy control animals. These findings suggest a relationship between the oestrous cycle and muscle size during atrophic pathologies. ABSTRACT: Recent efforts have focused on improving our understanding of female muscle physiology during exposure to muscle atrophic stimuli. A key feature of female rodent physiology is the oestrous cycle. However, it is not known how such stimuli interact with the oestrous cycle to influence muscle health. In this study, we investigated the impact of muscle atrophic stimuli on the oestrous cycle and how these alterations are correlated with musculoskeletal outcomes. A series of experiments were performed in female rodents, including hindlimb unloading (HU), HU followed by 24 h of reloading, HU combined with dexamethasone treatment, and Lewis lung carcinoma. The oestrous cycle phase was assessed throughout each intervention and correlated with musculoskeletal outcomes. Seven or 14 days of HU increased the duration in dioestrus or metoestrus (D/M; low hormones) and was negatively correlated with gastrocnemius mass. Time spent in D/M was also negatively correlated with changes in grip strength and bone density after HU, and with muscle recovery 24 h after the cessation of HU. The addition of dexamethasone strengthened these relationships between time in D/M and reduced musculoskeletal outcomes. However, in animals with Lewis lung carcinoma, oestrous cyclicity did not differ from that of control animals, and time spent in D/M was not correlated with either gastrocnemius mass or tumour burden. In vitro experiments suggested that enhanced protein synthesis induced by estrogen might protect against muscle atrophy. In conclusion, muscle atrophic insults are correlated with changes in the oestrous cycle, which are associated with deterioration in musculoskeletal outcomes. The magnitude of oestrous cycle alterations depends on the atrophic stimuli.

LSD2/KDM1B and its cofactor NPAC/GLYR1 endow a structural and molecular model for regulation of H3K4 demethylation.

Dynamic regulation of histone methylation represents a fundamental epigenetic mechanism underlying eukaryotic gene regulation, yet little is known about how the catalytic activities of histone demethylases are regulated. Here, we identify and characterize NPAC/GLYR1 as an LSD2/KDM1b-specific cofactor that stimulates H3K4me1 and H3K4me2 demethylation. We determine the crystal structures of LSD2 alone and LSD2 in complex with the NPAC linker region in the absence or presence of histone H3 peptide, at resolutions of 2.9, 2.0, and 2.25 Å, respectively. These crystal structures and further biochemical characterization define a dodecapeptide of NPAC (residues 214-225) as the minimal functional unit for its cofactor activity and provide structural determinants and a molecular mechanism underlying the intrinsic cofactor activity of NPAC in stimulating LSD2-catalyzed H3K4 demethylation. Thus, these findings establish a model for how a cofactor directly regulates histone demethylation and will have a significant impact on our understanding of catalytic-activity-based epigenetic regulation.

Effect of Natesto on Reproductive Hormones, Semen Parameters and Hypogonadal Symptoms: A Single Center, Open Label, Single Arm Trial.

PURPOSE: To evaluate the effect of short-acting 4.5% nasal testosterone gel (Natesto) on serum testosterone, gonadotropins, total motile sperm count, health related quality of life and sexual function. MATERIALS AND METHODS: This was a single institution, open label, single arm trial conducted between November 2017 and September 2019 at the University of Miami. Men 18 to 55 years old diagnosed with symptomatic hypogonadism (total testosterone less than 300 ng/dl on 2 occasions) were included. Men with azoospermia, vasectomy or a total motile sperm count less than 5 million were excluded. Enrolled patients were treated with Natesto, a short-acting nasal testosterone (125 µl per nostril, 11.0 mg testosterone per dose, TID) for 6 months. RESULTS: In total, 60 men were enrolled in the study. Of these, 44 and 33 patients were evaluated for testosterone at 3 and 6 months, respectively. A total of 31 patients (90.9%) reached a normal testosterone level (greater than 300 ng/dl) at 6 months. Follicle stimulating hormone and luteinizing hormone levels were maintained within the normal range in 81.8% and 72.7% of patients at 6 months, respectively. Total motile sperm count was maintained with total motile sperm count greater than 5 million over the treatment period in 88.4% of men at 3 months and 93.9% at 6 months. There were statistically significant improvements on International Index of Erectile Function sexual desire and overall satisfaction domains at 6 months. CONCLUSIONS: Natesto appears to increase testosterone while maintaining semen parameters in a majority of men. Natesto has the potential to be a safe and effective treatment for men with functional hypogonadism who wish to preserve semen parameters. Long-term studies beyond 6 months are needed before we can safely prescribe nasal testosterone gel for men interested in fertility.

GnRH Transactivates Human AMH Receptor Gene via Egr1 and FOXO1 in Gonadotrope Cells.

BACKGROUND/OBJECTIVES: Anti-Müllerian hormone (AMH) signaling is critical for sexual differentiation and gonadal function. AMH receptor type 2 (AMHR2) is expressed in extragonadal sites such as brain, and pituitary and emerging evidence indicates that AMH biological action is much broader than initially thought. We recently reported that AMH signaling enhances follicle-stimulating hormone synthesis in pituitary gonadotrope cells. However, mechanisms regulating AMHR2 expression in these extragonadal sites remain to be explored. METHOD/RESULTS: Here, we demonstrated in perifused murine LßT2 gonadotrope cells that Amhr2 expression is differentially regulated by GnRH pulse frequency with an induction under high GnRH pulsatility. Furthermore, we showed that GnRH transactivates the human AMHR2 promoter in LßT2 cells. Successive deletions of the promoter revealed the importance of a short proximal region (-53/-37 bp) containing an Egr1 binding site. Using site-directed mutagenesis of Egr1 motif and siRNA mediated-knockdown of Egr1, we demonstrated that Egr1 mediates basal and GnRH-dependent activity of the promoter, identifying Egr1 as a new transcription factor controlling hAMHR2 expression. We also showed that SF1 and ß-catenin are required for basal promoter activity and demonstrated that both factors contribute to the GnRH stimulatory effect, independently of their respective binding sites. Furthermore, using a constitutively active mutant of FOXO1, we identified FOXO1 as a negative regulator of basal and GnRH-dependent AMHR2 expression in gonadotrope cells. CONCLUSIONS: This study identifies GnRH as a regulator of human AMHR2 expression, further highlighting the importance of AMH signaling in the regulation of gonadotrope function.

Endocrine dysfunction induced by immune checkpoint inhibitors: Practical recommendations for diagnosis and clinical management.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. However, because ICIs block coinhibitory molecules on T cells and other immune cells, unleashing them to mediate tumor cell killing, they also can disrupt the maintenance of immunological tolerance to self-antigens. Compared with chemotherapy, ICIs have a different toxicity profile, especially the occurrence of autoimmune-like manifestations against multiple organ systems, including endocrine glands, commonly referred to as immune-related adverse events. The aim of this review was to provide practical recommendations regarding the proper assessment and clinical management related to the new onset of endocrinopathies after the use of ICIs in patients with cancer. Cancer 2018;124:1111-21. © 2018 American Cancer Society.

Central Precocious Puberty Caused by a Heterozygous Deletion in the MKRN3 Promoter Region.

CONTEXT: Loss-of-function mutations in the coding region of MKRN3, a maternally imprinted gene at chromosome 15q11.2, are a common cause of familial central precocious puberty (CPP). Whether MKRN3 alterations in regulatory regions can cause CPP has not been explored to date. We aimed to investigate potential pathogenic variants in the promoter region of MKRN3 in patients with idiopathic CPP. PATIENTS/METHODS: A cohort of 110 patients with idiopathic CPP was studied. Family history of precocious sexual development was present in 25%. Mutations in the coding region of MKRN3 were excluded in all patients. Genomic DNA was extracted from peripheral blood leukocytes, and 1,100 nucleotides (nt) of the 5'-regulatory region of MKRN3 were amplified and sequenced. Luciferase assays were performed in GT1-7 cells transiently transfected with plasmids containing mutated and wild-type MKRN3 promoter. RESULTS: We identified a rare heterozygous 4-nt deletion (c.-150_-147delTCAG; -38 to -41 nt upstream to the transcription start site) in the proximal promoter region of MKRN3 in a girl with CPP. In silico analysis predicted that this deletion would lead to the loss of a binding site for a downstream res-ponsive element antagonist modulator (DREAM), a potential transcription factor for MKRN3 and GNRH1 expression. Luciferase assays demonstrated a significant reduction of MKRN3 promoter activity in transfected cells with a c.-150_- 147delTCAG construct plasmid in both homozygous and heterozygous states when compared with cells transfected with the corresponding wild-type MKRN3 promoter region. CONCLUSION: A rare genetic alteration in the regulatory region of MKRN3 causes CPP.

S-Nitrosoglutathione Reductase (GSNOR) Deficiency Results in Secondary Hypogonadism.

BACKGROUND: Excess reactive oxygen species and reactive nitrogen species are implicated in male infertility and impaired spermatogenesis. AIM: To investigate the effect of excess reactive nitrogen species and nitrosative stress on testicular function and the hypothalamic-pituitary-gonadal axis using the S-nitrosoglutathione reductase-null (Gsnor-/-) mouse model. METHODS: Testis size, pup number, and epididymal sperm concentration and motility of Gsnor-/- mice were compared with those of age-matched wild-type (WT) mice. Reproductive hormones testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone were compared in Gsnor-/- and WT mice. Immunofluorescence for Gsnor-/- and WT testis was performed for 3ß-hydroxysteroid dehydrogenase and luteinizing hormone receptor (LHR) and compared. Human chorionic gonadotropin and gonadotropin-releasing hormone stimulation tests were performed to assess and compare testicular and pituitary functions of Gsnor-/- and WT mice. OUTCOMES: Evaluation of fertility and reproductive hormones in Gsnor-/- vs WT mice. Response of Gsnor-/- and WT mice to human chorionic gonadotropin and gonadotropin-releasing hormone to evaluate LH and T production. RESULTS: Gsnor-/- mice had smaller litters (4.2 vs 8.0 pups per litter; P < .01), smaller testes (0.08 vs 0.09 g; P < .01), and decreased epididymal sperm concentration (69 vs 98 × 106; P < .05) and motility (39% vs 65%; P < .05) compared with WT mice. Serum T (44.8 vs 292.2 ng/dL; P < .05) and LH (0.03 vs 0.74 ng/mL; P = .04) were lower in Gsnor-/- than in WT mice despite similar follicle-stimulating hormone levels (63.98 vs 77.93 ng/mL; P = .20). Immunofluorescence of Gsnor-/- and WT testes showed similar staining of 3ß-hydroxysteroid dehydrogenase and LHR. Human chorionic gonadotropin stimulation of Gsnor-/- mice increased serum T (>1,680 vs >1,680 ng/dL) and gonadotropin-releasing hormone stimulation increased serum LH (6.3 vs 8.9 ng/mL; P = .20) similar to WT mice. CLINICAL TRANSLATION: These findings provide novel insight to a possible mechanism of secondary hypogonadism from increased reactive nitrogen species and excess nitrosative stress. STRENGTHS AND LIMITATIONS: Limitations of this study are its small samples and variability in hormone levels. CONCLUSION: Deficiency of S-nitrosoglutathione reductase results in secondary hypogonadism, suggesting that excess nitrosative stress can affect LH production from the pituitary gland. Masterson TA, Arora H, Kulandavelu S, et al. S-Nitrosoglutathione Reductase (GSNOR) Deficiency Results in Secondary Hypogonadism. J Sex Med 2018;15:654-661.

Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating intragenic H3K4me2 methylation.

Dynamic histone H3K4 methylation is an important epigenetic component of transcriptional regulation. However, most of our current understanding of this histone mark is confined to the regulation of transcriptional initiation. We now show that human LSD2/KDM1b/AOF1, the human homolog of LSD1, is an H3K4me1/2 demethylase that specifically regulates histone H3K4 methylation within intragenic regions of its target genes. Genome-wide mapping reveals that LSD2 associates predominantly with the gene bodies of actively transcribed genes, but is markedly absent from promoters. Depletion of endogenous LSD2 results in an increase of H3K4me2 as well as a decrease of H3K9me2 at LSD2-binding sites and a consequent dysregulation of target gene transcription. Furthermore, characterization of the LSD2 complex reveals that LSD2 forms active complexes with euchromatic histone methyltransferases G9a and NSD3 as well as cellular factors involved in transcription elongation. These data provide a possible molecular mechanism linking LSD2 to transcriptional regulation after initiation.

High Frequency of MKRN3 Mutations in Male Central Precocious Puberty Previously Classified as Idiopathic.

BACKGROUND/AIMS: Recently, loss-of-function mutations in the MKRN3 gene have been implicated in the etiology of familial central precocious puberty (CPP) in both sexes. We aimed to analyze the frequency of MKRN3 mutations in boys with CPP and to compare the clinical and hormonal features of boys with and without MKRN3 mutations. METHODS: This was a retrospective review of clinical, hormonal and genetic features of 20 male patients with idiopathic CPP evaluated at an academic medical center. The entire coding regions of MKRN3, KISS1 and KISS1R genes were sequenced. RESULTS: We studied 20 boys from 17 families with CPP. All of them had normal brain magnetic resonance imaging. Eight boys from 5 families harbored four distinct heterozygous MKRN3 mutations predicted to be deleterious for protein function, p.Ala162Glyfs*14, p.Arg213Glyfs*73, p.Arg328Cys and p.Arg365Ser. One boy carried a previously described KISS1-activating mutation (p.Pro74Ser). The frequency of MKRN3 mutations among these boys with idiopathic CPP was significantly higher than previously reported female data (40 vs. 6.4%, respectively, p < 0.001). Boys with MKRN3 mutations had typical clinical and hormonal features of CPP. Notably, they had later pubertal onset than boys without MKRN3 abnormalities (median age 8.2 vs. 7.0 years, respectively, p = 0.033). CONCLUSION: We demonstrated a high frequency of MKRN3 mutations in boys with CPP, previously classified as idiopathic, suggesting the importance of genetic analysis in this group. The boys with CPP due to MKRN3 mutations had classical features of CPP, but with puberty initiation at a borderline age.