United States

Stress exerts a profound impact on reproductive function and disrupts pulsatile luteinizing hormone (LH) secretion. The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamus pituitary gonadal axis. Stress alters neuronal activity within the MePD, increasing the expression of Urocortin3 (Ucn3) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. Designer receptor exclusively activated by designer drugs (DREADDs) inhibition of MePD Ucn3 neurons prevents psychological stress-induced suppression of LH pulses in female mice. The neurochemical pathways in the MePD responsible for regulating the GnRH pulse generator in the presence of stress remain unknown. We investigate the neurotransmission involved in the neural circuitry responsible for the suppression of GnRH pulse generator activity by MePD Ucn3 neuronal activation. Ucn3-Cre-tdTomato female ovariectomised (OVX) mice were unilaterally injected with AAV-ChR2 and implanted with optofluid cannulae targeting the MePD. Firstly, we optically stimulated MePD Ucn3 neurons with blue light at 10 Hz, 10 mW and monitored the effect on LH pulses. Next, we combined optogenetic activation of MePD Ucn3 neurons with pharmacological antagonism of GABAA or GABAB receptors with bicuculline or CGP, respectively, and observed the effect on LH pulsatility. Finally, during optical activation of MePD Ucn3 neurons we administered a combination of NMDA and AMPA receptor antagonists, AP5 and CNQX respectively, and monitored the effect on pulsatile LH secretion. Optogenetic stimulation of MePD Ucn3 neurons suppressed pulsatile LH secretion compared to controls and administration of aCSF had no effect on LH pulse interval (MePD Ucn3 activation: 36.04 ± 2.49 vs. pre-treatment control period: 17.50 ± 0.67 vs control virus: 21.67 ± 1.67 min; mean ± SEM; p&lt;0.0001; n=12). Intra-MePD infusion of bicuculline (BIC) or CGP during optogenetic stimulation of MePD Ucn3 neurons completely blocked the suppressive effect of MePD Ucn3 neuronal activation on LH pulsatility (MePD Ucn3 activation and BIC: 20.50 ± 2.03 vs. pre-treatment control period: 17.58 ± 0.75 min; MePD Ucn3 activation and CGP: 16.30 ± 0.63 vs. pre-treatment control period: 17.50 ± 0.77 min; n=7-10). Intra-MePD infusion of AP5 + CNQX during optogenetic stimulation of MePD Ucn3 neurons similarly completely blocked the suppressive effect of MePD Ucn3 neuronal activation on LH pulsatility (MePD Ucn3 activation and AP5 + CNQX: 19.58 ± 1.42 min; n=6). Our findings show for the first time that optogenetic stimulation of MePD Ucn3 neurons inhibits GnRH pulse generator activity via GABA and glutamate signalling within the MePD.

ENDO 2022

Urocortin 3 Interaction with GABA and Glutamate in the Posterodorsal Subnucleus of The Medial Amygdala Mediates Suppression of GnRH Pulse Generator Frequency in Female Mice

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Stress exerts a profound impact on reproductive function and disrupts pulsatile luteinizing hormone (LH) secretion. The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamus pituitary gonadal axis. Stress alters neuronal activity within the MePD, increasing the expression of Urocortin3 (Ucn3) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. Designer receptor exclusively activated by designer drugs (DREADDs) inhibition of MePD Ucn3 neurons prevents psychological stress-induced suppression of LH pulses in female mice. The neurochemical pathways in the MePD responsible for regulating the GnRH pulse generator in the presence of stress remain unknown. We investigate the neurotransmission involved in the neural circuitry responsible for the suppression of GnRH pulse generator activity by MePD Ucn3 neuronal activation. Ucn3-Cre-tdTomato female ovariectomised (OVX) mice were unilaterally injected with AAV-ChR2 and implanted with optofluid cannulae targeting the MePD. Firstly, we optically stimulated MePD Ucn3 neurons with blue light at 10 Hz, 10 mW and monitored the effect on LH pulses. Next, we combined optogenetic activation of MePD Ucn3 neurons with pharmacological antagonism of GABAA or GABAB receptors with bicuculline or CGP, respectively, and observed the effect on LH pulsatility. Finally, during optical activation of MePD Ucn3 neurons we administered a combination of NMDA and AMPA receptor antagonists, AP5 and CNQX respectively, and monitored the effect on pulsatile LH secretion. Optogenetic stimulation of MePD Ucn3 neurons suppressed pulsatile LH secretion compared to controls and administration of aCSF had no effect on LH pulse interval (MePD Ucn3 activation: 36.04 ± 2.49 vs. pre-treatment control period: 17.50 ± 0.67 vs control virus: 21.67 ± 1.67 min; mean ± SEM; p<0.0001; n=12). Intra-MePD infusion of bicuculline (BIC) or CGP during optogenetic stimulation of MePD Ucn3 neurons completely blocked the suppressive effect of MePD Ucn3 neuronal activation on LH pulsatility (MePD Ucn3 activation and BIC: 20.50 ± 2.03 vs. pre-treatment control period: 17.58 ± 0.75 min; MePD Ucn3 activation and CGP: 16.30 ± 0.63 vs. pre-treatment control period: 17.50 ± 0.77 min; n=7-10). Intra-MePD infusion of AP5 + CNQX during optogenetic stimulation of MePD Ucn3 neurons similarly completely blocked the suppressive effect of MePD Ucn3 neuronal activation on LH pulsatility (MePD Ucn3 activation and AP5 + CNQX: 19.58 ± 1.42 min; n=6). Our findings show for the first time that optogenetic stimulation of MePD Ucn3 neurons inhibits GnRH pulse generator activity via GABA and glutamate signalling within the MePD.

technical paper

Kisspeptin neurons in the arcuate nucleus (ARC) of the hypothalamus are considered as the gonadotropin-releasing hormone (GnRH) pulse generator to control the cascade of hormone secretions that constitute the reproductive axis. These neurons typically co-express Neurokinin B and Dynorphin and are thus dubbed KNDy neurons. In addition, KNDy neuron kisspeptin is hypothesized to be a major sensor and regulator of metabolic homeostasis while relaying energy status to the hypothalamic-pituitary-gonad (HPG) axis. However, the direct metabolic impact of KNDy neuron kisspeptin has not been well-characterized. To explore this role, we examined the metabolic profile of our previously established KNDy neuron-specific kisspeptin knock-out mouse model (Pdyn-Cre/Kissfl/fl, or KO). To do so, we performed glucose tolerance tests, EchoMRI body composition analysis, and measured body weight in wild type (WT) control or KO mice fed with either regular chow or a high-fat diet (HFD, 60% kcal fat, Research Diets, New Brunswick, NJ) for 12 weeks post-weaning. At 4 weeks on the HFD, KO females weighed significantly more than HFD WT females, which continued through the remaining 8 weeks. Additionally, we found significantly decreased glucose tolerance and increased fat mass in HFD KO females compared to HFD WT females. However, KO males exhibited no significant differences in body weight, body composition, or glucose tolerance between the genotypes on either diet. This data suggests that KNDy neuron kisspeptin is critical for metabolic homeostasis and preventing metabolic dysfunction when challenged with a high-fat diet. Our findings further suggest a sexual dimorphism whereby KNDy neuron kisspeptin performs this action predominantly in females.

Absence of Kisspeptin in KNDy Neurons of Mice Causes Sexually Dimorphic Metabolic Dysfunction on a High-Fat Diet

Introduction: Loss-of-function (LOF) germline variants in the SDHx genes (SDHA, SDHB, SDHC, and SDHD), encoding the succinate dehydrogenase subunits, are a rare cause for pituitary tumors in the setting of various familial tumor syndromes. Occasional case reports have highlighted SDHx mutations in isolated pituitary tumors (including one case of Cushing's disease, CD). The actual frequency of SDHx variants as genetic drivers of pituitary tumors, however, remains elusive.
Aim: We sought to determine the frequency of germline SDHx variants in a large cohort of CD patients.
Methods: Of the 245 unrelated CD patients studied (139 females, 56.7%; 230 pediatric, 93.9%), 184 underwent whole-exome sequencing (WES) of peripheral blood and 27 of them underwent additional tumor WES. Forty-three additional germline samples and 92 tumor samples underwent Sanger sequencing of specific genes. Rare variants of uncertain significance (VUS), likely pathogenic or pathogenic variants of the SDHx genes were reviewed. Immunocytochemistry was used to analyze protein expression in tumor samples.
Results: Germline heterozygous variants of SDHA (three missense and one frameshift) and SDHD (two missense) were identified in six patients (four female; mean age of 8.8±2.2 years at onset of CD and 11.5±2.6 years at diagnosis). The variants were inherited in all 5 cases where parental germline samples were available. We found the following SDHA variants: Case 1 carried p.A21T (absent from gnomAD), Case 2 carried p.L74Ffs*9 (absent from gnomAD), Case 3 carried p.I235T (allele frequency, MAF=0.0100% in gnomAD exomes), and Case 4 carried p.H424Q (MAF=0.0056% in gnomAD exomes). Two patients (Cases 5 and 6) carried the SDHD missense VUS c.53C>T, p.A18V (MAF= 0.0068% in gnomAD exomes). Case 6 had a maternal family history of familial isolated pituitary adenomas, but an SDHD variant of paternal origin; disease presentation was apparently sporadic in the rest of cases. Case 2 displayed somatic loss of heterozygosity (LOH) for the SDHA variant, while no LOH was found in the three other cases tested. Somatic hotspot variants were absent in all tumors tested in USP8 (5/5 cases), USP48 (4/4 cases), and BRAF (4/4 cases). All patients carried microadenomas and achieved remission after one transsphenoidal surgery. Case 4 was diagnosed with a Crooke's cell corticotropinoma and also carried germline VUS in DICER1 and TSC2. In all three tumors tested, SDHx subunit expression was reduced: SDHB reduced in Cases 2, 4, and 5; SDHA reduced in Cases 2 and 4; absent SDHD in Case 2, and absent SDHC in case 5.
Conclusion: Altogether, SDHx variants were found in 2.4% of the cohort (2.6% of pediatric patients). Our findings suggest that germline SDHx variants are a rare but significant genotype associated with isolated sporadic Cushing's disease.
* JLM and CAS contributed equally.

Isolated Cushing’s Disease Associated With Rare Germline SDHx Variants

The pituitary gland controls responses to changing physiological requirements during growth, pregnancy, puberty, and stress. Synthesis and secretion of appropriate pituitary hormones in response to changing organismal demands is mediated through a remarkable capacity for cellular plasticity of the distinct hormone-producing cells. The mechanisms governing pituitary cellular plasticity are not fully understood. We hypothesize that plasticity in the adult tissue is mediated through similar mechanisms to those that occur during embryonic and postnatal pituitary development. The Prophet of Pit1(Prop1), a paired-like homeodomain transcription factor, is the earliest pituitary-specific transcription factor and is highly expressed in pituitary progenitor cells where it controls lineage commitment to the distinct hormone-producing cell types.  Our recent work has identified post-transcriptional regulation of pituitary gene expression in the adult tissue via the stem cell determinant, mRNA binding protein, Musashi. The full-length mouse Prop1 (mProp1) mRNA 3' untranslated region (UTR) contains 24 consensus binding elements for Musashi (MBEs) and demonstrates Musashi1-dependent translational activation in vitro. Specifically, the terminal mProp1 3' UTR MBE directs 40% of Musashi1-mediated translation activation, and mutational disruption of this terminal MBE abolishes all Musashi1-dependent translational activation. Immunoprecipitation of Musashi-mRNA complexes from adult mouse pituitaries confirmed Musashi1-mProp1 mRNA association in adult tissue. The gene sequence data for the human Prop1 (hPROP1) mRNA 3' UTR is incomplete in the Ensembl and RefSeq databases. Using published RNA sequencing datasets, we identified an expressed full-length hPROP1 mRNA 3' UTR of 6996 nucleotides with 54 predicted consensus MBEs, suggesting that the human PROP1 mRNA, like the murine Prop1 mRNA, may be subject to Musashi-dependent translational control. Our findings are consistent with the hypothesis that the Musashi protein exerts evolutionarily conserved control of Prop1 mRNA translation to facilitate plasticity of anterior pituitary function.

Regulation of the Pituitary Cell Lineage Regulator Prop1 by the Stem Cell Determinant Musashi

Heart Rate Variability (HRV) results from the autonomic nervous system activity, it is a non-invasive marker of cardiac autonomic function. Loss of parasympathetic function (PNS) and sympathetic (SNS) override, reflected in decreased HRV, is one of the earliest subclinical manifestations of cardiac autonomic dysfunction. Lower HRV is associated with increased risk of cardiac events in adults.
We aimed to characterize HRV in normal weight and overweight children with and without dysglycemia and to investigate the determinants of HRV in these youth, including body composition, glycemia measures, beta-cell function and inflammatory markers.
We evaluated 94 adolescents (50 males/44 females), age 15 ± 2.1 years; 21 normal weight with normal glucose tolerance (NW-NGT), 23 overweight with NGT (OW-NGT) and 50 overweight with impaired glucose regulation (OW-IGR) including prediabetes (n= 27) and type 2 diabetes (n= 23). They underwent assessment of anthropometrics, body composition (DXA scan), inflammatory markers (hs-CRP and TNF-α), fasting labs and 2-hour oral glucose tolerance test (OGTT) with determination of glucose and insulin concentration. EndoPat was used to measure HRV: frequency-domain indices [high-frequency (HF) reflecting PNS, low-frequency (LF) reflecting both PNS and SNS, and LF/HF (higher is worse) estimating the ratio between SNS and PNS] and time-domain indices [the inter-beat interval of normal sinus beats (NN) and the standard deviation of NN (SDNN) measuring overall HRV, the square root of the mean squared difference of successive NN (RMSSD) and the NN intervals differing by more than 50 milliseconds (NN50) measuring PNS activity].
LF/HF was higher in the OW-IGR group compared with NW and OW-NGT (p=0.005). After controlling for sex, race and Tanner stage, fasting glucose (FBG) negatively correlated with NN (r=-0.22,p=0.04), SDNN (r=-0.21,p=0.05), RMSSD (r=-0.3,p=0.004), NN50 (r=-0.27,p=0.01) and HF (r=-0.26,p=0.02). LF/HF was positively related to FBG (r=0.39,p<0.001), 2hr-glucose (r=0.31,p=0.004) and HbA1c (r=0.22,p=0.04), and negatively with the insulinogenic index (r=-0.27,p=0.02), but not fasting insulin or HOMA-IR; LF/HF also correlated with percent body fat (r=0.22,p=0.04), hs-CRP (r=0.33,p=0.002) and TNF-α (r=0.38,p=0.006).
In a linear regression model with LnLF/HF as the dependent variable and percent body fat, hs-CRP, FBG and HOMA-IR as the independent variables, FBG (beta=0.39,p=0.003) and hs-CRP (beta=0.21,p=0.09) were the significant determinants of LnLF/HF independent of age, sex, race and Tanner stage as covariates (R²= 0.23,p=0.013).
Youth with impaired glucose metabolism have evidence of early subclinical cardiac autonomic dysfunction with decreased HRV, loss of parasympathetic function and sympathetic overdrive as reflected by lower time-domain indices, lower HF and higher LF/HF, related to glycemia and systemic inflammation.

Hyperglycemia Is the Main Determinant of Cardiac Autonomic Dysfunction in Youth With Obesity Across the Spectrum of Glycemic Regulation.

Background: Pediatric obesity is a multifactorial disease characterized by a prolonged imbalance between energy intake and expenditure. In rare cases, it is caused by underlying medical disorders arising from disruptions in the leptin-melanocortin pathway which regulates satiety and energy expenditure.
Aim: To investigate and compare resting energy expenditure (REE) and body composition characteristics of children and adolescents with severe obesity with and without underlying medical causes.
Methods: This prospective observational study included pediatric patients who underwent an extensive diagnostic workup in our academic center in which endocrine, non-syndromic and syndromic genetic, hypothalamic, and medication-induced causes of obesity were evaluated. Patients in whom no underlying medical cause was identified were classified as multifactorial obesity. REE was assessed by indirect calorimetry; body composition by air displacement plethysmography. The ratio measured REE (mREE) vs predicted REE (Schofield equations) was expressed as REE%, with decreased mREE defined as REE% ≤90% and elevated mREE as ≥110%. Additionally, the ratio mREE vs fat-free-mass (FFM) was calculated.
Results: We included 292 patients, of which 218 (75%) patients had multifactorial obesity and 74 (25%) had an underlying medical cause: non-syndromic and syndromic genetic (n= 29 and 28, respectively), hypothalamic (n= 10), and medication-induced (n= 7) obesity. Mean age was 10.8 ± 4.3 years, 59% were female, mean BMI SDS was 3.8 ± 1.1, indicating severe obesity. Mean REE% was higher in children with non-syndromic genetic obesity (107.4% ± 12.7) and lower in children with hypothalamic obesity (87.6% ± 14.2) compared to multifactorial obesity (100.5% ± 12.6, both p<0.01). Measured REE was decreased in 60 (21%) patients (corresponding to an average overprediction of daily caloric needs of 341 kcal/day) and elevated in 69 (24%) patients. Only in hypothalamic obesity, a larger proportion of patients showed a decreased REE compared to multifactorial obesity (6/10 vs 41/218, p<0.01). FFM was higher in children with non-syndromic obesity compared to multifactorial obesity (+7.5kg, p<0.001), but lower in syndromic obesity (-5.2kg, p=0.03), hypothalamic obesity (-12.6kg, p<0.001), and similar in medication-induced obesity (+1.5kg FFM, p=0.80). Mean mREE/FFM was 46.5 ± 10.6 kcal/day/kg FFM and did not differ between patients with underlying medical causes compared to multifactorial obesity (all p>0.05).
Conclusion: In this cohort of children with severe obesity due to various etiologies, large inter-individual differences in mREE were found. Almost half of patients had decreased or elevated mREE. When relating mREE to FFM, no differences were found between children with underlying medical causes versus multifactorial obesity. Thus, our study underlines the importance of measuring REE and relating mREE to FFM in children with early-onset severe obesity with or without underlying medical causes. This knowledge is important for patient-tailored treatment, e.g. personalized dietary or physical activity interventions and consideration of pharmacotherapy affecting central energy expenditure regulation in children with decreased mREE.

Resting Energy Expenditure and Body Composition in Children and Adolescents with Genetic, Hypothalamic, Medication-Induced or Multifactorial Severe Obesity

Background: Turner syndrome (TS) is caused by the absence of whole/part of the second X-chromosome. Primary ovarian insufficiency (POI) is common in TS and serum anti-Mullerian hormone (AMH) of <4 pmol/L (0.56 ng/mL) is suggestive of POI and lack of spontaneous puberty.
Objective: To describe the distribution of AMH and spontaneous puberty in a cross-sectional cohort of patients in a newly-established pediatric TS specialty clinic.
Methods: Data on all TS patients seen in the multidisciplinary clinic between 1/1/2019 and 10/27/2021 were abstracted from retrospective chart review into a REDCap database. Descriptive analysis, one way ANOVA and two sample t-tests for pair-wise comparisons was completed (SPSSv28.0 and Excelv2102), with statistical significance set at p<0.05.
Results: Ninety-six patients (9% Asian, 13.5% African American and 37.5% Caucasian, and rest unknown/other self-reported race, and 29.2% Hispanic ethnicity) were enrolled in our clinic. While the majority were diagnosed postnatally (74%, median age 5 years), 6% were prenatal and 20% had positive prenatal screen with postnatal confirmation. Karyotype distribution: 41% non-mosaic monosomy X, 17% Xq (including 14% isochromosome Xq) and 14% Xp structural abnormalities, 10% ringX, 7% 45,X/46,XX mosaicism and 9% with Y-chromosome. Median age at first TS clinic visit was 10 years (IQR 9.7) with median height at 2%ile, weight 31%ile and BMI 73%ile. AMH was measured in 88 patients and only 33 (37.5%) had detectable (>0.015 ng/mL) AMH; 8 (9%) had AMH >0.56ng/mL. Mean AMH was significantly lower in 45, X monosomy (0.03+/- 0.14) vs. other karyotypes (0.44+/-1.13)(p=0.01). A one-way ANOVA revealed a statistically significant difference in AMH between 45,X, structural abnormalities in Xp/Xq and, mosaic 45,X/46,XX karyotypes (p<0.001). Pair-wise comparisons confirmed mean AMH in 45,X/46,XX was significantly higher (3.1ng/mL) (p<0.001) compared with 45, X (0.03ng/mL), Xq (0.02ng/mL) and Xp (0.08ng/mL) abnormalities but no other comparisons were statistically significant.
Spontaneous thelarche was reported in 23/58 (39.6%) pubertal patients with mean AMH (0.65ng/mL +/-1.3) compared to absent spontaneous thelarche (N=35, 0.02 +/- 0.04) (p=0.03). Spontaneous menarche was reported in 9 patients, with mean AMH 1.8+/- 1.7ng/mL; 4/9 had 45,X/46, XX karyotype and 1 each had 45,X, Xp, Xq, isoXq, and ringX karyotypes. The mean age at spontaneous thelarche was lower than age at thelarche with hormonal induction (11 + 1 years vs. 14 + 2 years, p < 0.001). Median age at estrogen initiation was 12 years (IQR 3); 86% received transdermal estradiol with menarche at a median age of 15 years. While 78/96 (81%) received fertility preservation counseling at a mean age of 11 + 6 years, only 4 utilized fertility preservation options (1 oocyte cryopreservation and 3 ovarian tissue cryopreservation).
Although cross-sectional data limit inferences on AMH as a marker of puberty, our data represents a large and diverse cohort of TS patients in the US.

AMH and Spontaneous Puberty in a Diverse US Turner Syndrome Clinic Cohort: A Cross-sectional study

Background
Pituitary stalk interruption syndrome (PSIS) may associate with brain midline defects and also with polydactyly (GLI2 mutations) but rarely with brain cortex anomalies.
Clinical Case
We report a boy referred at age 19 months because of growth failure. Bilateral postaxial polydactyly, syndactyly of the toes, and a nodule on the tongue were noted at term birth. The neonatal course was complicated by marked but transient hypoglycemia. During infancy, the acquisition of gross motor skills was slightly delayed. Upon referral, body length was 4.1 SD below the level of mid-parental height. Extremely low concentrations of circulating IGF-I (8 mcg/L) and IGFBP-3 (861 mcg/L) were suggestive of growth hormone (GH) deficiency which was corroborated by glucagon test (peak GH 3.3 mcg/L, peak cortisol 14.6 mcg/dL) in an euthyroid and normoprolactinemic state, and in the absence of polydipsia or polyuria. Brain MRI disclosed not only a PSIS triad (with a virtual absence of the pituitary stalk and the anterior pituitary, and with an ectopic position of the posterior pituitary) but also abnormal sulcation and polymicrogyria (pointing to abnormal lamination in the cortex) on both sides in the posterior cingulum. Familial history is positive for holoprosencephaly and polydactyly in two male relatives, who died neonatally.
Whole exome sequencing showed a rare maternally inherited variant in ZRSR2 on the X chromosome (c. 1207_1208delAG (p.Arg403Glyfs*24)) (OMIM 300028). ZRSR2 isdepleted from loss-of-function variants in the reference population, and has not been associated with congenital anomalies or with pituitary dysfunction. This ZRSR2 variant escapes nonsense mediated decay and segregates in this family according to an X-linked recessive pattern. X-inactivation studies and gene expression studies are ongoing to correlate the ZRSR2 variant to the patient's phenotype.
Conclusion
This case describes a toddler with polydactyly and short stature, based on GH deficiency due to PSIS, in combination with polymicrogyria. Studies are ongoing to link the patient's phenotype to a rare variant in ZRSR2.

Pituitary stalk interruption syndrome, polydactyly, polymicrogyria and a ZRSR2 variant

Weakening of the lower abdominal wall muscle via fibrosis causes visceral contents to bulge out, forming inguinal hernias. Humanized aromatase (Aromhum) transgenic male mice develop this weakness in the lower abdominal muscle (LAM) tissue, characterized by extensive fibrosis, muscle atrophy, and scrotal hernias. This mouse model incorporates the human version of the aromatase gene allowing for excessive estradiol (E2) production in skeletal muscle tissue. We have previously shown that the LAM fibroblasts are sensitive to local E2 production due to high levels of estrogen receptor alpha (ERα, Esr1)¹. Here, we explore the origins of estrogen-driven muscle fibrosis in the LAM tissue of Aromhum mice at a single-cell level.
Wild-type (WT) and Aromhum LAM tissues were processed via single-cell RNA sequencing (n = 3) and analyzed using Seurat. We observed a total of 22 UMAP clusters, including six fibroblast-like clusters. Five of these clusters express the fibro-adipogenic progenitor (FAP) marker Pdgfrα, and two of them are exclusively found in Aromhum LAM tissues. One Aromhum-enriched cluster highly expresses Esr1 and its downstream estrogen-responsive genes such as Pgr and Greb1. Pseudo-time analysis suggested that this cluster differentiates into a pathogenic fibroblast cluster characterized by high expression pro-inflammatory (Il33, Il6, Ccl8, C4b) and fibrosis-associated (Mmp3, Cthrc1, Saa3, Ptx3) genes. We further validated our findings via flow cytometry, immunocytochemistry, and western blots in LAM, upper abdominal muscle tissues, and quadriceps of both WT and Aromhum mice. We show that protein levels of Pdgfrα, ERα, Mmp3, C4b are greater in the fibroblasts freshly isolated from LAM tissues of Aromhum mice compared to WT mice. Moreover, Aromhum LAM tissue cell cycle analysis reveals that ~15% of all ERα+ cells are in the G2-S phase compared with <1% of ERα- cells. Overall, we provide an insight into the cell types, genes, and proteins that are dysregulated in the LAM tissues from Aromhum mice with hernias.
Inguinal hernias are found in ~25% of the elderly male population². Using an inguinal hernia mouse model that mimics the hernia pathophysiology of elderly men with estrogen excess and testosterone deficiency, we defined a specific population of estrogen-sensitive fibroblasts responsible for fibrosis and weakening of lower abdominal skeletal muscle tissue associated with hernias. We provide an insight into the specific disease-causing fibroblast populations and molecular targets that may be the basis for future therapeutics to prevent or treat inguinal hernias.
References:
1. Zhao H, Zhou L, Li L, et al. Shift from androgen to estrogen action causes abdominal muscle fibrosis, atrophy, and inguinal hernia in a transgenic male mouse model. Proceedings of the National Academy of Sciences. 2018;115(44): E10427-E10436.
2. Sazhin A, Zolotukhin I, Seliverstov E, et al. Prevalence and risk factors for abdominal wall hernia in the general Russian population. Hernia. 2019;23(6): 1237-1242.

A Unique Estrogen-Sensitive Fibroblast Population Drives Abdominal Muscle Fibrosis

Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. Estrogen receptor-alpha, ERɑ, functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the predominant naturally-occurring estrogen. This results in dimerization, DNA binding to cis-regulatory elements called enhancers, and gene regulatory function of ERɑ. In vivo, E2 production and action fluctuates during reproductive cycles and possibly even in a circadian manner. In tissues and cells, E2-regulated gene expression is dynamic, with rapid induction of enhancer formation and target gene expression (or repression), followed by enhancer "decommissioning" and cessation of the gene regulatory effects. The effects of sustained E2 production and signaling, fluctuations in E2 levels, and removal of E2 on estrogen-regulated gene expression are unknown. Classical experiments exploring the gene regulatory effects of E2 involve stimulation of cultured cells throughout the duration of the experiment with a continuous dose of hormone, with the cells harvested at the end of the stimulation period. While this has allowed us to understand the molecular underpinnings of signal-dependent transcription, it has not allowed analysis of physiological fluctuations in estrogen signaling involving periods of stimulation and non-stimulation. In our studies, we are applying a combination of next generation sequencing techniques and fluorescence microscopy to investigate signal-dependent responses to fluctuating E2 levels using ERɑ-positive MCF-7 human breast cancer cells after E2 treatment and removal. Our initial results suggest that different E2 target genes respond differently to E2 withdrawal. Moreover, for some E2 traget genes, sustained E2 treatment may not be required for a complete E2 transcriptional response. With these studies, we hope to connect physiological aspects of E2 production, release, and action with the molecular mechanisms of E2-dependent gene expression - an aspect of our understanding of estrogen signaling that has been lacking.
This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K.

Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers

Imidacloprid (IMI) is a neonicotinoid pesticide used for pest control and seed treatments. It mimics nicotine and selectively targets insect nicotinic acetylcholine receptors to induce paralysis and death. Popularly used worldwide, IMI residue is readily detectable in crops, the environment, and human samples. Gastrointestinal symptoms in patients as well as defective gut barrier and hepatoxicity in animal models have been reported subsequent to IMI exposure. However, the mechanism underlying IMI toxicity in mammals is unclear. Pesticide exposure frequently activates xenobiotic nuclear receptors, like constitutive androstane receptor (CAR), to induce detoxification phase I and phase II genes. We found that IMI exposure in wild-type (WT) mice induced hepatic expression of Cyp2b10, a bona fide target of CAR. This data led us to investigate the role of CAR in alleviating IMI toxicity. We dosed wild-type (WT) and Car-/- mice with 50mg/kg BW IMI, twice daily for 21 days and collected serum, liver, and intestine tissues. The absence of CAR resulted in a three-fold increased accumulation of IMI within the liver as well as a 6% increase in body weight. Intriguingly, IMI exposure resulted in hepatic inflammatory pockets in WT mice but did not induce serum liver injury markers (ALT and AST) in either WT or Car-/- mice. We quantified the expression of genes involved in detoxification and oxidative stress within the liver and ileum using qRT-PCR. We found Car-/-mice had significantly blunted expression of Cyp-P450 enzymes in the ileum. To compare gut function, we measured organ coefficient and examined ileal histology using Alcian Blue/PAS staining. We found the absence of CAR altered small intestine (SI) morphology independent of IMI exposure suggesting a basal role of CAR in maintaining overall SI morphology. Car-/-mice also exhibited increased SI weight and length and shorter villi height. AB-PAS staining showed IMI reduced ileal mucin production in WT mice but not Car-/- mice. To further examine CAR's role in the xenobiotic metabolism in the ileum, we will compare microbial diversity and bile acid pools with and without IMI exposure. Our results suggest IMI is not overtly toxic, but the absence of xenobiotic nuclear receptor CAR allows increased accumulation of IMI in the liver and disrupts the structure and Cyp gene expression in the intestine.

Urocortin 3 Interaction with GABA and Glutamate in the Posterodorsal Subnucleus of The Medial Amygdala Mediates Suppression of GnRH Pulse Generator Frequency in Female Mice

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