bluuebunny/crossref_metadata_2025_split huggingface dataset:
Musashi as a Regulator of the Follicle-Stimulating Hormone in the Gonadotropes
Authors: Silva Moreira Ana Rita , Lagasse Alexandra , Haney Anessa , Boehm Ulrich , Kharas Michael G , Lengner Christopher J , MacNicol Melanie C , MacNicol Angus M , Childs Gwen V , Odle Angela Katherine 
Published: May 2021

Abstract
The cyclic expression of gonadotropin releasing-hormone receptors (GnRHR), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by pituitary gonadotropes is critical in the female reproductive process. We have shown that the translational regulator Musashi (MSI) binds to Gnrhr mRNA and inhibits its translation, and the gonadotrope-specific deletion of Msi1 and Msi2 (Gon-Msi-null) leads to increased pituitary GnRHR protein levels. An in silico analysis of gonadotropin mRNAs revealed 5 different MSI binding elements in the 3’UTR of Fshb mRNA. We hypothesize that, in addition to Gnrhr, MSI may also bind and repress Fshb mRNA translation in the gonadotropes. To test if MSI does target the Fshb transcript in the pituitary, we performed RNA immunoprecipitation (IP) on pooled control female mouse pituitaries using a MSI1 antibody and measured Fshb mRNA by qRT-PCR. To study the in vivo effects of MSI on Fshb, we harvested the pituitaries of the Gon-Msi-null (MUT) female mice and their littermate controls (CTL) during the estrous cycle. We collected serum and protein for EIAs to measure the levels of FSH and LH, and RNA for Fshb qRT-PCR. We harvested proestrous ovaries and fixed them for embedding, sectioning, and H&E staining. Our RNA IP experiments show a 7-fold enrichment for Fshb with the MSI1 antibody. The Gon-Msi-null females have significantly higher pituitary FSH protein content than controls on estrous morning (MUT: 4.8±1.3 vs. CTL: 1.8±2.6 ng/ml/μg protein, p&lt;0.0001, n=9-10/group). These mice also have increased serum FSH levels (MUT: 56.9±6.4 vs. CTL: 44.5±9.6 ng/ml, p=0.0147, n=9-10/group). No changes at the Fshb mRNA level were detected. Analysis of Gon-Msi-null ovaries revealed a 50% decrease in the number of follicles, with significant decreases in the average numbers of maturing follicles (p&lt;0.0175) and corpora lutea (p&lt;0.0215). Interestingly, the LH levels in these mice were also altered. The Gon-Msi-null females show a decrease in the pituitary LH protein content in the evening of proestrus (MUT: 11.8±1.4 vs. CTL: 15.1±2.0 ng/ml/μg protein, p=0.0333, n=7/group), in addition to a delayed and blunted LH surge (MUT: 2.6±1.9 vs. CTL: 7.3±3.5 ng/ml, p=0.0089, n=7-11/group). Taken together, our data indicate that Fshb is a Musashi target in the gonadotropes. By deleting MSI from the pituitary gonadotropes, we observe an increase in FSH protein content and serum levels. These Gon-Msi-null female mice have significantly fewer maturing follicles and corpora lutea, which might suggest lower levels of estrogens and progesterone. This, together with the increased GnRHR pituitary protein content, affects LH secretion, leading to a blunted LH surge in the Gon-Msi-null females. Our studies thus reveal a novel translational regulatory mechanism to govern levels of critical reproductive hormones in the pituitary.
Neuroendocrine Basis for Disrupted Ovarian Cyclicity in Females During Chronic Undernutrition: A Mouse Model
Authors: Tadrousse Kirollos S , McCosh Richard B , Kreisman Michael J , Breen Kellie M 
Published: May 2021

Abstract
Chronic undernutrition is a type of metabolic stress that impairs reproduction across species and, in women, is implicated in the development of functional hypothalamic amenorrhea. Although the tight coupling of energy balance to reproductive capacity is recognized in principle, the neuroendocrine loci and molecular mechanisms that mediate ovarian cycle dysfunction during undernutrition remain poorly understood. Ovarian cyclicity is dependent on a population of kisspeptin (Kiss1) neurons in arcuate nucleus (ARCKiss1) for luteinizing hormone (LH) pulses and in the anteroventral periventricular nucleus (AVPVKiss1) for LH surge secretion. Here, we present a series of studies in which we tested the hypothesis that inhibition of both Kiss1 cell populations underlies the impairment of the cycle by undernutrition. During a baseline period, body weight, feed intake, and ovarian cycle stage (via vaginal cytology) were evaluated in female c57bl6 mice. Then, animals were randomly assigned into one of two groups (n=6-8/grp): 1) ad libitum fed controls or 2) feed restricted (70% of feed consumed during the baseline period). Control animals displayed clear and regular cycles throughout the 4-week treatment period. In contrast, feed restriction caused a significant and rapid cessation of ovarian cyclicity (4.8±0.3 vs. 1.5±0.5 estrus cycles/4 weeks; control vs. restricted, p&lt;0.05), causing all females to enter and remain mostly in diestrus. Based on these results, we conducted two experiments to directly test the hypothesis that undernutrition inhibits both modes of LH secretion (and both Kiss1 cell populations) using two well-defined estradiol (E) replacement paradigms. We first evaluated LH pulses in mice that were ovariectomized and implanted subcutaneously with a pellet containing a diestrus level of E (100 ng, OVX+LowE). Following 3 days of feed restriction or control diet (n=3/grp), serial blood samples were collected every 8 min for 88 min. Undernutrition prevented LH pulses and significantly reduced mean LH (5.2±0.6 vs. 0.6±0.2 ng/mL; control vs. restricted, p&lt;0.05). Fixed neural tissue was evaluated by immunohistochemistry to determine whether undernutrition impairs ARCKiss1 neuronal activation, using c-Fos as a marker. The percent of ARCKiss1 neurons expressing cFos was reduced by 90% (p&lt;0.05). We next evaluated the LH surge. After 3 days, control or feed restricted mice were OVX and implanted subcutaneously with a surge-inducing estradiol implant (OVX+HighE, 1 µg, n=3-4/grp). Undernutrition completely blocked the E-induced LH surge (1.9±0.3 vs. 0.2±0.02 ng/mL; control vs. restricted, p&lt;0.05) and diminished Kiss1 mRNA abundance in micropunches of the AVPV (42%, p&lt;0.05). Collectively, these studies clearly show that undernutrition impairs both ARCKiss1 control of LH pulses and AVPVKiss1 induction of the LH surge, via mechanisms that remain to be identified.
Paracrine Signalling From SOX2-Expressing Pituitary Embryonic Cells Is Required for Terminal Differentiation of Hormone-Producing Cells
Authors: Manshaei Saba , Willis Thea , Withers Dominic , Gil Jesus , Andoniadou Cynthia Lilian , Martinez-Barbera Juan Pedro 
Published: May 2021

Abstract
The pituitary gland is the master regulator of the endocrine system, housing six major hormone producing cell types. This gland is derived from Rathke’s Pouch, an invagination of the oral ectoderm. Hormone-producing pituitary cell lineages are derived from a population of embryonic cells expressing SOX2. ZFP36L1/Butyrate Response Factor 1 (BRF1) is an RNA binding protein that binds and targets mRNAs of various cytokines and chemokines for degradation prior to translation, attenuating secretion of inflammatory factors (Herranz et al. 2015). Here, we show that BRF1 is a novel marker expressed in SOX2+ cells in human and mouse pituitaries, suggesting that these cells may have a secretory profile. To investigate this possibility, we have combined molecular and genetic studies in vivo. We have used a novel mouse model, R26lsl-mBRF1 that allows the expression of a mutant, constitutively active BRF1 protein upon Cre-mediated recombination, alongside our lab’s models (Hesx1Cre/+ and Sox2CreERT2/+), to express mutant BRF1 in HESX1+ and SOX2+ cells during development and postnatally. This approach results in pituitary hypoplasia and severe hypopituitarism due to a failure of cell-lineage specified cells to differentiate into hormone-producing cells. Hormone production in these mutant cells, however, can be rescued in vitro through co-culture with WT pituitaries and in vivo in chimeric pituitaries, highlighting a cell non-autonomous mechanism underlying the phenotype. Single cell RNA sequencing of WT and Sox2CreERT2/+;R26lsl-mBRF1 murine embryonic pituitaries, as well as use publicly available human pituitary single cell datasets, have allowed us to identify specific cytokines and chemokines secreted by SOX2+ cells, as well as downstream intracellular signalling pathways in differentiating cells (Zhang et al. 2020), which may be responsible for controlling terminal differentiation of hormone-producing cells within the developing pituitary. Together with our recently published data, these results support the notion that SOX2+ pituitary stem cells play a critical paracrine role in controlling progenitor cell proliferation and terminal differentiation (Russell et al. 2021). References: Herranz, Nicolás et al. 2015. “MTOR Regulates MAPKAPK2 Translation to Control the Senescence-Associated Secretory Phenotype.” Nature Cell Biology 17(9): 1205–17. http://www.nature.com/doifinder/10.1038/ncb3225. Russell, John P et al. 2021. “Pituitary Stem Cells Produce Paracrine WNT Signals to Control the Expansion of Their Descendant Progenitor Cells.” eLife. Zhang, Shu et al. 2020. “Single-Cell Transcriptomics Identifies Divergent Developmental Lineage Trajectories during Human Pituitary Development.” Nature Communications.
Progesterone Might Be an Active Component of the Sleep-Wake Homeostatic Mechanism
Authors: Copinschi Georges , Caufriez Anne H 
Published: May 2021

Abstract
Background: Using a randomized, double blind, placebo-controlled, crossover protocol, we have shown previously that progesterone may prevent sleep disturbances but has no action on undisturbed sleep. In that study, 8 healthy postmenopausal women took daily at 2300 h for 3 wk a capsule of either 300 mg of progesterone or placebo. Sleep was polygraphically recorded during the last two nights and, during the second night, blood samples were collected at 15-min intervals using an iv catheter,. During the first night, sleep was normal under placebo and progesterone had no effect. During the second night, blood sampling procedure was associated under placebo with marked sleep disturbances, which were considerably reduced under progesterone: at the group level, mean ± SEM duration of wake after sleep onset (WASO) dropped from 152 ± 37 min to 71 ± 19 min (P = 0.01), and slow-wave sleep (SWS) duration increased from 53 ± 6 min to 79 ± 10 min (P = 0.04). Objective: To submit individual data collected in that study to new analyses designed to further investigate possible mechanism(s) of progesterone actions on sleep architecture. Results: Among individual subjects, no relation could be evidenced between progesterone levels and sleep variables, or between individual progesterone-associated improvements and absolute values of corresponding sleep variables under placebo. By contrast, for WASO and SWS, significant positive correlations (Spearman test) were evidenced between individual responses to progesterone (i.e. the difference, during night 2, between value under progesterone and value under placebo) and corresponding individual alterations caused under placebo by the blood sampling procedure (i.e. the difference, under placebo, between value during night 1 and value during night 2): WASO: rs = 0.74, P = 0.037, n = 8; SWS: rs = 0.86, P = 0.014, n = 7. (Pearson test yielded similar results: WASO: r = 0.85, P = 0.008; SWS: r = 0.76, P = 0.047). Conclusions: Although they obviously need to be confirmed by larger studies performed in a variety of clinical conditions, the present findings suggest that progesterone action on sleep architecture is specifically tailored to restore individual normality rather than group normality. Since SWS is mainly regulated by the sleep-wake homeostatic mechanism relating sleep pressure to the duration of prior wakefulness, it is tempting to speculate that progesterone might be an active component of this mechanism.
Reference Gene Optimization for Ganglia of the Sympathetic Nervous System in Male and Female Mice after Exposure to Cold Stress
Authors: Pandher Parleen Kaur , Short Landon Imre , Gray Sarah Louise 
Published: May 2021

Abstract
The sympathetic branch of the autonomic nervous system (ANS) regulates the body’s response to psychogenic and systemic stress. In response to stress, endocrine pathways are activated to rebalance physiological changes and restore homeostasis. Gene expression analysis through quantitative real-time PCR (qPCR) is a key method to assess differential gene expression in tissue and cell samples, with high sensitivity and specificity. To assess differential gene expression between samples, qPCR data must be normalized to two or more reference genes to account for technical variation in nucleic acid loading between samples, ensuring data represents target gene expression due to experimental treatments (1). Reference genes must be selected and tested for each experimental paradigm, showing stability of expression between samples and treatments. Reference genes are usually constitutively expressed genes required for cellular maintenance, such as well known “housekeeping” genes or genes encoding ribosomal RNAs. Ganglia of the sympathetic nervous system (SNS) are small in size, and thus total yields of RNA for qPCR experiments are limited. Our group has established a protocol for the isolation of adrenal glands, superior cervical, celiac, and stellate ganglia using a dissecting scope and anatomical landmarks in mice. Here we present optimization data assessing five commonly used reference genes for normalization of qPCR data in three ganglia of the SNS (superior cervical, stellate, and celiac) and adrenal glands. We have evaluated their suitability as reference genes in these tissues after acclimation to cold (4°C) stress compared to the same tissues from animals housed at thermoneutrality (30°C). Through qPCR reaction optimization and subsequent calculations of geNorm stability-measure M, we present optimized primer sequences and reaction conditions, and provide recommendations for combinations of three or more reference genes to use for normalization in gene expression analysis experiments in adrenal gland, superior cervical, stellate or celiac ganglia from male and female mice exposed to thermoneutrality or cold. This research will be valuable to groups interested in SNS physiology and facilitate a streamlined approach to gene expression analysis given the low RNA availability from these minute tissue samples, saving valuable sample, costs and time in carrying out qPCR experiments. Reference: (1) Bustin et al., Clin Chem. 2009 Jan 27;55(4):611-622.
Role of CORT Duration and Estradiol Dependence for Stress-level of CORT to Inhibit Pulsatile LH Secretion in Female Mice
Authors: Kreisman Michael J , McCosh Richard B , Breen Kellie M 
Published: May 2021

Abstract
Two common responses to stress include elevated circulating glucocorticoids and impaired luteinizing hormone (LH) secretion. We have shown that a chronic stress-level of corticosterone (CORT) can slow LH pulses in female mice and that this reduction required estradiol. In addition, the decrease in LH secretion was associated with a suppression in neuronal activation of arcuate kisspeptin (Kiss1) neurons, regarded as the LH pulse generator. Although the increment of the chronic CORT rise was physiological, the duration of the rise exceeded that of a normal CORT response to an acute stressor. Therefore, the goal of the current study was to investigate whether an acute, exogenous CORT rise is sufficient to inhibit LH pulses and arcuate Kiss1 expression, and whether estradiol is necessary for this inhibition. For this study, adult C57bl6 female mice were ovariectomized and implanted with either an implant filled with oil (OVX) or 100 ng estradiol (OVX+E), which approximates a diestrus level of estradiol. Blood samples to measure pulsatile LH were collected every 6 minutes for 90 minutes prior to and following the initiation of CORT or saline treatment. Animals were randomly assigned to receive one of 4 treatments: a single injection of CORT (0.6 mg/kg, i.p) or saline, or three successive injections separated by 30 minutes of CORT (3xCORT) or saline (3xSaline). This dose of CORT elicited a CORT elevation similar to the endogenous CORT rise induced by restraint stress. One injection of CORT elicited an elevation for 30 minutes; the three-injection paradigm elevated CORT for 90 minutes, the entirety of the post injection LH sampling period. Regardless of estradiol status, LH did not differ between the pre and post injection period in mice administered a single injection of CORT or saline. In contrast, in OVX+E mice, 3xCORT significantly slowed the frequency of LH pulses (pulses/90 min: 2.7±0.2 vs 1.4±0.3; pre vs post, p&lt;0.05) compared to mice given 3xSaline (pulses/90 min: 2.9±0.4 vs 2.8±0.4; pre vs post, p&gt;0.05). Interestingly, in OVX mice, there was no impairment of LH secretion in response to either 3xCORT (pulses/90 min: 4.9±0.4 vs 4.7±0.5; pre vs post, p&gt;0.05) or 3xSaline. Knowing that glucocorticoid receptors are present in arcuate Kiss1 cells, we investigated the effect of CORT on Kiss1 expression. Brains were collected 3 hrs following the initiation of the 3xSaline or 3xCORT treatment, and the arcuate nucleus was micropunched for measurement of Kiss1. In OVX+E mice, Kiss1 expression in the arcuate nucleus was suppressed 47% by 3xCORT compared to 3xSaline treated mice (p&lt;0.05). Collectively these data show that an acute CORT rise is sufficient to impair pulsatile LH secretion and Kiss1 expression, but the rise has to be sustained for more than 30 minutes and requires the presence of estradiol. Whether CORT is acting directly or indirectly on arcuate Kiss1 neurons to impair LH secretion remains to be determined.
Synergistic Control of KNDy Neuronal Influence on Energy Balance by Ghrelin and Estradiol
Authors: Conde Kristie M , Kulyk Danielle , Roepke Troy Adam 
Published: May 2021

Abstract
The gut peptide, ghrelin, mediates negative energy homeostasis and the neuroendocrine control of energy homeostasis by acting through its receptor, growth hormone secretagogue receptor (GHSR). GHSR, expressed in hypothalamic Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons in the arcuate (ARC), is well known to regulate energy balance. We have previously shown 17-beta-estradiol (E2) robustly increases Ghsr expression in KNDy neurons, enhancing their sensitivity to ghrelin. We hypothesize that E2-induced increase in GHSR expression augments KNDy sensitivity in a fasting state by elevating ghrelin to reduce energy expenditure in females. We developed a Kiss1-specific GHSR knockout to determine the role of GHSR in ARC KNDy neurons and fed them either a low-fat diet (LFD) or a high-fat diet (HFD). Knockout (experimental) females were resistant to HFD in terms of body weight gain, adiposity, and food intake compared to HFD-fed controls. HFD-fed experimental females also exhibited slower glucose clearance compared to HFD-fed controls. Experimental females, regardless of diet, exhibited elevated fasting (5h) glucose. Metabolic rates (V.O2, V.CO2) and energy expenditure (heat) were not different. Respiratory Exchange Ratio (RER) was elevated in LFD-fed females, indicating the utilization of carbohydrates over fat for energy. Further meal pattern analysis revealed a reduction in meal duration in HFD-fed females, but elevated meal frequency, while HFD-fed experimental females exhibited a reduced meal size. In two separate meal pattern experiments, experimental and control females were fasted for 24h and refed or injected with ghrelin (I.P. 1mg/kg) or saline. We observed a striking delay in refeeding behavior in experimental females compared to controls during the refeeding period after fasting. After injection, control females responded to ghrelin with a rapid and sustained increase in food intake which was blunted in experimentals. Collectively, these data suggest that GHSR activation in KNDy neurons modulates metabolism, glucose homeostasis, and feeding behavior, illustrating a novel mechanism for E2 and ghrelin to synergistically control KNDy neuronal output and their subsequent behavioral and physiological outcomes.
The Transcription Factor Ventral Anterior Homeobox 1 Modulates Circadian Time-Keeping and Fertility Through Direct Regulation of Vasoactive Intestinal Polypeptide Expression in the Suprachiasmatic Nucleus
Authors: Devries Brooke M , Breuer Joseph , Yaw Alexandra , Jackson Brooke , Nguyen Duong , Ho Emily V , Talluri Tulasi , Cui Laura , Gorman Michael , Sempere Lorenzo , Mellon Pamela L , Hoffmann Hanne Mette 
Published: May 2021

Abstract
Light provides the primary timing signal that enables fine-tuned behavioral and hormonal entrainment of circadian rhythms to the environment. Light is transmitted from the eye to the brain through the retinohypothalamic tract, where one target is the hypothalamic suprachiasmatic nucleus (SCN), which generates self-sustained circadian rhythms. The vasoactive intestinal polypeptide (VIP) expressing neurons of the SCN relay light information to peripheral cells and tissues through control of hormonal and nervous signals, allowing synchronization of molecular clocks located in individual cells throughout the body. Non-natural light cycles, ie shiftwork, and weakened SCN function through genetic manipulation, disrupt the body’s circadian rhythms, causing deregulated hormone release, metabolic disorders, and negative effects on reproductive systems such as irregular menstrual cycles and decreased sperm count. To further our understanding of how the SCN translates light information into neuroendocrine control of fertility, we conditionally deleted the SCN enriched transcription factor Ventral anterior homeobox 1 (Vax1) in post-developmental VIP neurons, generating Vax1-flox/flox:Vip-Cre+ (cKO) mice. To determine if the SCN timekeeping function was impacted in cKO mice, we single housed males and females with running wheels to examine activity during both 12-hour light/dark cycles and in constant darkness. Wheel-running behavior in constant darkness revealed a shortening of the endogenous free-running period (Tau) of the SCN. Aside from Tau, wheel running behaviors were comparable to controls. Weakened SCN output can negatively impact fertility. While on 12-hour light/dark cycles, we found a modest, but significant change in follicle stimulating hormone and estrogen in cKO females and a reduced sensitivity of GnRH neurons to kisspeptin in males. The changes in hormone release were associated with a slightly lengthened estrous cycle in cKO females and reduced sperm quality in cKO males. To identify the molecular origin of the shortened SCN period, we used immunohistochemistry and RNAscope to examine expression of Vip. We found that diestrus cKO females had a significant reduction in Vip expression at ZT16 and preliminary data suggest a reduction in the circadian clock gene Bmal1. Together, these studies identify a novel role of VAX1 in VIP neurons where VAX1 is required for VIP expression and circadian timekeeping. Loss of VAX1 in VIP neurons weakens SCN output, deregulating reproductive hormone release and modestly reducing reproductive function in both males and females.