bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Oral Concomitant Medications in Patients With Acromegaly
Authors: Petersenn Stephan , Houchard Aude , Schneider Maria del Pilar , Ribeiro-Oliveira Antonio , Neggers Sebastian Jcmm
Published: May 2021

Background: Acromegaly is a rare condition characterized by excess GH and IGF-I levels.1 Injectable somatostatin analogs (SSAs) are recommended as first-line medical treatment.2 Comorbidities including diabetes, hypertension and hypopituitarism reduce quality of life in patients with acromegaly; 3,4 treatment for these includes oral medications. We assessed the most common oral medications used by patients with acromegaly during treatment with lanreotide autogel 120 mg (LAN).

Methods: This post-hoc analysis reports data from the PRIMARYS5 (NCT00690898) and LEAD6 (NCT00701363) open-label trials in which adult patients with acromegaly received LAN. PRIMARYS patients were SSA/surgery-naïve with uncontrolled acromegaly, LEAD patients were previously treated with octreotide long-acting release with normal age-adjusted IGF-I levels for two consecutive measurements.

Results: PRIMARYS included 90 patients: mean (standard deviation [SD]) age 49.5 (12.4) years; 47.8% male; mean (SD) baseline IGF-I 809.9 (300.0) µg/L, 97.8% had IGF-I >1.3xULN. LEAD included 124 patients: mean (SD) age 55.4 (10.9) years; 37.1% male. Median (range) time since diagnosis was 99.0 (27.0-1,261.0) days (PRIMARYS) and 7.1 (0.5-27.0) years (LEAD). 51.1%, 24.4% and 3.3% of participants in PRIMARYS had a history of cardiovascular disorders (including hypertension), diabetes/glucose intolerance, and hypopituitarism/hypothalamic disorders; the corresponding values in LEAD were 69.4%, 33.9% and 18.5%. 83.3% of participants in PRIMARYS and 86.3% in LEAD used ≥1 concomitant oral medication with LAN. Of these participants, >50% used >3 types (PRIMARYS: 53.3%; LEAD: 61.7%). Out of participants in both studies who used concomitant oral medication with LAN, this was commonly indicated for cardiovascular disorders including: renin-angiotensin agents (PRIMARYS: 33.3%; LEAD: 45.8%); antithrombotic agents (PRIMARYS: 10.7%; LEAD: 19.6%); beta-blocking agents (PRIMARYS: 22.7%; LEAD: 19.6%) and calcium channel blockers (PRIMARYS: 16.0%; LEAD: 22.4%). Diuretics (PRIMARYS: 16.0%; LEAD: 20.6%) and lipid modifying agents (PRIMARYS: 20.0%; LEAD: 39.3%) were also common concomitant oral medications, as were medications used in diabetes (PRIMARYS: 17.3%; LEAD: 20.6%) and for thyroid therapy (PRIMARYS: 28.0%; LEAD: 31.8%).

Conclusions: In this analysis, most patients with acromegaly receiving LAN, regardless of prior therapy, used ≥1 concomitant oral medication. An awareness of the ongoing burden of comorbidities requiring oral medications should help physicians in managing acromegaly.

Funding: This study was sponsored by Ipsen.

1. Lavrentaki A Pituitary 2017;20:4-9

2. Melmed S Nat Rev Endocrinol. 2018;14:552-61

3. Lesén E Eur J Endocrinol. 2017:176:203-12

4. Vilar L Pituitary 2017;20:22-32

5. Caron PJ J Clin Endocrinol Metab. 2014;99:1282-90

6. Neggers SJ Eur J Endocrinol. 2015;173:313-23
Age and Parity Effects in the Hypothalamic Pituitary Adrenal Axis’ Response to Multimodal Stress in Female Mice
Authors: Sheng Julietta A , Heck Ashley L , Miller Alex M , Bales Natalie J , Myers Sage A , Tan Sarah M L , Daniels Renata M , Handa Robert J
Published: May 2021

The hypothalamic pituitary adrenal (HPA) axis responds to environmental perturbations to maintain homeostasis. Pregnancy demands extensive modifications in HPA axis function to prepare for increased energy and metabolic demands required to meet the needs of mother and offspring. Short-term effects of pregnancy on the HPA axis have been shown, but data is lacking regarding the long-term effects in middle-aged female mice no longer breeding. Since changes of the HPA axis are further found with age, in this study, we examined both parity- and age-related interactions on the HPA axis in female mice. Wildtype C57bl/6N females were divided into nulliparous young (NY) (3-6 mo) and nulliparous middle-aged (NM) or multiparous retired-breeder middle-aged (RBM) (8-10 mo) groups. RBM mice were killed at least 4 weeks after their last litter was weaned. Control mice were euthanized directly out of the home cage and experimental groups were euthanized at 0 min, 30 min, or 90 min recovery (n=8-10/ group) after 2h of multi-modal stress (MMS; restraint, noise, shaker, light). (Paraventricular nucleus of the hypothalamus (PVN) neuronal activity was quantified by c-FOS immunoreactivity (-ir), and plasma corticosterone (CORT) levels were measured by radioimmunoassay. Corticotropin releasing hormone (CRH) mRNA was assayed by in situ hybridization. Two-way ANOVA showed effects of age (p<0.0248), parity (p<0.0021), time (p<0.0001), and interaction (p<0.0009) on CORT levels. Specifically, basal CORT levels were reduced in NM/RBM versus NY mice. In all groups, CORT levels were significantly elevated by MMS. There was no difference between CORT levels immediately after MMS in NY or NM groups, but CORT levels after 30- and 90- min recovery from MMS remained elevated in NM, indicating reduced negative feedback with age. Additionally, RBM plasma CORT was further reduced in all time groups versus NM, accompanied by a return to baseline CORT after 90 min recovery, suggesting a parity-dependent effect on the HPA axis. Changes in CORT levels were correlated with c-FOS-ir. MMS increased PVN c-FOS-ir in all groups compared to controls and c-FOS-ir in NM was significantly greater than PVN c-FOS of RBM. Further, while c-FOS-ir in the NY females was reduced to baseline 30 min after MMS, the return to baseline was more gradual in NM. No effect of parity or age was seen in Crh mRNA. Collectively, our findings show that activation of the HPA axis in females involves interactions between age- and parity- dependent function. Our findings further show activation and inhibition of the HPA axis in females involving long-term changes that occur after pregnancy, which may increase risk for stress- or postpartum- related disorders. Supported by NIDDK 1-R01 DK105826
Antisense Oligonucleotides as a Novel Therapy for Cushing’s Disease
Authors: Eltumi Hanan , Whatmore Jacob , Kemp Elizabeth Helen , Newell-Price John
Published: May 2021

Background: Cushing’s disease (CD) is caused by high levels of blood cortisol resulting from excess secretion of ACTH from a corticotroph adenoma in the anterior pituitary gland. Clinical features include hypertension, diabetes, osteoporosis, and depression. If untreated CD has an increased mortality of five-fold owing to cardiovascular comorbidities, stroke or raised vulnerability to infection. Transsphenoidal surgery is considered the first-line treatment but remission is achieved in only 65% of cases and the relapse rate is high. Furthermore, medical treatments are often accompanied by unpleasant side-effects. Antisense therapy is a technique for suppressing gene expression at the level of translation using antisense oligonucleotides (ASOs) against the mRNA of interest. Aims: To investigate antisense therapy as a treatment for CD by targeting ASOs against ACTH-encoding POMC mRNA thereby reducing secretion of the hormone. To transfect mouse AtT20 cells (cells that secrete high levels of ACTH) with ASOs against POMC at varying doses to determine which is the most effective at reducing ACTH secretion. Methods: AtT-20 cells that secrete high levels of ACTH were used as the model system. ASOs were designed to specifically target exon 3 of the POMC gene. Transfection of AtT-20 was carried out using Lipofectamine. FACS was used to determine transfection efficiency. ACTH levels secreted by AtT-20 cells were determined by immunoassay. Statistical analysis was done using ANOVA with P values < 0.05 considered significant. Results: ASOs that targeted POMC exon 3 (ASO-2 and ASO-3) were transfected into AtT-20 cells at 10 and 100 nM. Control ASOs were ASO-1 (matched to POMC sense strand) and ASO-4 (a scrambled version of ASO-3). Experiments included untreated AtT-20 cells and AtT-20 cells treated with transfection reagent or ASOs alone. The results of six experiments indicated that ACTH secretion from AtT-20 cells was reduced after transfection with ASO-2 and ASO-3 at 100 nM (ANOVA, P = < 0.05) and 10 nM (ANOVA, P < 0.05) when compared with untreated AtT20 cells. ASO-1 and ASO-4 had no effect on ACTH secretion by AtT-20 cells (ANOVA, P > 0.05). Conclusions: Initial experiments have shown that ASOs against POMC can reduce ACTH secretion from AtT-20 cells and may be useful as a novel therapy for CD.
Chronic Undernutrition Inhibits KNDy Neurons in Ovariectomized Ewe Lambs
Authors: Harlow KaLynn , Griesgraber Max , Seman Andrew , Shuping Sydney , Sommer Jeffrey , Hileman Stanley , Nestor Casey
Published: May 2021

Undernutrition negatively impacts reproductive success, at least in part, through a central suppression of GnRH secretion. Given that GnRH neurons are devoid of receptors for peripheral metabolic hormones such as leptin and insulin, nutritional regulation of GnRH secretion must be through afferent input. Neurons which co-express kisspeptin, neurokinin B (NKB), and dynorphin, termed KNDy neurons, are a unique population of cells in the arcuate nucleus (ARC) of the hypothalamus and are believed to play a critical role in GnRH/LH pulse generation. With our recent evidence that chronic feed restriction reduced kisspeptin and NKB in young, castrated male sheep, we hypothesized that nutrient restriction would inhibit expression of the KNDy neuron peptides kisspeptin and NKB, while increasing dynorphin expression in young, ovariectomized female sheep. Fifteen ewe lambs were ovariectomized and were fed to maintain body weight (n=7; Fed) or feed-restricted to lose 20% of pre-study body weight (FR; n=8). Blood samples were taken weekly every 12 minutes for 4.5 hours via jugular venipuncture and plasma was stored at -20°C until assessment of LH using radioimmunoassay. Body weights were recorded weekly and feed amounts were adjusted to achieve desired body weights. Following blood collection at Week 13, animals were euthanized, brain tissue was perfused with 4% paraformaldehyde, and tissue containing the hypothalamus was collected for assessment of KNDy neuropeptide mRNA abundance by in situ hybridization (RNAscope). At Week 13, the average percent change in body weight was clearly evident (Fed, 7.35 +/- 2.2% vs FR, -21.36 +/- 0.9%), and mean LH concentrations were lower in FR ewes (10.70 +/- 3.1 ng/ml) compared to Fed controls (20.98 +/- 3.8 ng/ml). Data analyzed to date for kisspeptin, NKB, and dynorphin in the ARC show that feed restriction reduced the number of kisspeptin mRNA-expressing cells (Fed, 165 +/- 25 vs FR, 9.25 +/- 6), the number of NKB mRNA-expressing cells (Fed, 141 +/- 28 vs FR, 24 +/- 5), and the number of dynorphin mRNA-expressing cells (Fed, 109 +/- 41 vs FR, 29.5 +/- 24). Together, these findings demonstrate that chronic feed restriction suppresses KNDy neurons and supports a role for these key reproductive neurons in the central mechanism governing GnRH/LH secretion during undernutrition in female sheep.
Pro-Opiomelanocortin Neural Activation and Sexual Interest in Male Mice
Authors: Harberson Mitchell T , Hill Jennifer W
Published: May 2021

Pro-opiomelanocortin (POMC) neurons in the hypothalamus play a role in both the control of metabolic state and sexual behavior. Along with the fast neurotransmitters glutamate and/or GABA, POMC neurons secrete cocaine- and amphetamine-regulated transcript (CART) and products of the POMC gene, including β-endorphin and α-melanocortin stimulating hormone (α-MSH). Published data from our lab demonstrate a lack of sexual interest in male mice when both the leptin receptor and insulin receptor are deleted from POMC neurons. Furthermore, this absence of interest correlates with a decrease in the POMC product α-MSH. However, it is not known whether these effects correspond to an increase in POMC neural activation. We hypothesized that activation of POMC neurons in male mice would lead to improved sexual interest. We have crossed mice with cre-dependent expression of the excitatory designer receptor, hM3Dq, with mice expressing cre under control of the POMC promoter. When these mice are administered intraperitoneal clozapine-N-oxide (CNO), POMC neurons exhibit increased activation. We completed a comprehensive mating analysis to measure the sexual desire and erectile and ejaculatory capabilities of these male mice under CNO or saline administration. Additionally, we sacrificed the mice after injection of CNO or saline to perform immunostaining for the protein c-fos as an indicator of neural activation. As expected, activation of POMC neurons with CNO increased c-fos expression, while the impact on male sexual interest was more nuanced. These experiments emphasize the need to investigate the specific neuropeptide and transmitter output by POMC neurons that influences sexual behavior and function.
Dissecting the Involvement of Arcuate Nucleus Kisspeptin Neurons in Puberty Onset and LH Secretion
Authors: Coutinho Eulalia A , Esparza Lourdes A , Steffen Paige H , Bolleddu Shreyana , Kauffman Alexander S
Published: May 2021

Puberty is a crucial period of transition to adulthood, marked by an increased activation of gonadotropin-releasing hormone (GnRH) neurons that drives increased pulsatile secretion of pituitary luteinizing hormone (LH). The mechanisms governing GnRH neuron activation at puberty remain unclear but are likely due to enhanced signaling from upstream neuron populations, including kisspeptin neurons. Kisspeptin (encoded by Kiss1) directly stimulates GnRH neurons to drive GnRH release and downstream LH secretion. Humans and animals with Kiss1 mutations fail to reach puberty, demonstrating kisspeptin’s importance in puberty onset. Nonetheless, the specific brain area(s) from where kisspeptin signaling arises to trigger puberty remain undetermined. Kisspeptin is primarily expressed in two hypothalamic areas, the arcuate nucleus (ARC) and anteroventral periventricular (AVPV) region. ARC Kiss1 neurons are known to drive pulsatile GnRH/LH secretion in both sexes whereas AVPV Kiss1 neurons are sexually dimorphic and mediate the preovulatory GnRH/LH surge in females. We previously showed that Kiss1 gene expression increases in both the ARC and AVPV across the peri-pubertal period, yet it still remains to be determined whether just one or both of these populations is essential for proper pubertal timing. Indeed, the relative involvement of either ARC or AVPV Kiss1 neurons in the pubertal process still remains unknown. Here, we hypothesized that ARC Kiss1 neurons are required for normal puberty timing in both sexes and, conversely, AVPV Kiss1 neurons are not sufficient on their own to trigger normal puberty. To test this hypothesis, we used transgenic mice expressing diphtheria toxin receptor (DTR) exclusively in Kiss1 cells (Kiss Cre/iDTR flox) and took advantage of the differential ontogeny of ARC and AVPV Kiss1 neurons to selectively ablate ARC kisspeptin neurons before puberty, while leaving AVPV neurons intact. We found that targeted deletion of just ARC Kiss1 neurons during the juvenile period (which does not alter AVPV Kiss1 cell number) significantly delays puberty onset in both sexes, as measured by vaginal opening, first estrous, and preputial separation. In addition, these mice also exhibit decreased basal and pulsatile LH secretion in adulthood, further supporting a role for ARC kisspeptin neurons in GnRH pulse generation. By contrast, females with ablated ARC Kiss1 cells still exhibit full estradiol-induced LH surges, ruling out a necessary role of ARC kisspeptin neurons in that process and further supporting AVPV kisspeptin as the primary regulator of the surge. Collectively, our findings demonstrate that ARC Kiss1 neurons are required for both properly timed activation of the reproductive axis during puberty and proper pulsatile LH secretion in adulthood, while AVPV Kiss1 neurons are not sufficient to drive normal puberty onset but are sufficient for the preovulatory LH surge.
Kisspeptin Neurons Excitability Is Not Modulate by Fasting
Authors: Da Silva Mansano Naira , Frazao Renata
Published: May 2021

Kisspeptin is the most important neuromodulator of gonadotrophin releasing hormone neurons. Hypothalamic Kiss1-expressing neurons can be found in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and in the arcuate nucleus of the hypothalamus (ARH). Food intake and energy balance are modulated by leptin, which acts primarily in the ARH. Leptin acts inhibiting orexigen neurotransmitters and stimulating anorectic neurotransmitters. Few ARH kisspeptin cells (10-15%) coexpress the leptin receptor and have their resting membrane potential (RMP) depolarized by leptin. Since the reproductive axis is sensitive to metabolic disorders, in the present study we used immunohistochemical and electrophysiological approach to understand the effects of fasting on kisspeptin neurons activity. To determine whether AVPV/PeN and ARH kisspeptin neurons activity are modulated by fasting, Kiss1/hrGFP female mice were fed regular chow ad libitum or fasted for 24 hours. Mice were euthanized after 24h. Fasting induced a significant increase of Fos protein on the ARH nucleus, as expected (rostral level of ARH, control: 19.3 ± 3.5 vs fasted: 81.3 ± 3.5 cells; caudal level, control: 15.7 ± 1.3 vs fasted: 103.3 ± 6.2 cells, P <0.0001, n=3/6 mice per group). Despite the significant increase of Fos-immunoreactive in the ARH nucleus, kisspeptin neurons did not co-express this neuronal marker. Next, we determined the RMP of kisspeptin cells obtained from control or fasted mice. Compared to the control group (AVPV/PeN, -56.5 ± 2.0 mV, n=13 cells from 4 animals; ARH, - 49.8 ± 2.4 mV, n= 9 cells from 6 animals), fasting was not sufficient to induce changes in RMP (AVPV/PeN, -56.1 ± 4.6 mV, n=6 cells from 3 animals, P=0.95; ARH, - 50.9 ± 2.6 mV, n=9 cells from 3 animals, P=0.11). The frequency (freq) and amplitude (amp) of the excitatory postsynaptic currents acting on kisspeptin neurons was further investigated. No significant difference was observed by comparing data obtained from control (AVPV/PeN: freq 0.76 ± 0.1 Hz, n=19; amp, 28.6 ± 1.8 pA, n=16; ARH: freq 0.36 ± 0.1 Hz, n=15; amp, 28.5 ± 2 pA, n=15) and fasted mice (AVPV/PeN: freq 0.7 ± 0.1 Hz, n=18; amp, 28.5 ± 1.1 pA; n=18; ARH: freq 0.5 ± 0.2 Hz, n=24; amp, 27.9 ± 1.3 pA; n=24; P > 0.5, 7/9 animals per group). Considering that 24hr fasting is not enough to inhibit estrous cyclicity, even though it is sufficient to induce a significant reduction of hypothalamic Kiss1 mRNA expression (unpublished data), our results suggest that prolonged periods of food restriction may be required to disturb excitatory inputs into kisspeptin cells.
LC-MS/MS for Ultra-Sensitive Quantification of Multiple Estrogens in the Blood and Brain
Authors: Jalabert Cecilia , Shock Maria A , Ma Chunqi , Soma Kiran K
Published: May 2021

Estrogens are steroid hormones that affect many aspects of brain function, including cognition, social behavior, and neuroprotection. It is well-known that estrogens are synthesized in the ovaries. Estrogens are also synthesized in the brain, where aromatase is expressed in specific regions. Importantly, estrogens play crucial roles in the brain, even at extremely low levels. Current assays lack the necessary sensitivity and/or specificity to measure brain-synthesized estrogens. Furthermore, current methods focus on only 17β-estradiol and generally disregard other estrogens that are synthesized in the brain. Here, we developed a method to measure several estrogens simultaneously, with high sensitivity and specificity. To improve sensitivity, we derivatized estrogens with 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to examine a panel of eight estrogens: 17β-estradiol, 17α-estradiol, estrone, estriol, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 4-methoxyestradiol. After derivatization, we have improved sensitivity 20-fold, detecting as little as 0.01 pg per sample, demonstrating that our method is extremely sensitive. For each analyte, we have identified a distinct retention time as well as 2 scheduled multiple reaction monitoring (sMRM) transitions that were used as quality control criteria for clear identification. Therefore, we are able to distinguish each estrogen (even stereoisomers) by the chromatographic separation and the sMRM, demonstrating that our method is highly specific.

This method has been applied to microdissected brain samples. Initially, we used a songbird model because songbirds have high levels of aromatase and 17β-estradiol in specific brain regions. We were able to simultaneously quantify multiple estrogens in small amounts of brain sample (1-2 mg). We examined seasonal changes of estrogens in the brain and blood. Future work will apply this method to mouse, rat, and human samples and expand the panel of estrogens examined. Our ultra-sensitive assay is essential for small animal models, where estrogen measurement is extremely challenging because of the limited amount of brain tissue. This novel technique will also have wide-ranging applications for basic research and clinical testing, including estrogen measurement in humans with low estrogen levels, such as men, pre-pubertal children, and post-menopausal women.
Macroprolactin Molecular Heterogeneity and Variable Immunoassay Reactivity
Authors: Hashim Ibrahim A , Nguyen Khanh Q , Langevin Rachel H , McPhaul Michael John
Published: May 2021

Circulating prolactin (PRL) exhibits molecular heterogeneity the clinical significance of which is not known. Macroprolactin (mPRL) is widely reported, however, its heterogeneity is unknown. In addition to determining its molecular nature, the relative impact of the various forms on routinely available immunoassays needs to be examined. This study, applied various proteomics techniques to define the nature of mPRL and examined the effect of those forms on its measurement. Methods: Samples suspected of false hyperprolactinemia were subjected to; 1) precipitation by polyethylene glycol, 2) gel permeation chromatography, 3) protein-G affinity chromatography to identify presence of PRL-antibody complex and, 4) samples negative for antibody by step (3) were applied onto a lectin concanavalin A column to examine the presence of glycosylated PRL forms. Analysis was according to manufacturer’s instructions. All study samples and their chromatography fractions were measured using highly sensitive ELISA (DuoSet, R&D Systems, MN). Samples identified above as containing different mPRL forms were analyzed using four immunoassay analyzers in routine clinical use, namely; Advia Centaur® and Atellica® (Siemens, PA), Alinity-ci (Abbott Laboratories, IL), and Cobas 6000® (Roche Diagnostics, IN). Results: A total of 13 samples were entered into the study. PRL levels ranged from 21.4 to 1,469 ng/mL (median 48.8). Samples positive for mPRL (n=8) (with <40% recovery by PEG) exhibited predominant (52.3 to 95.0%) PRL activity in the (H) range (≥150kDa), with significant but relatively lower amount (3.6 to 34.1%) in the (M) range (≥30<150kDa) and (1.4 to 34.5%) at the (L) range (<30kDa). Samples negative for mPRL exhibited little PRL activity (1.2 to 5.1%) in (H) range, predominantly (60.0 to 79.4%) in the (M) range and moderate presence (15.4 to 38.9 %) in the (L) range. Two samples indeterminate for mPRL contained prolactin forms at all molecular weight levels, (H) (7.9, 27.1%), (M) (67.0, 40.7%), and (L) (5.9, 51.4%). Samples with mPRL exhibited significant binding to protein G affinity column indicating presence of PRL-antibody complex. Samples with (M) range mPRL forms exhibited significant lectin affinity binding. Samples with (H) mPRL showed markedly high PRL by two of the analyzers (Atellica®, Abbott Laboratories) and Cobas 6000®, (Roche Diagnostics). The deviation was more marked when using the Atellica® compared to Cobas 6000®. Samples with (M) mPRL showed marked deviation using the Roche Cobas 6000® compared to the others. In conclusion: Macroprolactin is heterogenous with antibody-PRL complex and aggregated glycosylated forms. Those forms exhibit variable immunoassay reactivity in different routinely used assays. Knowledge of circulating form as well as of their immunoreactivity is important when suspecting false hyperprolactinemia due to macroprolactin.
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

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<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<0.0175) and corpora lutea (p<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

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<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<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<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<0.05) and diminished Kiss1 mRNA abundance in micropunches of the AVPV (42%, p<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

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

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

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

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<0.05) compared to mice given 3xSaline (pulses/90 min: 2.9±0.4 vs 2.8±0.4; pre vs post, p>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>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<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

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

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.