bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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&lt;0.0248), parity (p&lt;0.0021), time (p&lt;0.0001), and interaction (p&lt;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

Abstract
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 &lt; 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 = &lt; 0.05) and 10 nM (ANOVA, P &lt; 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 &gt; 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

Abstract
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

Abstract
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

Abstract
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

Abstract
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 &lt;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 &gt; 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

Abstract
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

Abstract
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 &lt;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&lt;150kDa) and (1.4 to 34.5%) at the (L) range (&lt;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.