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Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.

Background: Granulocyte-macrophage colony stimulating factor (GM-CSF) inhalation may alleviate pulmonary inflammation caused by viral pneumonia. To investigate this, we evaluated its efficacy on COVID-19 pneumonia., Methods: This double-blind, randomised, placebo-controlled study (ClinicalTrials.gov: NCT04642950) evaluated patients in the first half of 2021 at seven Japanese hospitals. Hospitalised patients with COVID-19 pneumonia with moderate hypoxaemia inhaled sargramostim or placebo for 5 days. The primary endpoint was days to achieve a ≥ 2-category improvement from baseline on a modified 7-category ordinal scale. Secondary endpoints included degree of oxygenation, defined by amount of oxygen supply, and serum CCL17 level., Results: Seventy-five patients were randomly assigned in a 2:1 ratio to receive sargramostim or placebo, of which 47 and 23 were analysed, respectively. No difference was observed between groups regarding the primary endpoint (8.0 and 7.0 days for sargramostim and placebo, respectively) or in the secondary endpoints, except for CCL17. A post hoc sub-analysis indicated that endpoint assessments were influenced by concomitant corticosteroid therapy. When the cumulative corticosteroid dose was ≤500 mg during Days 1-5, recovery and oxygenation were faster in the sargramostim group than for placebo. Bolus dose corticosteroids were associated with temporarily impaired oxygenation and delayed clinical recovery. The increase in serum CCL17, a candidate prognostic factor, reflected improvement with sargramostim inhalation. The number of adverse events was similar between groups. Two serious adverse events were observed in the sargramostim group without causal relation., Conclusions: Inhaled sargramostim was likely to be effective for COVID-19 pneumonia unless the concomitant corticosteroid dose was high.

Background: In the ovarian follicle, the Theca Cells (TCs) have two main functions: preserving morphological integrity and, importantly, secreting steroid androgen hormones. TCs express the essential enzyme 17α-hydroxylase/17,20-desmolase (CYP17), which permits the conversion of pregnenolone and progesterone into androgens. Dysregulation of CYP17 enzyme activity due to an intrinsic ovarian defect is hypothesized to be a cause of hyperandrogenism in women. Androgen excess is observed in women with polycystic ovary syndrome (PCOS) resulting from excess endogenous androgen production, and in transgender males undergoing exogenous testosterone therapy after female sex assignment at birth. However, the molecular and morphological effects of Cyp17 overexpression and androgen excess on folliculogenesis is unknown., Methods: In this work, seeking a comprehensive profiling of the local outcomes of the androgen excess in the ovary, we generated a transgenic mouse model (TC17) with doxycycline (Dox)-induced Cyp17 overexpression in a local and temporal manner. TC17 mice were obtained by a combination of the Tet-dependent expression system and the Cre/LoxP gene control system., Results: Ovaries of Dox-treated TC17 mice overexpressed Cyp17 specifically in TCs, inducing high testosterone levels. Surprisingly, TC17 ovarian morphology resembled the human ovarian features of testosterone-treated transgender men (partially impaired folliculogenesis, hypertrophic or luteinized stromal cells, atretic follicles, and collapsed clusters). We additionally assessed TC17 fertility denoting a perturbation of the normal reproductive functions (e.g., low pregnancy rate and numbers of pups per litter). Finally, RNAseq analysis permitted us to identify dysregulated genes (Lhcgr, Fshr, Runx1) and pathways (Extra Cellular Matrix and Steroid Synthesis)., Conclusions: Our novel mouse model is a versatile tool to provide innovative insights into study the effects of Cyp17 overexpression and hyperandrogenism in the ovary., (© 2021. The Author(s).)

Type I diabetes (T1D) is one of the most common childhood diseases and Type 2 diabetes (T2D) is increasing at alarming rates. Given that children spend a great percentage of their time in school, this setting is a critical environment for models of care that lead to better management of this and other health conditions. The School Nurses Managing Diabetes Care ECHO was offered to Colorado school nurses to build their capacity in providing evidence-based management of T1D. The purpose of this effort was to (1) determine whether or not the model could be used as a tool of collaboration and dissemination for school nurses across Colorado and (2) assess the effectiveness of the "School Nurses Managing Diabetes Care" ECHO learning series. Post-series survey results demonstrated a 25% increase in self-efficacy ratings, moving learners from "average among my peers" toward "competent." Additionally, all respondents planned to make one or more practice changes to improve care for students with T1D. Expanding the use of the ECHO model to implement intensive management of children and youth with T1D is critically important as rates of this and other chronic conditions continue to increase.

Background: Adult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary characterized by elevated estrogen levels. aGCTs ubiquitously harbor a somatic mutation in FOXL2 gene, Cys134Trp (c.402C < G); however, the general molecular effect of this mutation and its putative pathogenic role in aGCT tumorigenesis is not completely understood. We previously studied the role of FOXL2 C134W , its partner SMAD3 and its antagonist FOXO1 in cellular models of aGCT., Methods: In this work, seeking more comprehensive profiling of FOXL2 C134W transcriptomic effects, we performed an RNA-seq analysis comparing the effect of FOXL2 WT /SMAD3 and FOXL2 C134W /SMAD3 overexpression in an established human GC line (HGrC1), which is not luteinized, and bears normal alleles of FOXL2., Results: Our data shows that FOXL2 C134W /SMAD3 overexpression alters the expression of 717 genes. These genes include known and novel FOXL2 targets (TGFB2, SMARCA4, HSPG2, MKI67, NFKBIA) and are enriched for neoplastic pathways (Proteoglycans in Cancer, Chromatin remodeling, Apoptosis, Tissue Morphogenesis, Tyrosine Kinase Receptors). We additionally expressed the FOXL2 antagonistic Forkhead protein, FOXO1. Surprisingly, overexpression of FOXO1 mitigated 40% of the altered genome-wide effects specifically related to FOXL2 C134W , suggesting it can be a new target for aGCT treatment., Conclusions: Our transcriptomic data provide novel insights into potential genes (FOXO1 regulated) that could be used as biomarkers of efficacy in aGCT patients.

Introduction: Our previous clinical studies have demonstrated the short-term efficacy and safety of the sirolimus gel for patients with tuberous sclerosis complex (TSC). However, long-term clinical evidence is lacking. Our objective was to assess the safety and efficacy of long-term treatment with the sirolimus gel for the skin lesions of TSC patients., Methods: We conducted a multicenter, open-label, uncontrolled clinical trial in 94 Japanese patients with TSC. Patients applied the 0.2% sirolimus gel on their face or head twice daily for > 52 weeks (maximum 136 weeks for safety). The safety endpoints were the rate of adverse event (AE)-caused discontinuation (primary endpoint) and the incidence of AEs. The efficacy endpoint was the response rate of angiofibromas, cephalic plaques, and hypomelanotic macules., Results: Among 94 enrolled patients (mean age, 21 years; range 3-53 years), the rate of AE-caused discontinuation was 2.1% (2/94 patients). Although application site irritation and dry skin occurred relatively frequently, none of the drug-related AEs were serious; most of the drug-related AEs resolved rapidly. The major drug-related AEs (≥ 5% in incidence) were application site irritation (30.9%), dry skin (27.7%), acne (20.2%), eye irritation (8.5%), pruritus (8.5%), erythema (7.4%), dermatitis acneiform (6.4%), and dermatitis contact (5.3%). The response rates of angiofibromas, cephalic plaques, and hypomelanotic macules were 78.2% [95% confidence interval (CI) 68.0-86.3%], 66.7% (95% CI 51.1-80.0%), and 72.2% (95% CI 46.5-90.3%), respectively., Conclusions: The gel was well tolerated for a long time by patients with TSC involving facial skin lesions and continued to be effective., Trial Registration: ClinicalTrials.gov identifier: NCT02634931.

Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structure N ω -(carboxymethyl)arginine (CMA) is produced by oxidation in glycated collagen, accounting for one of the major proteins detected in biological samples. In this study, we investigated the mechanism by which CMA is generated in collagen and detected CMA in collagen-rich tissues. When various protein samples were incubated with glucose, the CMA content, detected using a monoclonal antibody, increased in a time-dependent manner only in glycated collagen, whereas the formation of N ε -(carboxymethyl)lysine (CML), a major antigenic AGE, was detected in all glycated proteins. Dominant CMA formation in glycated collagen was also observed by electrospray ionization-liquid chromatography-tandem mass spectrometry (LC-MS/MS). During incubation of glucose with collagen, CMA formation was enhanced with increasing glucose concentration, whereas it was inhibited in the presence of dicarbonyl-trapping reagents and a metal chelator. CMA formation was also observed upon incubating collagen with glyoxal, and CMA was generated in a time-dependent manner when glyoxal was incubated with type I-IV collagens. To identify hotspots of CMA formation, tryptic digests of glycated collagen were applied to an affinity column conjugated with anti-CMA. Several CMA peptides that are important for recognition by integrins were detected by LC-MS/MS and amino acid sequence analyses. CMA formation on each sequence was confirmed by incubation of the synthesized peptides with glyoxal and ribose. LC-MS detected CMA in the mouse skin at a higher level than other AGEs. Furthermore, CMA accumulation was greater in the human aorta of older individuals. Overall, our study provides evidence that CMA is a representative AGE structure that serves as a useful index to reflect the oxidation and glycation of collagen., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2019 Sho Kinoshita et al.)

Nurses and caseworkers engage in assessments with the families they serve. Nurse home visitors from Nurse-Family Partnership (NFP) improve maternal-child health outcomes with first-time low-income mothers through care, education and support. In the United States, Child Protective Services (CPS) are state-level governmental agencies that protect children, including responding to reports of child maltreatment. This paper aimed to characterise similarities and differences in risk assessment practices between NFP nurses and CPS caseworkers in Colorado, United States. Using a grounded theory approach, we conducted in-depth qualitative interviews with 112 NFP and CPS workers from seven Colorado NFP sites from 2013 to 2015. Study sites were purposefully selected based on size, structure, geography and degree of collaboration with CPS. We conducted interviews first with NFP sites and used snowball sampling to recruit CPS workers. Interviews were recorded, transcribed, validated and then coded in NVivo 10. Memo writing was conducted to organise and link concepts within the theme of risk assessment. NFP and CPS workers emphasised the importance of risk assessment in their respective practices. Although there were similarities in the types of risks assessed, we found variations in work processes, operational definitions and methods of risk assessment between the two organisations that impacted inter-organisational collaboration to serve high-risk mothers and their children. NFP and CPS workers may have different roles and responsibilities but their underlying goals are the same - to keep children and their families safe and healthy. By understanding these similarities and differences in practice, there lies potential to improve collaboration between home visiting programmes and child welfare to provide integrated service delivery of high-risk families and prevention of future child maltreatment., (© 2019 John Wiley & Sons Ltd.)

Background: In February 2018, President Trump signed into law the Family First Prevention Act, legislation in the United States aimed at providing prevention services for families at risk of entering the child welfare system. The effectiveness of these prevention efforts is dependent on the formation of collaborative relationships between prevention-programs and child welfare., Objective: To identify factors that influence the ability of the Nurse-Family Partnership (NFP) and Child Protective Services (CPS) to collaborate in serving high-risk mothers and their children., Participants: 123 NFP, CPS workers, and community partners., Setting: Seven sites in the U.S. state of Colorado selected to include an array of community sizes, geographies, apparent levels of collaboration, and variations in internal structures and practices., Methods: Using an adapted grounded theory approach, we conducted semi-structured interviews with frontline NFP and CPS workers and supervisors. Interviews were recorded, transcribed, validated, and coded in NVivo 10., Results: Alignment of core organizational mission and methods was key in determining collaboration levels between NFP and CPS. Only when workers perceived there to be alignment in organizational mission, did other factors such as program eligibility, communication channels, and risk and safety assessment practices influence the perceived benefits and efforts undertaken to enhance collaboration., Conclusions: High-risk families frequently require services that go beyond the scope of any one organization. As programs that serve high-risk families refine their efforts to serve them effectively, collaborative efforts should focus on examining opportunities and challenges involved in creating greater mission alignment., (Copyright © 2019. Published by Elsevier Ltd.)

Adult granulosa cell tumor (aGCT) is a rare type of ovarian cancer characterized by estrogen excess. Interestingly, only the single somatic mutation FOXL2 C134W was found across virtually all aGCTs. We previously reported that FOXL2 C134W stimulates CYP19 transcription synergistically with SMAD3, leading to elevated estradiol synthesis in a human granulosa cell line (HGrC1). This finding suggested a key role for FOXL2 C134W in causing the typical estrogen overload in patients with aGCTs. We have now investigated the effect of FOXO1, a tumor suppressor, on CYP19 activation by FOXL2 C134W in the presence of SMAD3. Intriguingly, FOXO1 antagonized the positive, synergistic effect of FOXL2 C134W and SMAD3 on CYP19 transcription. Similar to FOXL2 C134W , FOXO1 binds SMAD3 but not the proximal FOXL2 C134W binding site (-199 bp) of the CYP19 promoter identified in our earlier studies. The results of a competitive binding assay suggested a possible underlying mechanism in which FOXO1 sequesters SMAD3 away from FOXL2 C134W , thereby negating the cooperative action of FOXL2 C134W and SMAD3 in inducing CYP19 expression. To our knowledge, this study is the first to demonstrate the ability of FOXO1 to restore an altered CYP19 expression by FOXL2 C134W and SMAD3 and provides insight as to why FOXO1 deficiency promotes GCT development in mice., (Copyright © 2019 Endocrine Society.)

Introduction: Project Extension for Community Health Outcomes (ECHO) was originally developed by the University of New Mexico's Health Science Center (UNMHSC) to build the capacities of primary-care providers and to increase specialty-care access to rural and underserved populations. ECHO Colorado, a replication site at the University of Colorado Anschutz Medical Campus (CUAMC), was developed with the same purpose and to help build the health workforce of Colorado. The CUAMC and its community-based partners recognized that by reducing unnecessary referrals to the medical campus and building primary-care capacity in communities, both would increase their scope and expand overall capacity. This study examines the key factors that influence participant engagement, how participants value the ECHO experience, and the utility of the ECHO Colorado experience according to participants., Methods: This study used a mixed-methods approach including 42 interviews and 34 completed surveys. Transcribed interview recordings were coded in NVivo 11, and codes were queried in NVivo and Excel to identify key themes. Survey responses were analyzed in SPSS. Data were examined between and across four attendance groups and triangulated to assess the reliability of the data and validity of overall findings., Findings: Key factors increasing registrant engagement included relevant and practical curriculum content; strong and supportive relationships among learners, ECHO faculty, and workplace colleagues; and innovative learning approaches that included opportunities for active, virtual participation through technology, participant management activities, and ECHO's unique curriculum design., Conclusion: Findings from this study validated many of the important elements of ECHO Colorado that make it unique from other iterations of the model being implemented nationally and internationally and identified participant-driven strategies for further amplifying its impact., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Germline knockout studies in female mice demonstrated an essential role for forkhead box L2 (FOXL2) in early follicle development, whereas an inducible granulosa cell (GC)-specific deletion of Foxl2 in adults has shown ovary-to-testis somatic sex reprogramming. In women, over 120 different germline mutations in the FOXL2 gene have been shown to cause blepharophimosis/ptosis/epicantus inversus syndrome associated with or without primary ovarian insufficiency. By contrast, a single somatic mutation (FOXL2C134W) accounts for almost all adult-type GC tumors (aGCTs). To test the hypothesis that FOXL2C134W differentially regulates the expression of aGCT markers, we investigated the effect of FOXL2C134W on inhibin B and P450 aromatase expression using a recently established human GC line (HGrC1), which we now show to bear two normal alleles of FOXL2. Neither FOXL2wt nor FOXL2C134W regulate INHBB messenger RNA (mRNA) expression. However, FOXL2C134W selectively displays a 50-fold induction of CYP19 mRNA expression dependent upon activin A. Mechanistically, the CYP19 promoter is activated in a similar way by FOXL2C134W interaction with SMAD3, but not by FOXL2wt. SMAD2 had no effect. Moreover, FOXL2C134W interactions with SMAD3 and with the FOX binding element located at -199 bp upstream of the ATG initiation codon of CYP19 are more sustainable than FOXL2wt. Thus, FOXL2C134W potentiates CYP19 expression in HGrC1 cells via enhanced recruitment of SMAD3 to a proximal FOX binding element. These findings may explain the pathophysiology of estrogen excess in patients with aGCT.

Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted factors with a leading role in the control of ovarian function in female reproduction, modulating both the cell fate of the somatic granulosa cells and the quality and developmental competence of the egg. This short review aims to consolidate the molecular aspects of GDF9 and BMP15 and their integral actions in female fertility to understand particularly their effects on oocyte quality and fetal growth. The significant consequences of mutations in the GDF9 and BMP15 genes in women with dizygotic twins as well as the clinical relevance of these oocyte factors in the pathogenesis of primary ovarian insufficiency and polycystic ovary syndrome are also addressed., (© 2018 Elsevier Inc. All rights reserved.)

Recent studies report the involvement of intra-ovarian factors, such as inflammation and oxidative stress, in the pathophysiology of polycystic ovary syndrome (PCOS), the most common endocrine disorder of reproductive age women. Endoplasmic reticulum (ER) stress is a local factor that affects various cellular events during a broad spectrum of physiological and pathological conditions. It may also be an important determinant of pro-fibrotic remodeling during tissue fibrosis. In the present study, we showed that ER stress was activated in granulosa cells of PCOS patients as well as in a well-established PCOS mouse model. Pharmacological inducers of ER stress, tunicamycin and thapsigargin, were found to increase the expression of pro-fibrotic growth factors, including transforming growth factor (TGF)-β1, in human granulosa cells, and their expression also increased in granulosa cells of PCOS patients. By contrast, treatment of PCOS mice with an ER stress inhibitor, tauroursodeoxycholic acid or BGP-15, decreased interstitial fibrosis and collagen deposition in ovaries, accompanied by a reduction in TGF-β1 expression in granulosa cells. These findings suggest that ER stress in granulosa cells of women with PCOS contributes to the induction of pro-fibrotic growth factors during ovarian fibrosis, and that ER stress may serve as a therapeutic target in PCOS.

Problem: Plasminogen activator inhibitor-1 (PAI-1) is elevated in women with polycystic ovary syndrome (PCOS), but the regulation in granulosa cells (GCs) is unclear., Method of Study: PAI-1 expression in PCOS ovaries was investigated immunohistologically. PAI-1 expressions in HGrC1, a human GC cell line, were investigated at mRNA and activity levels. The expressions of TGF-β and TNF-α in peritoneal fluid mononuclear cells (PFMCs) were measured with quantitative PCR., Results: Little PAI-1 expression is observed in healthy GCs, whereas GCs of PCOS and atretic follicle exhibit distinct expression in vivo. In vitro study using HGrC1 shows that TGF-β and TNF-α increase PAI-1 mRNA and its activity, and both together exhibit a synergistic effect. The expression of PAI-1 mRNA is suppressed by simvastatin. Moreover, insulin-sensitizing drugs (metformin, pioglitazone, and rosiglitazone) suppress LPS-induced TGF-β and TNF-α mRNA expression in PFMC., Conclusion: Statin and insulin-sensitizing drugs may provide a potential therapy for PCOS via down-regulation of PAI-1 expression in GCs and down-regulation of TGF-β and TNF-α expression in PFMC, respectively., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Factors that differentially regulate oocyte and granulosa cell growth within the early preantral follicle and how these factors differ at each stage of follicle growth remain poorly understood. The aim of this study was to isolate and evaluate the effect of recombinant growth and differentiation factor 9 (GDF9) on oocyte and granulosa cell growth at the primary and early secondary stages of preantral follicle growth during in vitro culture., Methods: Primary stage follicles (diameters of 50-89 μm) and early secondary stage follicles (diameters of 90-120 μm) were isolated from immature mice, and individual, intact follicles were cultured in vitro in the presence and absence of recombinant GDF9. The effects of GDF9 on follicle growth were determined by the assessment of changes in the follicle volume during culture. The growth of the granulosa cell and oocyte compartments of the follicles was evaluated separately at each stage., Results: GDF9 significantly increased the growth of isolated follicles at both the primary and early secondary follicle stages. Independent evaluation of the granulosa cell and oocyte compartments revealed that, while GDF9 promoted granulosa cell growth at both stages of folliculogenesis, oocyte growth was stage specific. GDF9 promoted growth of the oocyte at the primary, but not the early secondary, follicle stage., Conclusions: These findings demonstrate a stage-specific role for GDF9 in the regulation of oocyte and granulosa cell growth at the primary and early secondary stages of preantral follicle development.

Objective: To investigate practices, knowledge, attitudes, and beliefs regarding infant sleep among adolescent mothers, a demographic at high risk for sudden unexpected infant death, and to identify novel public health interventions targeting the particular reasons of this population., Study Design: Seven targeted focus groups including 43 adolescent mothers were conducted at high school daycare centers throughout Colorado. Focus groups were recorded, transcribed, validated, and then analyzed in NVivo 10. Validation included coding consistency statistics and expert review., Results: Most mothers knew many of the American Academy of Pediatrics recommendations for infant sleep. However, almost all teens reported bedsharing regularly and used loose blankets or soft bedding despite being informed of risks. Reasons for nonadherence to recommendations included beliefs that babies are safest and sleep more/better in bed with them, that bedsharing is a bonding opportunity, and that bedsharing is easier than using a separate sleep space. The most common justifications for blankets were infant comfort and concern that babies were cold. Participants' decision making was often influenced by their own mothers, with whom they often resided. Participants felt that their instincts trumped professional advice, even when in direct contradiction to safe sleep recommendations., Conclusions: Among focus group participants, adherence with safe sleep practices was poor despite awareness of the American Academy of Pediatrics recommendations. Many mothers expressed beliefs and instincts that infants were safe in various unsafe sleep environments. Future study should investigate the efficacy of alternative educational strategies, including education of grandmothers, who have significant influence over adolescent mothers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3β signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice. Pubertal female C57BL/6N mice were treated for 5 wk with LET, which resulted in increased serum testosterone and normal diestrus levels of estradiol, similar to the hyperandrogenemia and follicular phase estrogen levels of PCOS women. As in PCOS, ovaries from LET mice were larger, polycystic, and lacked corpora lutea versus controls. Most LET females were acyclic, and all were infertile. LET females displayed elevated serum LH levels and higher Lhb mRNA in the pituitary. In contrast, serum FSH and Fshb were significantly reduced in LET females, demonstrating differential effects on gonadotropins, as in PCOS. Within the ovary, LET females had higher Cyp17, Cyp19, and Fsh receptor mRNA expression. In the hypothalamus, LET females had higher kisspeptin receptor mRNA expression but lower progesterone receptor mRNA levels. LET females also gained more weight than controls, had increased abdominal adiposity and adipocyte size, elevated adipose inflammatory mRNA levels, and impaired glucose tolerance, mirroring the metabolic phenotype in PCOS women. This is the first report of a LET paradigm in mice that recapitulates both reproductive and metabolic PCOS phenotypes and will be useful to genetically probe the PCOS condition., (© 2015 by the Society for the Study of Reproduction, Inc.)

Objective: To determine whether granulosa cells contribute to excess androgen production, by assessing inhibin B (Inh B) responses to hCG in women with polycystic ovary syndrome (PCOS) and in normal women., Design: Prospective study., Setting: Academic medical center., Patient(s): Twenty women with PCOS and 16 normal women., Intervention(s): Blood samples obtained before and 24 hours after injection of 25 μg recombinant hCG (r-hCG)., Main Outcome Measure(s): Basal and stimulated Inh B, E2, androstenedione (A), and T responses after r-hCG administration., Result(s): In normal and PCOS women, r-hCG induced a significant reduction of Inh B levels. Lowered Inh B responses were not related to body mass index, PCOS status, or age by multivariate regression. Recombinant hCG significantly increased serum A and E2 in both normal and PCOS women., Conclusion(s): In normal and PCOS women, Inh B production was decreased following r-hCG administration. These findings strongly suggest that in PCOS women androgen excess is not enhanced by LH-stimulated Inh B production., Clinical Trial Registration Number: NCT00747617., (Published by Elsevier Inc.)

Objective: To test whether and to what extent inhibin mediates Cyp17 messenger RNA (mRNA) expression in theca cells (TCs) in response to FSH stimulation of granulosa cells (GCs)., Design: Ex vivo and in vitro experimental study., Setting: University., Animal(s): Immature female Sprague Dawley rats., Intervention(s): Ovarian tissue explants and isolated theca cell preparations with or without GCs were treated with FSH, inhibin, inhibin antibody, or β-glycan antibody., Main Outcome Measure(s): As a key enzyme in androgen production, Cyp17 mRNA levels were measured by real-time reverse transcription-polymerase chain reaction., Result(s): After 24 hours, Cyp17 mRNA expression was dose-dependently increased by FSH in ovarian tissue explants and theca cells, suggesting that paracrine factor(s) secreted from GCs in response to FSH mediates Cyp17 mRNA expression in TCs. Antibodies against inhibin and inhibin coreceptor, β-glycan, blocked the stimulatory effect of FSH on Cyp17 mRNA expression. However, inhibin alone did not increase Cyp17 mRNA level to the same extent., Conclusion(s): These findings suggest a role for inhibin in the paracrine regulation of TC Cyp17 mRNA expression by GCs influenced by FSH; however, other paracrine factors produced by GCs by virtue of FSH seem to be required., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The FOXL2(C134W) mutation has been identified in virtually all adult granulosa cell tumors (GCTs). Here we show that the exogenous FOXL2 expression is necessary for GDF-9 stimulation of follistatin transcription in the human GCT cell line, COV434 that lacks endogenous FOXL2 expression. Interestingly, in the presence of Smad3 co-expression, FOXL2(C134W) negated GDF-9 stimulation of follistatin transcription. However, mutation of the Smad binding element (SBE) located in the intronic enhancer elements in the follistatin gene restored normal FOXL2 activity to FOXL2(C134W), thus the altered activity of FOXL2(C134W) is dependent on the ability of Smad3 to directly bind the SBE. Mutation of the FOXL2 binding element (FBE) or the FBE and SBE completely prevented GDF-9 activity, suggesting that the FBE is essential for GDF-9 stimulation in COV434. Overall, our study supports the view that altered interaction of FOXL2(C134W) with co-factors may underlie the pathogenesis of this mutation in GCTs., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

A single somatic FOXL2 mutation (FOXL2(C134W)) was identified in almost all granulosa cell tumor (GCT) patients. In the pituitary, FOXL2 and Smad3 coordinately regulate activin stimulation of follistatin transcription. We explored whether a similar regulation occurs in the ovary, and whether FOXL2(C134W) has altered activity. We show that in primary granulosa cells, GDF-9 and activin increase Smad3-mediated follistatin transcription. In contrast to findings in the pituitary, FOXL2 negatively regulates GDF-9 and activin-stimulated follistatin transcription in the ovary. Knockdown of endogenous FOXL2 confirmed this inhibitory role. FOXL2(C134W) displayed enhanced inhibitory activity, completely ablating GDF-9 and activin-induced follistatin transcription. GDF-9 and activin activity was lost when either the smad binding element or the forkhead binding element were mutated, indicating that both sites are required for Smad3 actions. This study highlights that FOXL2 negatively regulates follistatin expression within the ovary, and that the pathogenesis of FOXL2(C134W) may involve an altered interaction with Smad3., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

To compare and evaluate the bioavailability for intravenous fosphenytoin sodium with that of intravenous phenytoin sodium in Japanese subjects. In study 1, healthy Japanese male volunteers received a 30-min infusion of 375 mg fosphenytoin sodium or an equimolar dose of 250 mg phenytoin by a double-blind, crossover method. In study 2, other healthy Japanese male volunteers received a 30-min or 10-min infusion of 563 mg fosphenytoin sodium, followed by a dose of 750 mg after 2 weeks in an unblinded manner. Comparing with 250 mg phenytoin sodium, 375 mg fosphenytoin sodium exhibited lower total plasma phenytoin C max, whereas the geometric mean ratio of the AUC of total and free phenyotoin for fosphenytoin sodium at a dose of 375 mg was very similar to phenytoin sodium at a equimolar dose of 250 mg (AUC0-t ratio: 0.98 and 1.02, respectively). Therefore, fosphenytoin is almost completely converted to phenytoin in subjects. Fosphenytoin sodium was rapidly converted to phenytoin at doses of 375, 563, and 750 mg. The maximum concentration (C max) of total plasma phenytoin increased in a dose-dependent manner. The area under the plasma concentration-time curve (AUC) increased slightly more than proportionally with the administered dose, and clearance (CL) decreased with increasing dose. Pain and other infusion-site reactions were reported by all 12 subjects with phenytoin sodium, whereas very few symptoms were observed with fosphenytoin sodium. In conclusion, fosphenytoin sodium is considered to be a useful substitute for phenytoin sodium with almost no associated injection-site reactions.

Study Question: What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster?, Summary Answer: The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis., What Is Known Already: The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report., Study Design, Size, Duration: We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes., Participants/materials, Setting, Methods: We used quantitative reverse transcription-polymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression., Main Results and the Role of Chance: We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes., Limitations, Reasons for Caution: While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these non-testicular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary., Wider Implications of the Findings: The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and oogenesis. Our results also raise the possibility that modulation of RHOX gene levels could correct some cases of human infertility and that their encoded proteins are candidate targets for contraceptive drug design.