Edition 76 - November 2019 / Bibliographic Reviews

Bibliographic Review – Ed. 76

Marco A. Rivarola and Alicia Belgorosky.
Hospital de Pediatria Garrahan, Buenos Aires, Argentina.

For this issue Bibliographic Review Section, we have selected the following publications:

Gene Expression and Behavioral Alterations in Mice with Conditional Knockout of GR, MR, or Both GR and MR in the Hippocampus.
Article Extracted from The Journal of the Endocrine Society (Supplement_1), April 2019, DOI: 10.1210/js.2019-SAT-005
Robert Oakley1, Shannon Whirledge1, Natallia Riddick2, John Lydon3, Francesco DeMayo F3, John Cidlowski4.
1Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, 2Comparative Medicine Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, 3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, 4Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC.

Glucocorticoids are primary stress hormones that regulate brain function. Aberrant levels of these steroids have been implicated in the pathogenesis of cognitive impairments and psychiatric disorders. Glucocorticoids exert their effects on cells via binding the glucocorticoid receptor (GR) and the closely related mineralocorticoid receptor (MR). A distinctive feature of the hippocampus is that it expresses high levels of both GR and MR. However; the specific roles played by these receptors in mediating the direct actions of glucocorticoids in the hippocampus are poorly understood. To elucidate the in vivo function of hippocampal GR and MR, we employed emx1-cre mice to ablate GR (hippocampal GRKO), MR (hippocampal MRKO), or both GR and MR (hippocampal GRMRdKO) in the hippocampus. The single and double knockout mice were born at the expected Mendelian ratio and survived normally through 12 months of age. Small reductions in body weight were observed for the hippocampal MRKO and hippocampal GRMRdKO mice, but not the hippocampal GRKO mice. RNA sequencing performed on hippocampal RNA revealed major differences in both the basal and glucocorticoid regulated transcriptome in the single and double knockout mice. In behavioral assays, all three mutant mice exhibited reduced anxiety in the marble-burying test. However, only the hippocampal MRKO and hippocampal GRMRdKO mice showed decreased anxiety in the elevated plus-maze and open field tests. A forced swim test revealed no genotype differences in depression-like behavior. In a conditioned fear test, the hippocampal GRKO mice showed a reduction in context-dependent learning whereas the hippocampal MRKO and hippocampal GRMRdKO mice showed an increase in cue-dependent learning. These findings demonstrate that GR and MR regulate both common and unique signaling pathways in the hippocampus that influence anxiety and learning and memory.

Adjuvant Radiation Improves Recurrence-Free Survival and Overall Survival in Adrenocortical Carcinoma.
Article Extracted from The J Clin Endocrinol Metabol, Volume 104, Issue 9, 3743-3750. DOI: 10.1210/jc.2019-00029.
Gharzai LA1Green MD1Griffith KA2Else T3Mayo CS1Hesseltine E3Spratt DE1Ben-Josef E4Sabolch A5Miller BS6Worden F7Giordano TJ8Hammer GD3Jolly S1. 1Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.2Department of Biostatistics, University of Michigan, Ann Arbor, Michigan. 3Department of Endocrinology, University of Michigan, Ann Arbor, Michigan. 4Department of Radiation Oncology, University of Pennsylvania, Philadelphia, Pennsylvania.5Department of Radiation Oncology, Kaiser Permanente, Portland, Oregon. 6Department of Surgery, University of Michigan, Ann Arbor, Michigan.7Department of Internal Medicine, Division of Medical Oncology, University of Michigan, Ann Arbor, Michigan, 8Department of Pathology, University of Michigan, Ann Arbor, Michigan.

CONTEXT: Adrenocortical carcinoma (ACC) is a rare malignancy with high rates of recurrence and poor prognosis. The role of radiotherapy (RT) in localized ACC has been controversial, and RT is not routinely offered. OBJECTIVE: To evaluate the benefit of adjuvant RT on outcomes in ACC. DESIGN: This is a retrospective propensity-matched analysis. SETTING: All patients were seen through the University of Michigan’s Endocrine Oncology program, and all those who underwent RT were treated at the University of Michigan. PARTICIPANTS: Of 424 patients with ACC, 78 were selected; 39 patients underwent adjuvant radiation. INTERVENTION: Adjuvant RT to the tumor bed and adjacent lymph nodes. MAIN OUTCOME MEASURES: Time to local failure, distant failure, or death. RESULTS: Median follow-up time was 4.21 years (95% CI, 2.79 to 4.94). The median radiation dose was 55 Gy (range, 45 to 60). The 3-year overall survival estimate for patients improved from 48.6% for patients without RT (95% CI, 29.7 to 65.2) to 77.7% (95% CI, 56.3 to 89.5) with RT, with a hazard ratio (HR) of 3.59 (95% CI, 1.60 to 8.09; P = 0.002). RT improved local recurrence-free survival (RFS) from 34.2% (95% CI, 18.8 to 50.3) to 59.5% (95% CI, 39.0 to 75.0), with an HR of 2.67 (95% CI, 1.38 to 5.19; P = 0.0035). RT improved all RFS from 18.3% (95% CI, 6.7 to 34.3) to 46.7% (95% CI, 26.9 to 64.3), with an HR 2.59 (95% CI, 1.40 to 4.79; P = 0.0024). CONCLUSIONS: In the largest single-institution study to date, adjuvant RT after gross resection of ACC improved local RFS, all RFS, and overall survival in this propensity-matched analysis. Adjuvant RT should be considered a part of multidisciplinary management for patients with ACC.

Excerpts selected from this article:
The standard of care for localized adrenocortical carcinoma (ACC) is surgical resection. Prognoses are poor after gross total resection, and there is a high failure rate with this treatment. Loco-regional failures are a common component of this treatment, with estimated rates of local recurrence as high as 65% with associated poor long-term survivorship. Distant failures are also quite high. Therefore, there is a need for adjuvant therapy intensification to improve patient outcomes. Adjuvant therapy, including mitotane, systemic chemotherapy, and radiotherapy (RT), have been explored, each being used in various settings. The utility of adjuvant RT after gross resection in ACC is controversial, therefore it is infrequently used as a treatment option in the United States. The Endocrine Oncology Program at the University of Michigan is a major tertiary referral center for ACC. In this analysis, Gharzai et al. updated the ACC database with patients who received adjuvant radiation with modern radiation techniques compared with those who were not treated with adjuvant radiation after gross total resection.
Patients who received adjuvant RT were then matched to control subjects who received surgery with curative intent but without adjuvant RT.
Control subjects were selected from an internal database of 424 patients with ACC seen by the Endocrine Oncology Program at the University of Michigan and were selected by propensity matching methods There was an average of 64 days from the date of surgery to initiation of RT (range, 25 to 147 days).
The evolution of the literature on adjuvant radiation in ACC has shown a clear arc in the efficacy of treatment with RT as radiation techniques have improved. Improvements in local control with RT have translated to improvements in OS in other cancer types [breast cancer, rectal cancer, head and neck cancers, although studies typically required large patient populations to show this advantage. The present findings demonstrate that adjuvant RT is important for local tumor control for patients with ACC. Current recommendations for adjuvant RT in ACC suggest consideration of RT in high-risk patients, including those with large size, incomplete resection, or stage III disease. These data suggest that adjuvant RT provides substantial improvements in loco-regional control regardless of margin status. Therefore, resection followed by adjuvant RT should be considered for all patients with ACC.

Adrenal Steroids in Female Hypothyroid Neonates: Unraveling an Association Between Thyroid Hormones and Adrenal Remodeling.
Article Extracted from: J Clin Endocrinol Metab. 2019 Feb 20. DOI: 10.1210/jc.2018-02013.
Sofia Galanou1 Giorgos Chouliaras1 Panagiotis Girginoudis2 Chryssanthi Mengreli,2 Amalia Sertedaki1 Maria Dracopoulou1 Ioanna Farakla1 Dimitris Platis2 Alexandra Iliadi1,2 George P. Chrousos1 Catherine Dacou-Voutetakis1 Emanuil Zoumakis1 Alexandra-Maria Magiakou1 Christina Kanaka-Gantenbein1 and Antonis Voutetakis1
1Division of Endocrinology, Metabolism, and Diabetes, 1st Department of Pediatrics, Medical School, National and Kapodistrian University of Athens, Athens  Greece; and 2Department of Biochemistry, Institute of Child Health, “Aghia Sophia” Children’s Hospital, Athens, Greece. ORCiD numbers: 0000-0003-1663-6831 (A. Voutetakis).

CONTEXT: The adrenal gland undergoes substantial remodeling during the neonatal period, an essential developmental process that remains incompletely understood. With respect to control over the remodeling process and, specifically, the role of thyroid hormones (THs), no human studies have been published. The effects of both hypo- and hyperthyroidism have only been evaluated in adults, focusing on the mature adrenal. Recent studies have identified the expression of the TH receptor b1 in the mouse adrenal X-zone and have demonstrated that TH administration could alter the postnatal adrenal remodeling process. OBJECTIVE: To address whether THs influence adrenal steroid profiles and adrenal remodeling during the neonatal period. METHODS: Authors compared the adrenal steroid profile of a naturally occurring prototype, female neonates, with severe congenital hypothyroidism (CH) (n = 522, upon diagnosis of CH), with that of euthyroid neonates (n = 520). Male neonates were not included in the study to avoid confounding hormonal effects due to circulating testicular steroids, especially during mini-puberty. RESULTS: Significant higher levels of adrenal steroids (17-OH-progesterone, dehydroepiandrosterone sulfate, D4-androstenedione, and testosterone) were measured in neonates with severe CH compared with euthyroid neonates and returned to within normal range after the euthyroid state had been established on L-thyroxine replacement therapy, whereas cortisol levels did not differ. TSH values in the CH group were positively correlated with circulating adrenal steroids, whereas free T4 levels were negatively correlated with these steroids. CONCLUSIONS: The hormonal profile of female neonates with severe CH suggests a more active adrenal fetal zone compared with control subjects. These data indirectly associate THs with the adrenal remodeling and maturation process in humans. Based on these results, it is suggested that severe hypothyroidism decelerates the involution of the adrenal fetal zone that normally occurs postnatally.

Excerpts selected from this article:
In humans, the enlarged adrenal fetal zone, mostly producing dehydroepiandros- terone sulfate (DHEAS) during gestation, rapidly involutes postnatally due to increased cellular apoptosis. At the same time, the zona fasciculata and zona glomerulosa, are quickly established to ensure adequate secretion of glucocorticoids and mineralocorticoids. During childhood, the zona reticularis begins to form (leading to clinical adrenarche) and continues to expand throughout puberty and until early adulthood. The complex regulatory mechanisms leading stepwise from the adrenogonadal primordium and the fetal adrenal gland to the adult adrenal cortex (and especially the control over the adrenal remodeling process) are not fully understood. Moreover, genetic and molecular mechanisms governing adrenal zonation also remain poorly understood. Finally, emerging evidence points out to epigenetics as another regulatory mechanism involved (Baquedano MS and Belgorosky A, Horm Res Paediatr, 2018).
In a recent elegant mouse study, Huang et al. (Endocrinology, 2015) showed that the thyroid hormone has a direct role in adrenocortical development and function. Specifically, they identified an adrenal cortical cell population, including but not limited to the cells occupying the mouse X-zone, which expresses thyroid hormone receptor [THR]b1. Moreover, they demonstrated that T3 administration could alter the postnatal adrenal remodeling process involving the X-zone and change the differentiation status of this adrenal cell subpopulation of mice to become similar to zona fasciculata cells.
The observations of Huang et al. raise the question of whether a link between THs and the adrenal fetal zone and adrenal remodeling also exists in humans. In this publication, authors prospectively studied a naturally occurring clinical model: female neonates with severe congenital hypothyroidism (CH). Results clearly show an altered adrenal steroid profile in neonates with CH compared with euthyroid neonates. Based on their findings, they hypothesized that subnormal TH levels may decelerate the involution of the fetal adrenal zone and delay postnatal adrenal maturation.
The mammalian adrenal gland remains a developmentally dynamic organ after birth because it adapts to homeostatic and age-related challenges. These necessary adjustments, achieved through fundamental cell population changes, not only support transition but also are characteristic of specific life periods, especially the neonatal, adrenarche, and adrenopause periods.
During the neonatal period, the enlarged fetal adrenal zone involutes, leading to a substantial decrease of DHEAS levels.  During adrenarche, the zona reticularis forms and expands, leading to a gradual increase of DHEAS and adrenal androgens.
In this study, the effect of hypothyroidism during the human neonatal period, a critical adrenal remodeling window, is reported. Specifically, adrenal steroid levels were prospectively evaluated in 22 full-term female neonates with severe CH (very high TSH levels) and in a control group of 20 full-term euthyroid female neonates without hypoyhyroidism (normal TSH levels). Significant higher levels of adrenal steroids (17-OH progesterone, DHEAS, D4-androstenedione, and testosterone) were observed in neonates with severe CH compared with euthyroid neonates.
Authors, therefore, hypothesized that severe hypothyroidism during the first days of life may affect the rate of adrenal maturation by delaying the adrenal remodeling process and specifically the involution of the fetal zone. In summary, the adrenal steroid hormone profile of neonates with severe CH points to a persistently active adrenal fetal zone compared with control subjects.

Germline USP8 Mutation Associated With Pediatric Cushing Disease and Other Clinical Features: A New Syndrome.
Article extracted from:  J Clin Endocrinol Metab. 2019 Oct 1;104(10):4676-4682. DOI: 10.1210/jc.2019-00697.
Michal Cohen1,2, Rebecca Persky3 Rachel Stegemann4, Laura C. Hernández-Ramirez3, Deena Zeltser3, Maya B. Lodish3, Anlu Chen5, Margaret F. Keil3 Christina Tatsi3, Fabio R. Faucz3, David A. Buchner3,4,5, Constantine A. Stratakis3, and Dov Tiosano1,2
1Pediatric Endocrinology Unit, Ruth Rappaport Children’s Hospital, Rambam Health Care Campus, Haifa, Israel; 2The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel; 3Section on Genetics and Endocrinology, Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland 20892; 4Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio; 5Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio; and 6Research Institute for Children’s Health, Case Western Reserve University, Cleveland, Ohio, ORCiD numbers: 0000-0002-4058-5520 (C. A. Stratakis).

BACKGROUND: Somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene are common in corticotropinomas of children with Cushing disease (CD). We report a unique patient with a germline USP8 mutation who presented with CD and a constellation of other findings that constitute an intriguing genetic syndrome. CASE DESCRIPTION: Authors describe a 16-year-old female with CD, developmental delay, dysmorphic features, ichthyosiform hyperkeratosis, chronic lung disease, chronic kidney disease, hyperglycemia, dilated cardiomyopathy with congestive heart failure, and previous history of hyperinsulinism and partial GH deficiency. She was diagnosed with CD at 14 years old and underwent transsphenoidal surgery. Despite the initial improvement, she developed recurrent CD. Methods: DNA was extracted from peripheral blood and tumor DNA; whole-exome and Sanger confirmatory sequencing were performed. Immunohistochemistry was performed on the resected adenoma. Results: A de novo germline heterozygous USP8 mutation (c.2155T.C, p.S719P) in the critical 14-3-3 binding motif hot spot locus of the gene was identified in both the peripheral blood and tumor DNA. Histopathologic evaluation of the resected tumor confirmed an ACTH-secreting adenoma. Conclusion: Somatic USP8 mutations are common in adenomas causing CD, but to date, no germline defects have been reported. We describe a patient with a de novo germline USP8 mutation with recurrent CD and multiple other medical problems. This unique patient informs us of the multitude of signaling events that may be controlled by USP8. (J Clin Endocrinol Metab 104: 4676–4682, 2019).

Excerpts selected from this article:
USP8 regulates epidermal growth factor receptor (EGFR) signaling by controlling its deubiquitination. Specifically, gain-of-function USP8 somatic mutations in corticotropinomas increase its deubiquitinating effect and thus the overall EGFR signaling activation, leading to enhanced POMC expression and ACTH secretion. Notably, all USP8 somatic mutations associated with CD are located in the 14-3-3 binding motif (between amino acids 713 and 720). In this report, the authors describe a unique patient that presented with developmental delay (DD), dysmorphic features, and other manifestations. She was also diagnosed with CD and multiple complications of hypercortisolemia, which only transiently improved after transsphenoidal surgery (TSS); she is currently under medical treatment.
WES analysis of the patient’s peripheral blood-derived DNA identified the c.2155T.C, p.S719P variant in the USP8 gene. This variant was heterozygous.
As expected, from the known involvement of USP8 in sporadic corticotropinomas causing CD, the patient also developed a severe form of CD, which was not surgically cured. The p.S719P USP8 variant identified in this patient lies at the 14-3-3 binding motif of the gene, and its pathogenic effects have been tested in vitro. Specifically, although p.S719P mutant protein is expressed at similar levels as the wild-type protein, it fails to bind 14-3-3 proteins; these proteins normally lead to USP8 phosphorylation and inactivation. Thus, it presents as a gain-of-function mutation with increased deubiquitination and decreased degradation of its substrate (EGFR). These effects are similar to those of other frequent USP8 pathogenic mutations previously identified in corticotropinomas (Ma ZY et al., Cell Res. 2015;25(3):306–317).
This publication confirms that germline USP8 defects constitute a cause of CD in pediatric patients. This patient also presented with additional clinical features that together may characterize an intriguing genetic syndrome. 0

Uterine Glucocorticoid Receptors are Critical for Fertility in Mice through Control of Embryo Implantation and Decidualization.
Extracted from: Proc Natl Acad Sci U S A, 2015 Dec 8;112(49):15166-71. DOI: 10.1073/pnas.1508056112.
Whirledge SD1Oakley RH1Myers PH2Lydon JP3DeMayo F3Cidlowski JA4
1Pediatric Endocrinology Unit, Ruth Rappaport Children’s Hospital, Rambam Health Care Campus, Haifa, Israel, 2The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel.3Section on Genetics and Endocrinology, Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, 4Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio, 5Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 6Research Institute for Children’s Health, Case Western Reserve University, Cleveland, Ohio.

In addition to the well-characterized role of the sex steroid receptors in fertility and reproduction, organs of the female reproductive tract are also regulated by the hypothalamic-pituitary-adrenal axis. These endocrine organs are sensitive to stress-mediated actions of glucocorticoids, and the mouse uterus contains high levels of the glucocorticoid receptor (GR). Although the presence of GR in the uterus is well established, uterine glucocorticoid signaling has been largely ignored in terms of its reproductive and/or immunomodulatory functions on fertility. To define the direct in vivo function of glucocorticoid signaling in adult uterine physiology, we generated a uterine-specific GR knockout (uterine GR KO) mouse using the PR(cre) mouse model. The uterine GR KO mice display a profound subfertile phenotype, including a significant delay to first litter and decreased pups per litter. Early defects in pregnancy are evident as reduced blastocyst implantation and subsequent defects in stromal cell decidualization, including decreased proliferation, aberrant apoptosis, and altered gene expression. The deficiency in uterine GR signaling resulted in an exaggerated inflammatory response to induced decidualization, including altered immune cell recruitment. These results demonstrate that GR is required to establish the necessary cellular context for maintaining normal uterine biology and fertility through the regulation of uterine-specific actions.
Significance. Glucocorticoids are primary stress and immune mediators of several fundamental processes, including reproduction and development. However, the direct actions of glucocorticoid signaling in the uterus are largely unknown. We have discovered that the glucocorticoid receptor (GR) in the mouse uterus is essential for normal fertility. Female mice lacking GR specifically in the uterus are subfertile due to defects in implantation related to inadequate remodeling of the endometrial stroma. Moreover, the deficiency in uterine glucocorticoid signaling leads to aberrant gene regulation during tissue remodeling events. Our findings demonstrate previously unrecognized functions of GR in both uterine physiology and fertility.

Sex Hormone Phenotypes in Young Girls and the Age at Pubertal Milestones.
Extracted from: J Clin Endocrinol Metab. 2019 Dec 1;104(12):6079-6089. DOI: 10.1210/jc.2019-00889. Fassler CS1Gutmark-Little I2,3Xie C1Giannini CM1Chandler DW4Biro FM2,3Pinney SM1. 1
1Department of Environmental Health1, University of Cincinnati College of Medicine, Cincinnati, Ohio; 2 Cincinati Children´s HospitalMedical Center, Cincinati, Ohio; 3Department of Pediatrics University of Cincinnati College of Medicine, Cincinnati, Ohio; 4 Endocrine Sciences, LabCorp, Calabazas Hill, California.

CONTEXT: The age of pubertal onset is influenced by many variables in young girls. Previous studies have not examined sex hormones longitudinally around the time of breast development and their relationship to pubertal onset. OBJECTIVE: We sought to use an unbiased statistical approach to identify phenotypes of sex hormones in young girls and examine their relationship with pubertal milestones. DESIGN AND SETTING: Longitudinal observational study. PARTICIPANTS AND MAIN OUTCOME MEASURES: In 269 girls, serum concentrations of steroid sex hormones [estradiol (E2), estrone, testosterone, and dehydroepiandrosterone sulfate] were measured by HPLC-mass spectrometry at time points before, at, and after thelarche. Girls were classified into four hormone phenotypes using objective principal components and cluster analyses of longitudinal hormone data. The association between the identified phenotypes and age of pubertal milestones was estimated using Cox proportional hazards modeling. RESULTS: Mean ages at thelarche, pubarche, and menarche were 9.02, 9.85, and 12.30 years, respectively.
(Translator Note. PHENOTYPE 1: high serum DHEAS, T, and E1; PHENOTYPE 2: high E2, T, and E1; PHENOTYPE 3a: low E1, E2, and T; PHENOTYPE 3b: all four hormones low)
Girls with low levels of all four hormones, phenotype 3b, were youngest at thelarche (8.67 years); those in phenotype 2, with the highest E2 levels and E2 surge 6 months after thelarche, were youngest at menarche (11.87 years) with shortest pubertal tempo. When controlling for race, maternal age of menarche, caregiver education, and body mass, different phenotypes were associated with the age of pubertal events. CONCLUSIONS: Hormone phenotypic clustering can identify clinically relevant subgroups with differing ages of thelarche, pubarche, and menarche. These findings may enhance the understanding of the timing of pubertal milestones and the risk of adult disease.

Excerpts selected from this article:
Although female sex hormones change dramatically throughout puberty, previous studies have not been able to assess patterns of changes considering multiple hormones in a longitudinal cohort. Most studies examining hormone values have been cross-sectional in nature, reporting as chronologic age regardless of pubertal status or as relative to having achieved a specific pubertal milestone. The objective of this study was to identify groups of girls with similar patterns of changes in multiple hormones over multiple time periods during puberty.
The identification of hormone phenotypes of girls and the quantification of their risk of earlier pubertal timing may also produce a better understanding of girls at risk for adult-onset diseases, such as polycystic ovarian syndrome (PCOS) or breast cancer. This longitudinal prospective cohort was comprised of girls from the Greater Cincinnati area, recruited at age 6 to 7 years. Samples later identified as those obtained at time points around thelarche were sent to the laboratory and analyzed for concentrations of estradiol (E2), estrone (E1), testosterone (T), and dehydroepiandrosterone sulfate (DHEA-S). All participants were members of the Cincinnati site of the Puberty Cohort Study of Breast Cancer and the Environment Research Program. Authors used an agnostic approach to examine only hormone values through principal component analysis (PCA) and cluster analysis (CA), without any attempt to associate the hormones with age of pubertal milestones or characterize them further by variables, such as race or body mass.
The primary endpoints of age at the pubertal milestone, measured in months from birth, include thelarche, pubarche, and menarche. Trained and certified female staff assessed pubertal maturation. Both thelarche and pubarche were based on reaching Tanner sexual maturation stage 2 or greater. The tempo was defined as the interval in months between thelarche and menarche. The pubertal pathway described the sequence of initiation of secondary sex characteristics and was categorized as pubarche before thelarche or thelarche before pubarche or synchronous.
The results of the final models were expressed as hazard ratios (HRs) with Wald’s 95% CIs. An HR 5 1 indicates no relationship between the predictor and the age of reaching the pubertal milestone. An HR, -1 indicates a reduced risk of reaching the pubertal milestone early, in other words, the age of reaching the pubertal milestone is late. An HR +1 indicates an increased risk of reaching the pubertal milestones.
The authors concluded that with the use of PCA-CA on longitudinal sex hormones, values related to the time of thelarche identified four distinct heterogeneous phenotypes. Girls within each phenotype varied along with hormone values at the time points, changes in the hormones between time points, age of pubertal milestones, and other demographic characteristics. These findings highlight the need to understand better the impact of these hormones on adult related morbidity.

Sexual Dimorphism of Microglia and Synapses During Mouse Postnatal Development.
Extracted from: Dev Neurobiol 2018 Jun;78(6):618-626. DOI: 10.1002/dneu.22568
Laetitia Weinhard,* Urte Neniskyte,* Auguste Vadisiute, Giulia di Bartolomei, Nil Aygun, Laurie Riviere, Francesca Zonfrillo, Susan Dymecki, Cornelius Gross
1Epigenetics and Neurobiology Unit, European Molecular Biology Laboratory (EMBL), Monterotondo, Italy, 2Department of Genetics, Harvard Medical School, Boston, Massachusetts

Microglia participate in synapse remodeling in the cortex and hippocampus during mouse postnatal development. Although sex differences in microglia activity during embryonic development have been reported in these regions, it remains unexplored whether microglia show sexually dimorphic features during the early postnatal period, a critical window for synapse formation and maturation.
Here, Laetitia Weinhard et al. investigated morphological and functional features of microglia across early postnatal development as well as morphological features of both pre- and postsynaptic neuronal compartments in the mouse hippocampus. They found a sex-dependent shift in microglia volume and phagocytic capacity across the first four postnatal weeks. Measurements of synaptic features revealed sex differences in the density of synaptic spines and boutons during the second postnatal week. These data are consistent with the precocious development of both microglia and synapses in the female brain. They further hypothesize that this bias may contribute to sex-specific brain wiring.

Excerpts selected from this article:
Microglia are the resident immune cells of the brain. They subsequently proliferate and mature from an amoeboid, phagocytic type to a ramified, surveilling state. As a result of the plethora of receptors they express to sense their environment, microglia are highly sensitive to any disturbance of brain homeostasis. Microglia have been recently demonstrated to play an important role in synaptic circuit remodeling through the formation, elimination, and maturation of synapses.
The importance of microglia in brain development was further demonstrated in several studies that associated aberrant microglia function with neurodevelopmental diseases, such as autism and schizophrenia. Intriguingly, microglia were shown to be involved in sexual differentiation of brain regions that control sexually dimorphic behaviors, while sex differences in microglia morphology and function were recently reported in the mouse brain at perinatal and late postnatal stages.
They investigated microglia morphology across postnatal stages together with the morphology and density of pre- and postsynaptic structures to identify a sexual dimorphism in microglia and synapse development in the postnatal hippocampus. They observed a transient peak in microglia volume and CD68 immuno-colocalization, a measure of phagocytic capacity, during the second postnatal week in both sexes. Microglia volume and phagocytic capacity increased and decreased earlier in females than males. This shift was paralleled by a difference in the density of neuronal spines and boutons. The pattern of differences in microglia and synaptic features is consistent with the precocious development of microglia and synapses in the female brain.
Numerous data suggest that the hippocampus is sexually dimorphic, with sex differences reported in neurotransmitter activity, synaptic function, and hippocampus-dependent behaviors, such as spatial learning, leaving open the possibility that the transient differences reported underlie persistently sex-dependent effects in the adult hippocampus.

Deletion of the Cardiomyocyte Glucocorticoid Receptor Leads to Sexually Dimorphic Changes in Cardiac Gene Expression and Progression to Heart Failure.
Extracted from: J Am Heart Assoc. 2019 Aug 6;8(15):e011012. DOI: 10.1161/JAHA.118.011012.
Cruz-Topete D1,2Oakley RH3Carroll NG1He B3Myers PH4Xu X5Watts MN6Trosclair K7Glasscock E7,2Dominic P6,2Cidlowski JA3.
1Department of Molecular and Cellular Physiology LSU Health Sciences Center Shreveport LA. 2Center for Cardiovascular Diseases and Sciences LSU Health Sciences Center Shreveport LA. 3Signal Transduction Laboratory National Institute of Environmental Health Sciences National Institutes of Health Department of Health and Human Services Research Triangle Park NC. 4Comparative Medicine Branch National Institute of Environmental Health Sciences National Institutes of Health Department of Health and Human Services Research Triangle Park NC 5Laboratory of Integrative Bioinformatics National Institute of Environmental Health Sciences National Institutes of Health Department of Health and Human Services Research Triangle Park NC. 6Department of Cardiology LSU Health Sciences Center Shreveport LA. 7Department of Cellular Biology and Anatomy LSU Health Sciences Center Shreveport LA.

BACKGROUND: The contribution of glucocorticoids to sexual dimorphism in the heart is essentially unknown. Therefore, the authors sought to determine the sexually dimorphic actions of glucocorticoid signaling in cardiac function and gene expression. To accomplish this goal, they conducted studies on mice lacking glucocorticoid receptors (GR) in cardiomyocytes (cardioGRKO mouse model). METHODS AND RESULTS: The deletion of cardiomyocyte GR leads to an increase in mortality because of the development of spontaneous cardiac pathology in both male and female mice; however, females are more resistant to GR signaling inactivation in the heart. Male cardioGRKO mice had a median survival age of 6 months. In contrast, females had a median survival age of 10 months. Transthoracic echocardiography data showed phenotypic differences between male and female cardioGRKO hearts. By 3 months of age, male cardioGRKO mice exhibited left ventricular systolic dysfunction. Conversely, no significant functional deficits were observed in female cardioGRKO mice at the same time point. The functional sensitivity of male hearts to the loss of cardiomyocyte GR was reversed following gonadectomy. RNA-Seq analysis showed that deleting GR in the male hearts leads to a more profound dysregulation in the expression of genes implicated in heart rate regulation (calcium handling). In agreement with these gene expression data, cardiomyocytes isolated from male cardioGRKO hearts displayed altered intracellular calcium responses. In contrast, female GR-deficient cardiomyocytes presented a response comparable with controls. CONCLUSIONS: These data suggest that GR regulates calcium responses in a sex-biased manner, leading to sexually distinct responses to stress in male and female mice hearts, which may contribute to sex differences in heart disease, including the development of ventricular arrhythmias that contribute to heart failure and sudden death.

Excerpts selected from this article:
A global analysis comparison between male versus female cardioGRKO hearts revealed that gene networks involved in heart failure, hypertrophy, and dilated cardiomyopathy were activated in male cardioGRKO hearts; in contrast, these gene networks were not significantly altered in the hearts of female cardioGRKO mice at the same time point. Visualization of the genes involved in heart failure revealed dysregulation in the expression of genes involved in cardiomyocyte contractility, mitochondrial ADP/ATP transport, and sarcomere structure. Among the genes involved in cardiac contractility, they found Ryr2 and Atp2a2 (Serca2). The expression of these two genes was significantly dysregulated in male cardioGRKO hearts; in contrast, their levels were normal in female hearts. Cardiac Ryr2 is an intracellular receptor that modulates the release of Ca2+ from the sarcoplasmic reticulum leading to cardiomyocyte contraction. Atp2a2 encodes the Serca2 pump which is involved in the transport of calcium ions from the cytoplasm into the sarcoplasmic reticulum. Reductions in Ryr2 and Serca2 levels lead to perturbations in intracellular Ca2+ cycling. Elegant studies by Rog-Zielinska et al. showed that GR regulates the expression of Ryr2 and Serca2 in cardiomyocytes. Therefore, defects in the expression of these 2 genes may explain in part the early deterioration of cardiac function observed in cardioGRKO males as compared with their female counterparts. Functional data showed that reductions in Ryr2 expression in male cardioGRKO hearts correlate with abnormal calcium handling. Male cardiomyocytes isolated from cardioGRKO hearts exhibited a decreased sensitivity to caffeine-induced intracellular calcium responses as compared with their female counterparts. Studies have shown that estrogen can protect the female heart from pathological cardiac hypertrophy by inducing rapid intracellular Ca2+ mobilization through the regulation of L-type voltage-operated Ca2+ channels and Atp2a2 and via indirect interactions with the Ryr2s.

Clinical Perspective. What Is New?
Stress is an emerging risk factor for heart disease, yet the sex‐specific effects of stress on the heart are unknown. Using mice lacking the glucocorticoid receptor (GR) in cardiomyocytes, these authors investigated the contribution of GR signaling to maintain cardiac homeostasis in a sex‐specific manner. Their findings showed that the ablation of GR signaling in the heart leads to sex‐specific changes in cardiac gene expression and function. They found that GR plays sex‐specific effects in preserving intracellular calcium handling in ventricular cardiomyocytes, and the mechanisms underlying these effects may result from cardiac GR signaling crosstalk with sex hormones signaling.

Posted in Edition 76 - November 2019, Bibliographic Reviews
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