Extraction of RNA and Analysis of Estrogen-Responsive Genes by RT-qPCR.

Reverse transcription-quantitative RT-PCR (RT-qPCR) is a powerful tool for assessing gene transcription levels. The technique is especially useful for measuring estrogen receptor transcript levels as well as gene expression changes in response to estrogen stimulation as it is quick, accurate, and robust and allows the measurement of gene expression in a variety of tissues and cells. This chapter describes the protocols used for RNA extraction and analysis as well as for RT-qPCR assay using hydrolysis (TaqMan-type) probes.

Protocol for DNA Microarrays on Glass Slides.

The DNA microarray is a powerful, flexible, nonbiased discovery technology. Microarrays can be used to assess processes from gene expression to long noncoding RNAs to specific pathologies, as well as many others. This chapter describes the protocol for DNA microarray analysis of differential gene expression using DNA sequences spotted on microscope slides.

Strain-specific differences in brain gene expression in a hydrocephalic mouse model with motile cilia dysfunction.

Congenital hydrocephalus results from cerebrospinal fluid accumulation in the ventricles of the brain and causes severe neurological damage, but the underlying causes are not well understood. It is associated with several syndromes, including primary ciliary dyskinesia (PCD), which is caused by dysfunction of motile cilia. We previously demonstrated that mouse models of PCD lacking ciliary proteins CFAP221, CFAP54 and SPEF2 all have hydrocephalus with a strain-dependent severity.

Murine Myocardial Transcriptome Analysis Reveals a Critical Role of COPS8 in the Gene Expression of Cullin-RING Ligase Substrate Receptors and Redox and Vesicle Trafficking Pathways.

The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases. Supported primarily by the decrease of substrate receptor (SR) proteins of CRLs in cells deficient of a CSN subunit, CSN-mediated cullin deneddylation is believed to prevent autoubiquitination and self-destruction of the SR in active CRLs. However, it is unclear whether the decrease in SRs is solely due to protein destabilization.

Mifepristone increases mRNA translation rate, triggers the unfolded protein response, increases autophagic flux, and kills ovarian cancer cells in combination with proteasome or lysosome inhibitors.

The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity.

Assessing Direct Vascular Actions of Estrogens.

Estrogens are known to affect vascular function. In order to decipher the underlying mechanisms, it is essential to study the direct actions of estrogenic substances on blood vessels. There are two widely used approaches to assess the effects of estrogenic substances directly on blood vessels, the isolated perfused intact mesenteric vascular bed (McGregor preparation) and the isolated perfused/pressurized vessel approach.

DNA Microarray Analysis of Estrogen-Responsive Genes.

DNA microarray is a powerful, non-biased discovery technology that allows the analysis of the expression of thousands of genes at a time. The technology can be used for the identification of differential gene expression, genetic mutations associated with diseases, DNA methylation, single-nucleotide polymorphisms, and microRNA expression, to name a few. This chapter describes microarray technology for the analysis of differential gene expression in response to estrogen treatment.

The Use of Real-Time Reverse Transcription-PCR for Assessing Estrogen Receptor and Estrogen-Responsive Gene Expression.

Real-time reverse transcription-polymerase chain reaction (RT-PCR), also known as quantitative RT-PCR (qRT-PCR), is a powerful tool for assessing gene transcription levels. The technique is especially useful for measuring estrogen receptor transcript levels as well as gene expression changes in response to estrogen stimulation as it is quick, accurate, robust, and allows the measurement of gene expression in a variety of tissues and cells. This chapter describes the protocols used for the real-time RT-PCR assay using hydrolysis (TaqMan-type) probes.

The Estrogen Receptors: An Overview from Different Perspectives.

The estrogen receptors, ERα, ERβ, and GPER, mediate the effects of estrogenic compounds on their target tissues. Estrogen receptors are located in the tissues of the female reproductive tract and breast as one would expect, but also in tissues as diverse as bone, brain, liver, colon, skin, and salivary gland. The purpose of this discussion of the estrogen receptors is to provide a brief overview of the estrogen receptors and estrogen action from perspectives such as the historical, physiological, pharmacological, pathological, structural, and ligand perspectives.

Retained Foreign Body: "Needle in a Haystack".

Retained foreign bodies remain an area of potential patient harm. This case describes a retained needle from distant surgery discovered at the time of the needle count after myomectomy.

Effects of estradiol on transcriptional profiles in atherosclerotic iliac arteries in ovariectomized cynomolgus macaques.

Objective: This study aimed to assess the in vivo effects of estradiol treatment on arterial gene expression in atherosclerotic postmenopausal female monkeys.

Methods: Eight ovariectomized cynomolgus monkeys were fed atherogenic diets for 6.5 years.

What happened to WHI: menopausal hormonal therapy in 2012.

Menopause is characterized by amenorrhea for 1 year due to the cessation of ovarian function. The hormonal treatment of menopause has significantly altered since the publication of initial results from the Women's Health Initiative continuous, combined, conjugated equine estrogen with medroxyprogesterone acetate study arm in 2002. Current studies suggest that treatment should be individualized and that the lowest dose of estrogen providing relief should be used for the shortest period of time in menopausal women who experience vasomotor symptoms or urogenital atrophy.

Contrasting hippocampal and amygdalar expression of genes related to neural plasticity during escape from social aggression.

Social subjugation has widespread consequences affecting behavior and underlying neural systems. We hypothesized that individual differences in stress responsiveness were associated with differential expression of neurotrophin associated genes within the hippocampus and amygdala. To do this we examined the brains of hamsters placed in resident/intruder interactions, modified by the opportunity to escape from aggression.

Müllerian agenesis with hypohidrotic ectodermal dysplasia syndrome.

Objective: To describe the association of müllerian agenesis with hypohidrotic ectodermal dysplasia.

Design: Case report.

Setting: University medical center.

Regional differences in sexually dimorphic protein expression in the spontaneously hypertensive rat (SHR).

Hypertension is sexually dimorphic and modified by removal of endogenous sex steroids. This study tested the hypothesis that endogenous gonadal hormones exert differential effects on protein expression in the kidney and mesentery of SHR. At ~5 weeks of age male and female SHR underwent sham operation, orchidectomy, or ovariectomy (OVX).

Dysfunctional hormonal regulation of metabolism in obesity.

A complex network of hormones from the pancreas, adipose tissue, stomach, intestines and the central nervous system coordinates regulation of metabolism and energy balance. Obesity disrupts this regulatory network. This paper reviews the anorexigenic and orexigenic hormones and their dysfunctional regulation in obesity.

Estrogens and selective estrogen receptor modulators regulate gene and protein expression in the mesenteric arteries.

Estrogen has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Gene targets of estrogen and SERMs in the vasculature are not well-known.

Gene expression signatures differ with extent of atherosclerosis in monkey iliac artery.

Objective: The aim of this study was to evaluate global gene expression patterns in the common iliac arteries of monkeys with a varied extent of atherosclerosis.

Methods: The left common iliac artery was removed from ovariectomized cynomolgus monkeys (n = 12) after 6.5 years of consuming a diet containing fat and cholesterol at levels comparable with those consumed in Western populations.

Hypercalcemia: an unusual etiology of a common menopausal symptom.

Objective: To describe atypical vasomotor symptoms that were secondary to primary hyperparathyroidism.

Design: Case report.

Setting: University medical center.

Blood pressure and mesenteric vascular reactivity in spontaneously hypertensive rats 7 months after gonadectomy.

Objective: Sexual dimorphism in the degree of high blood pressure (BP) has been observed in both animal and human hypertension. However, the mechanisms are still poorly understood. We tested the hypothesis that long-term loss of sex steroids promotes changes in mesenteric vascular reactivity that impact the maintenance of hypertension in the spontaneously hypertensive rats (SHR).

Reciprocal communication between endometrial stromal cells and macrophages.

This study tested the hypothesis that reciprocal communication occurs between macrophages and cultured human endometrial stromal cells and that this communication may contribute to the pathology of endometriosis. An endometrial stromal cell line (telomerase-immortalized human endometrial stromal cell [T-HESC]) was treated with macrophage-conditioned medium (CM) +/- estradiol + progesterone. Macrophages were treated without or with T-HESC CM.

Management of endometriosis-associated pain.

Endometriosis, a common cause of morbidity in reproductive-age females, results in pelvic pain and infertility. Endometriosis-associated pain can be approached with surgical or medical therapies. Conservative surgery maintains reproductive organs and is effective in the treatment of endometriosis-associated pain.

Genetics and genomics of endometriosis.

Endometriosis is a common cause of morbidity in women with an unknown etiology. Studies have demonstrated the familial nature of endometriosis and suggest that inheritance occurs in a polygenic/multifactorial fashion. Studies have attempted to define the gene or genes responsible for endometriosis through association or linkage studies with candidate genes or DNA mapping technology.

Two-way communication between endometrial stromal cells and monocytes.

Immune system cells and cells of the endometrium have long been proposed to interact in both physiological and pathological processes. The current study was undertaken to examine communication between cultured monocytes and endometrial stromal cells and also to assess responses of endometrial stromal cells for treatment with estradiol (E) in the absence and presence of medroxyprogesterone acetate (P). A telomerase-immortalized human endometrial stromal cell (T-HESC) line and the U937 monocyte cell line were used.