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). At 20-23 weeks of age, mean arterial pressure (MAP) was measured in conscious rats. The mesenteric arterial tree and kidneys were collected, processed for Western blots, and probed for Cu Zn superoxide dismutase (SOD1), soluble epoxide hydrolase (sEH), and Alpha 2A adrenergic receptor (A2AR) expression. MAP was unaffected by ovariectomy (Sham 164 ± 4: Ovariecttomy 159 ± 3 mm Hg). MAP was reduced by orchidectomy (Sham 189 ± 5:Orchidectomy 167 ± 2 mm Hg). In mesenteric artery, SOD1 expression was greater in male versus female SHR. Orchidectomy increased while ovariectomy decreased SOD1 expression. The kidney exhibited a different pattern of response. SOD1 expression was reduced in male compared to female SHR but gonadectomy had no effect. sEH expression was not significantly different among the groups in mesenteric artery. In kidney, sEH expression was greater in males compared to females. Ovariectomy but not orchidectomy increased sEH expression. A2AR expression was greater in female than male SHR in mesentery artery and kidney. Gonadectomy had no effect in either tissue. We conclude that sexually dimorphic hypertension is associated with regionally specific changes in expression of three key proteins involved in blood pressure control. These data suggest that broad spectrum inhibition or stimulation of these systems may not be the best approach for hypertension treatment. Instead regionally targeted manipulation of these systems should be investigated.

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. Future studies into different delivery mechanisms such as transdermal applications and different agents, such as tibolone and raloxifene, will help refine the treatment of menopause.

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.

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). METHODS: Male SHR were sham operated (M-SHAM) or castrated (M-CX), and female SHR were sham-operated (F-SHAM) or ovariectomized (F-OVX) at 3 weeks of age. Seven months later, BP was measured in anesthetized rats, and vascular responsiveness was evaluated in the isolated perfused mesentery. RESULTS: Mean arterial BP (mm Hg) was significantly greater in M-SHAM (186 ± 6) compared with F-SHAM (159 ± 5). Gonadectomy reduced BP in male SHR (M-CX: 160 ± 4) but had no significant effect in female SHR (F-OVX: 153 ± 7). Norepinephrine-induced constriction was similar in all groups. Gonadectomy attenuated serotonin-induced vasoconstriction in the mesentery. Acetylcholine (ACh)- and isoproterenol (ISO)-induced vasodilation was greater in female than male SHR. Ovariectomy of female SHR blunted ACh and ISO dilatory responses. ISO dose-response curves were shifted to the left in castrated male SHR. CONCLUSIONS: Gonadectomy exerts long-term effects on mesenteric vascular reactivity and hypertension in the SHR.

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.

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. A number of genomics studies have demonstrated significant alterations in gene expression in endometriosis. A more thorough understanding of the genetics and genomics of endometriosis will facilitate understanding the basic biology of the disease and open new inroads to diagnosis and treatment of this enigmatic condition.

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. A more radical surgical approach of hysterectomy with bilateral salpingo-oophorectomy remains a mainstay of therapy for patients who have completed childbearing. Current medical therapies rely upon interruption of normal cyclic ovarian hormone production resulting in an environment not conducive to the growth of endometriosis. Genomics promises to further characterize endometriosis and tailor therapies based on a woman's symptoms and reproductive goals.

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.

Pioglitazone administration alters ovarian gene expression in aging obese lethal yellow mice.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) are often treated with insulin-sensitizing agents, e.g. thiazolidinediones (TZD), which have been shown to reduce androgen levels and improved ovulatory function. Acting via peroxisome proliferator-activated receptor (PPAR) gamma, TZD alter the expression of a large variety of genes. Lethal yellow (LY; C57BL/6J Ay/a) mice, possessing a mutation (Ay) in the agouti gene locus, exhibit progressive obesity, reproductive dysfunction, and altered metabolic regulation similar to women with PCOS. The current study was designed to test the hypothesis that prolonged treatment of aging LY mice with the TZD, pioglitazone, alters the ovarian expression of genes that may impact reproduction. METHODS: Female LY mice received daily oral doses of either 0.01 mg pioglitazone (n = 4) or an equal volume of vehicle (DMSO; n = 4) for 8 weeks. At the end of treatment, ovaries were removed and DNA microarrays were used to analyze differential gene expression. RESULTS: Twenty-seven genes showed at least a two-fold difference in ovarian expression with pioglitazone treatment. These included leptin, angiopoietin, angiopoietin-like 4, Foxa3, PGE1 receptor, resistin-like molecule-alpha (RELM), and actin-related protein 6 homolog (ARP6). For most altered genes, pioglitazone changed levels of expression to those seen in untreated C57BL/6J(a/a) non-mutant lean mice. CONCLUSION: TZD administration may influence ovarian function via numerous diverse mechanisms that may or may not be directly related to insulin/IGF signaling.

Effect of ovariectomy on blood pressure and venous tone in female spontaneously hypertensive rats.

BACKGROUND: Venous capacitance plays an important role in circulatory homeostasis. A number of reports have suggested an effect of estrogen on venous function. This study tested the hypothesis that ovariectomy would increase venous tone in the female spontaneously hypertensive rat (SHR) via autonomic mechanisms. METHODS: Five-week-old female SHR were subjected to sham operation (Sham) or ovariectomy (OVX). At 10 weeks of age, the rats were instrumented for the measurement of arterial and venous pressure. A balloon catheter was advanced into the right atrium. Mean circulatory filling pressure (MCFP), an index of venous tone, was calculated. Mean arterial pressure (MAP), heart rate (HR), and MCFP were recorded from conscious rats. Postsynaptic adrenergic responsiveness was assessed by constructing cumulative dose-response curves to norepinephrine (NE). RESULTS: MAP was not significantly affected by ovariectomy (Sham 127 +/- 6 mm Hg vs. OVX 130 +/- 3 mm Hg). HR also was not different between groups (Sham 409 +/- 11 bpm vs. OVX 399 +/- 12 bpm). Conversely, MCFP was significantly, but moderately, increased in OVX SHR (Sham 5.2 +/- 0.2 mm Hg vs. OVX 5.9 +/- 0.2 mm Hg). Ganglionic blockade produced marked decreases in MAP, HR, and MCFP in both groups; however, the responses were not different between groups. Infusion of NE caused dose-dependent increases in MAP and MCFP. There were no statistically significant differences in these responses between Sham and OVX SHR. CONCLUSION: Endogenous ovarian hormones effect a small reduction in MCFP. This effect does not appear to be mediated by adrenergic mechanisms.

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. While morphological defects are more severe on the C57BL/6J (B6) background than 129S6/SvEvTac (129), cerebrospinal fluid flow is perturbed on both backgrounds, suggesting that abnormal cilia-driven flow is not the only factor underlying the hydrocephalus phenotype. Here, we performed a microarray analysis on brains from wild type and nm1054 mice lacking CFAP221 on the B6 and 129 backgrounds. Expression differences were observed for a number of genes that cluster into distinct groups based on expression pattern and biological function, many of them implicated in cellular and biochemical processes essential for proper brain development. These include genes known to be functionally relevant to congenital hydrocephalus, as well as formation and function of both motile and sensory cilia. Identification of these genes provides important clues to mechanisms underlying congenital hydrocephalus severity.

New paradigms in signal transduction.

Signal transduction is a dynamic field in which established pathways evolve and new pathways emerge. The purpose of this commentary is to highlight new paradigms of signal transduction that have developed over the past few years. This discussion proposes a third member of the generic models of membrane receptors in addition to the 7-transmembrane pass receptor and the enzyme-linked receptor: the non-enzymatic nucleating receptor. Also discussed are the new paradigms of signal transduction by proteolysis which includes signaling by Notch, signaling through the Hedgehog and Wnt pathways, signaling through histidine phosphorylation, and reactive oxygen species in signal transduction.

The effects of estrogen and testosterone on gene expression in the rat mesenteric arteries.

A dramatic difference exists in the timing of development of cardiovascular disease in men vs. women. The primary candidates underlying the cause of this gender difference are the sex steroids, estrogen and testosterone. The vasculature is considered to be a site of action of these steroids. In spite of these concepts there is little data on the direct effects of estrogen and testosterone on gene expression in the vasculature. In this study, ovariectomized Sprague Dawley rats were treated for 4 days with vehicle (sesame oil), estradiol benzoate (0.15 mg/kg/day), or testosterone (1 mg/kg/day). The mesenteric arteries were obtained, total RNA was extracted, and CodeLink Uniset Rat I DNA microarrays were used to identify differential gene expression. Seven genes were identified as differentially expressed from the DNA microarray data and confirmed by real time RT-PCR. The expression of D site albumin promoter binding protein and fatty acid synthase were increased in response to both estrogen and testosterone. 3 alpha-hydroxysteroid dehydrogenase, interleukin 4 receptor, JunB and c-Fos expression were increased by estrogen but not by testosterone. Aryl hydrocarbon nuclear translocator-like gene was reduced by testosterone. These data identify genes not previously known to be responsive to estrogen and testosterone in the vasculature.

The membrane and lipids as integral participants in signal transduction: lipid signal transduction for the non-lipid biochemist.

Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction. However, lipids serve a variety of roles in signal transduction. They act as ligands that activate signal transduction pathways as well as mediators of signaling pathways, and lipids are the substrates of lipid kinases and lipid phosphatases. Cell membranes are the source of the lipids involved in signal transduction, but membranes also constitute lipid barriers that must be traversed by signal transduction pathways. The purpose of this review is to explore the magnitude and diversity of the roles of the cell membrane and lipids in signal transduction and to highlight the interrelatedness of families of lipid mediators in signal transduction.

Career counseling: 101+ things you can do with a degree in biology.

Biology is the science of life and of how living things work. Our students choose to major in biology in college because of a fascination with understanding how living things function, but often they have difficulty in identifying a career that uses their foundation in biology despite the variety of biology-based careers available. The purpose of this discussion is to assist biology students and the career counselors who work with them in identifying satisfying careers that build upon their interest and foundation in biology. The categories of career options include research, healthcare, teaching, science writing, administration/management, government, industry, and miscellaneous careers that do not fit into the other categories.

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.

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 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. Moreover, our prior studies have demonstrated that cardiac specific knockout of Cops8 (Cops8-CKO) impairs autophagosome maturation and causes massive necrosis in cardiomyocytes but the underlying mechanism remains poorly understood. Given that Cops8 is nucleus-enriched and a prior report showed its binding to the promoter of several genes and association of its ablation with decreased mRNA levels of these genes, we sought to determine the dynamic changes of myocardial transcriptome in mice with perinatal Cops8-CKO and to explore their functional implications. Methods and Results: Myocardial transcriptomes of Cops8flox/flox , Cops8flox/+::Myh6-Cre, and Cops8flox/flox::Myh6-Cre littermate mice at postnatal 2 and 3 weeks were analyzed. The data were imported into an in-house analysis pipeline using Bioconductor for quantile normalization and statistical analysis. Differentially expressed genes (DEGs) between groups at each time point or between time points within the group were revealed by t-test. Genes with p < 0.05 after Benjamini and Hochberg false discovery rate correction for multiple hypothesis testing were considered as significant DEGs. We found that (1) the Ingenuity Pathway Analysis (IPA) revealed significant enrichment of DEGs in multiple pathways, especially those responding to oxidative stress, in homozygous Cops8-CKO hearts at both 2 and 3 weeks, corroborating the occurrence of massive cardiomyocyte necrosis at 3 weeks; (2) the decreases in multiple CRL SR proteins were associated with decreased transcript levels; and (3) enrichment of DEGs in the chromatin remodeling pathway and the microtubule motility and vesicle trafficking pathways. Conclusions: Our data are consistent with the notion that Cops8/CSN plays a role in the transcriptional regulation of CRL SRs and in the redox and vesicle trafficking pathways.

A review of current management of endometriosis in 2006: an evidence-based approach.

Endometriosis, a common cause of morbidity in reproductive age females, results in pelvic pain and infertility. Effective, evidence-based treatments of endometriosis-associated infertility include conservative surgical therapy and assisted reproductive technologies. In early stage endometriosis ovulation induction, with or without intrauterine insemination, improves pregnancy rates. In early stage disease in vitro fertilization reduces time to pregnancy as compared to controls, but does not increase the chance of pregnancy after three years. Endometriosis-associated pain can be approached with surgical or medical therapies. Conservative surgery maintains the reproductive organs and is an effective mode of treatment for endometriosis-associated pain. A more radical surgical approach of hysterectomy with bilateral salpingo-oophorectomy, not investigated in randomized controlled trials, remains a mainstay of therapy for endometriosis-associated pain in patients who have completed child-bearing. Current medical therapies rely upon interruption of normal cyclic, ovarian hormone production resulting in an environment not conducive to the growth of endometriosis. The current accepted therapies for endometriosis include danazol, progestational agents, oral contraceptive agents, and gonadotropin-releasing hormone analogues which all function similarly in relieving pain. The new era of genomics promises to help characterize endometriosis and allow one to tailor therapies based on a woman's symptoms and reproductive goals.

The Estrogen Receptors: An Overview from Different Perspectives.

The estrogen receptors, ERα, ERß, 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.

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.