Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.

/pdf/accurate-normalization-of-real-time-quantitative-rt-pcr-data-34rwzsvnu5.pdf

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes

Recent discoveries of endogenous negative regulators of angiogenesis, thrombospondin, angiostatin and glioma-derived angiogenesis inhibitory factor, all associated with neovascularized tumours, suggest a new paradigm of tumorigenesis. It is now helpful to think of the switch to the angiogenic phenotype as a net balance of positive and negative regulators of blood vessel growth. The extent to which the negative regulators are decreased during this switch may dictate whether a primary tumour grows rapidly or slowly and whether metastases grow at all.

Angiogenesis in cancer, vascular, rheumatoid and other disease

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. Keywords: extracellular vesicle; exosome; microvesicle; standardization; isolation (Published: 27 May 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 20360 - http://dx.doi.org/10.3402/jev.v2i0.20360

/pdf/standardization-of-sample-collection-isolation-and-analysis-4om42nmomj.pdf

Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

Significant advances have taken place in our knowledge of the enzymes involved in steroid hormone biosynthesis since the last comprehensive review in 1988. Major developments include the cloning, identification, and characterization of multiple isoforms of 3β-hydroxysteroid dehydrogenase, which play a critical role in the biosynthesis of all steroid hormones and 17β-hydroxysteroid dehydrogenase where specific isoforms are essential for the final step in active steroid hormone biosynthesis. Advances have taken place in our understanding of the unique manner that determines tissue-specific expression of P450aromatase through the utilization of alternative promoters. In recent years, evidence has been obtained for the expression of steroidogenic enzymes in the nervous system and in cardiac tissue, indicating that these tissues may be involved in the biosynthesis of steroid hormones acting in an autocrine or paracrine manner. This review presents a detailed description of the enzymes involved in the biosynthe...

https://www.researchgate.net/profile/Dale_Buck_Hales/publication/8144948_Overview_of_steroidogenic_enzymes_in_the_pathway_from_cholesterol_to_active_steroid_hormones/links/02e7e5249ac2529059000000.pdf

Overview of Steroidogenic Enzymes in the Pathway from Cholesterol to Active Steroid Hormones

Effects of heat-stress on production in dairy cattle.

The mammalian placenta is the organ through which respiratory gases, nutrients, and wastes are exchanged between the maternal and fetal systems. Thus, transplacental exchange provides for all the metabolic demands of fetal growth and development. The rate of transplacental exchange depends primarily on the rates of uterine (maternal placental) and umbilical (fetal placental) blood flows. In fact, increased uterine vascular resistance and reduced uterine blood flow can be used as predictors of high risk pregnancies and are associated with fetal growth retardation. The rates of placental blood flow, in turn, are dependent on placental vascularization, and placental angiogenesis is therefore critical for the successful development of viable, healthy offspring. Recent studies, including gene knockouts in mice, indicate that the vascular endothelial growth factors represent a major class of placental angiogenic factors. Other angiogenic factors, such as the fibroblast growth factors or perhaps the angiopoietins, also may play important roles in placental vascularization. In addition, recent observations suggest that these angiogenic factors interact with the local vasodilator nitric oxide to coordinate placental angiogenesis and blood flow. In the future, regulators of angiogenesis that are currently being developed may provide novel and powerful methods to ensure positive outcomes for most pregnancies.

https://bioone.org/journals/biology-of-reproduction/volume-64/issue-4/biolreprod64.4.1033/Angiogenesis-in-the-Placenta1/10.1095/biolreprod64.4.1033.pdf

Angiogenesis in the Placenta

In adult tissues, capillary growth (angiogenesis) occurs normally during tissue repair, such as in healing of wounds and fractures. Rampant capillary growth is associated with various pathological conditions, including tumor growth, retinopathies, hemangiomas, fibroses and rheumatoid arthritis. The female reproductive organs (i.e., ovary, uterus, and placenta) exhibit dynamic, periodic growth and regression accompanied by equally dramatic changes in rates of blood flow. It is not surprising, therefore, that they are some of the few adult tissues in which angiogenesis occurs as a normal process. Thus, the female reproductive system provides a unique model for studying regulation of angiogenesis during growth and differentiation of normal adult tissues. Ovarian, uterine, and placental tissues recently have been shown to contain and produce angiogenic and anti-angiogenic factors. This review discusses the current state of knowledge regarding angiogenic processes and their regulation in female reproductive tissues. In addition, implications of this research for regulation of fertility as well as for control of angiogenesis in other normal and pathological processes are discussed.

Angiogenesis in the female reproductive system.

The rate of fetal growth and subse- quent birth weight are major determinants of postna- tal survival and growth. Because the placenta is the organ through which respiratory gases, nutrients, and wastes are transported between the maternal and fetal systems, its primary function is to supply the metabolic substrates necessary to support fetal growth. Placental growth and development, therefore, are critical for normal fetal growth and development. During the last half of gestation in mammals, growth of the fetus is exponential, whereas utero-placental growth slows or ceases. Nevertheless, unless placental transport capacity keeps pace with the continually increasing demands of the fetus, fetal growth will be compromised. Studies over the last two decades have shown that placental transport capacity does indeed keep pace with fetal growth. This increase in placental function can be accounted for primarily by continual increases in placental (uterine and umbilical) blood flows, associated with increased placental vascularity. Placental vascular growth and development, in turn, are probably regulated by angiogenic factors produced by the placental tissues themselves. These placental angiogenic factors are produced primarily by the maternal placental tissues, are heparin-binding, and seem to be related to the fibroblast growth factor family. Further elucidation of the factors responsible for placental growth and vascular development is critical for an improved understanding of utero- placental-fetal interactions, which result in delivery of a healthy offspring.

Utero-placental vascular development and placental function.

The placenta is the organ that transports nutrients, respiratory gases, and wastes between the maternal and fetal systems. Consequently, placental blood flow and vascular development are essential components of normal placental function and are critical to fetal growth and development. Normal fetal growth and development are important to ensure optimum health of offspring throughout their subsequent life course. In numerous sheep models of compromised pregnancy, in which fetal or placental growth, or both, are impaired, utero-placental blood flows are reduced. In the models that have been evaluated, placental vascular development also is altered. Recent studies found that treatments designed to increase placental blood flow can ‘rescue’ fetal growth that was reduced due to low maternal dietary intake. Placental blood flow and vascular development are thus potential therapeutic targets in compromised pregnancies.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2005.104430

Evidence for altered placental blood flow and vascularity in compromised pregnancies.

In adult tissues, capillary growth (angiogenesis) occurs normally during tissue repair, such as in the healing of wounds and fractures. Inappropriate capillary growth is associated with various pathological conditions, including tumour growth, retinopathies, haemangiomas, fibroses and rheumatoid arthritis in the case of rampant capillary growth, and nonhealing wounds and fractures in the case of inadequate capillary growth. The female reproductive organs exhibit marked, periodic growth and regression, accompanied by equally striking changes in their rates of blood flow. It is not surprising, therefore, that they are some of the few adult tissues in which angiogenesis occurs as a normal process. Ovarian follicles and corpora lutea have been shown to contain and produce angiogenic factors. These angiogenic factors appear to be heparin-binding and to belong to the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) families of proteins. In addition, factors regulating gap junctional communication may play a critical role in coordinating the interactions between luteal vascular and nonvascular tissues. Further elucidation of the specific physiological roles of these factors in follicular and luteal growth, development and function will ultimately lead to improved methods for regulating fertility in mammals.

Lawrence P. Reynolds

Papers

Angiogenesis in the Placenta

Angiogenesis in the female reproductive system.

Utero-placental vascular development and placental function.

Evidence for altered placental blood flow and vascularity in compromised pregnancies.

Angiogenesis in the ovary