Steroidogenic pathway

Steroid isolation , depending on context, is the isolation of chemical matter required for chemical structure elucidation, derivitzation or degradation chemistry, biological testing, and other research needs (generally milligrams to grams, but often more [37] or the isolation of "analytical quantities" of the substance of interest (where the focus is on identifying and quantifying the substance (for example, in biological tissue or fluid). The amount isolated depends on the analytical method, but is generally less than one microgram. [38] [ page needed ] The methods of isolation to achieve the two scales of product are distinct, but include extraction , precipitation, adsorption , chromatography , and crystallization . In both cases, the isolated substance is purified to chemical homogeneity; combined separation and analytical methods, such as LC-MS , are chosen to be "orthogonal"—achieving their separations based on distinct modes of interaction between substance and isolating matrix—to detect a single species in the pure sample. Structure determination refers to the methods to determine the chemical structure of an isolated pure steroid, using an evolving array of chemical and physical methods which have included NMR and small-molecule crystallography . [2] :10–19 Methods of analysis overlap both of the above areas, emphasizing analytical methods to determining if a steroid is present in a mixture and determining its quantity. [38]

The enzymes and pathways of steroidogenesis are familiar to most endocrinologists, but the biochemistry and molecular biology of these processes are still being studied. This chapter outlines current knowledge about each enzyme. The quantitative regulation of steroidogenesis occurs at the first step, the conversion of cholesterol to pregnenolone. Chronic regulation is principally at the level of transcription of the gene for P450 side chain cleave (P450scc), which is the enzymatically rate-limiting step. Acute regulation is mediated by steroidogenic acute regulatory protein, which facilitates the rapid influx of cholesterol into mitochondria, where P450scc resides. Qualitative regulation, determining the class of steroid produced, is principally determined by P450c17. In the absence of P450c17 in the zona glomerulosa, C21 deoxy steroids are produced, leading to the mineralocorticoid aldosterone. In the presence of the 17alpha-hydroxylase but not the 17,20 lyase activity of P450c17 in the zona fasciculata, C21, 17-hydroxy steroids are produced, leading to the glucocorticoid cortisol. When both the 17alpha-hydroxylase and 17,20 lyase activities of P450c17 are present in the zona reticularis, the androgen precursor dehydroepiandrosterone is produced. The discrimination between 17alpha-hydroxylase and 17,20 lyase activities is regulated by two posttranslational events, the serine phosphorylation of P450c17 and the allosteric action of cytochrome b5, both of which act to optimize the interaction of P450c17 with its obligatory electron donor, P450 oxidoreductase.

The focus on the refinement, reduction and replacement of animal use in toxicity testing requires the development of cell-based systems that mimic the effects of xenobiotics in human tissues. The human adrenocortical carcinoma cell line, H295R, has been proposed as a model for studies on adrenal steroidogenesis and its disruption. In this study, expression profiles for nine adrenal steroidogenic genes were characterized in H295R cells using real-time RT-PCR. Treatment with forskolin increased cortisol secretion and stimulated transcription of all the steroidogenic genes except SULT2A1. The transcript profile from H295R cells in the presence and absence of forskolin was compared with the transcript profile from human adrenal glands. The gene expression pattern observed in the forskolin-treated H295R cells was more similar to that in the human adrenal gland, than the expression pattern in untreated cells. To examine H295R cells as a possible in vitro system for the assessment of adrenal disruption using molecular endpoints, the insecticide lindane (gamma-hexachlorocyclohexane) was used. In vivo, lindane has been shown to inhibit testicular, ovarian and adrenal steroidogenesis. It was demonstrated that lindane reduced cortisol secretion, downregulated the expression of a subset of the genes encoding steroidogenic enzymes and repressed transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene promoter. Thus the H295R cell line provides a good in vitro system for the analysis of the human adrenal steroidogenic pathway at the level of hormone production and gene expression. This in vitro test can be used for the rapid detection of adrenal endocrine disruption and as a tool for mechanistic studies.

Studies have also suggested that BPA interferes with germ cell nest breakdown in animal models. In neonatally exposed lambs, low-dose BPA was reported to increase the incidence of multioocyte follicles ( Rivera et al. 2011 ). Similarly, in gestationally exposed macaques, dietary low-dose BPA exposure increased the number of oocytes present in secondary and antral follicles at birth, and continuous BPA exposure (measured < 1 ng/mL in maternal serum) increased the incidence of unenclosed oocytes ( Hunt et al. 2012 ). Further, in gestationally exposed CD-1 mice, low-dose BPA increased the number of unenclosed oocytes, whereas it decreased the number of primordial follicles in a dose-dependent manner ( Zhang HQ et al. 2012 ). In another study, Veiga-Lopez et al. (2013) reported that prenatal BPA exposure altered the fetal ovarian steroidogenic gene and microRNA expression that mediate gonadal differentiation and folliculogenesis in sheep. Collectively, these studies provide strong evidence that gestational BPA exposure—across multiple exposure routes, doses, and species—impairs proper germ cell nest breakdown, leading to the formation of multioocyte follicles. The presence of multioocyte follicles is of concern because they are considered a pathologic condition that may lead to ovulatory problems ( Iguchi et al. 1990 ).

Steroidogenic pathway

steroidogenic pathway

Studies have also suggested that BPA interferes with germ cell nest breakdown in animal models. In neonatally exposed lambs, low-dose BPA was reported to increase the incidence of multioocyte follicles ( Rivera et al. 2011 ). Similarly, in gestationally exposed macaques, dietary low-dose BPA exposure increased the number of oocytes present in secondary and antral follicles at birth, and continuous BPA exposure (measured < 1 ng/mL in maternal serum) increased the incidence of unenclosed oocytes ( Hunt et al. 2012 ). Further, in gestationally exposed CD-1 mice, low-dose BPA increased the number of unenclosed oocytes, whereas it decreased the number of primordial follicles in a dose-dependent manner ( Zhang HQ et al. 2012 ). In another study, Veiga-Lopez et al. (2013) reported that prenatal BPA exposure altered the fetal ovarian steroidogenic gene and microRNA expression that mediate gonadal differentiation and folliculogenesis in sheep. Collectively, these studies provide strong evidence that gestational BPA exposure—across multiple exposure routes, doses, and species—impairs proper germ cell nest breakdown, leading to the formation of multioocyte follicles. The presence of multioocyte follicles is of concern because they are considered a pathologic condition that may lead to ovulatory problems ( Iguchi et al. 1990 ).

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