The androgen testosterone is essential for the Sertoli cell to support the maturation of male germ cells and the production of spermatozoa (spermatogenesis). In the classical view of androgen action, binding of androgen to the intracellular androgen receptor (AR) produces a conformational change in AR such that the receptor-steroid complex has high affinity for specific DNA regulatory elements and is able to stimulate gene transcription. Here, we demonstrate that testosterone can act by means of an alternative, rapid, and sustainable mechanism in Sertoli cells that is independent of AR-DNA interactions. Specifically, the addition of physiological levels of testosterone to Sertoli cells stimulates the mitogen-activated protein kinase signaling pathway and causes phosphorylation of the cAMP response element binding protein transcription factor on serine 133, a modification known to be required for Sertoli cells to support spermatogenesis. Androgenmediated activation of mitogen-activated protein kinase and cAMP response element binding protein occurs within 1 min, extends for at least 12 h and requires AR. Furthermore, androgen induces endogenous cAMP response element binding protein-mediated transcription in Sertoli cells. These newly identified mechanisms of androgen action in Sertoli cells suggest new targets for developing male contraceptive agents.testis ͉ spermatogenesis ͉ nongenomic ͉ signal transduction A ndrogen actions are critical in the testis for the maturation of male germ cells into spermatozoa (spermatogenesis). In the absence of relatively high levels of testosterone (Ͼ70 nM) in the testis, spermatogenesis is halted before the completion of meiosis so that few if any spermatozoa are produced (1, 2). The molecular mechanisms by which testosterone regulates spermatogenesis are not well understood, but like other steroid hormones, androgens exert many of their actions by diffusing into target cells and binding specific intracellular receptor proteins that are located in the cytoplasm and the nucleus (3). The importance of the androgen receptor (AR) for maintaining spermatogenesis is confirmed by mutations that eliminate AR activity. These mutations result in the testicular feminization phenotype (tfm) and the absence of mature male germ cells (4). In the classical view of androgen action, binding of androgen to its receptor produces a conformational change in AR such that the receptor-steroid complex has high affinity for specific DNA regulatory elements and is able to stimulate gene transcription (5). The entire process required to initiate gene expression by means of this classical mechanism takes at least 30-45 min (6, 7), and the length of time required to produce significant levels of nascent proteins is in the order of hours.

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Patterning of cells is critical to the formation and function of the normal organ, and it appears to be dependent upon internal and external signals. Additionally, the formation of most tissues requires the interaction of several cell types. Indeed, both extracellular matrix (ECM) components and cellular components are necessary for three-dimensional (3-D) tissue formation in vitro. Using 3-D cultures we demonstrate that ECM arranged in an aligned fashion is necessary for the rod-shaped phenotype of the myocyte, and once this pattern is established, the myocytes were responsible for the alignment of any subsequent cell layers. This is analogous to the in vivo pattern that is observed, where there appears to be minimal ECM signaling, rather formation of multicellular patterns is dependent upon cell-cell interactions. Our 3-D culture of myocytes and fibroblasts is significant in that it models in vivo organization of cardiac tissue and can be used to investigate interactions between fibroblasts and myocytes. Furthermore, we used rotational cultures to examine cellular interactions. Using these systems, we demonstrate that specific connexins and cadherins are critical for cell-cell interactions. The data presented here document the feasibility of using these systems to investigate cellular interactions during normal growth and injury.

Aims Cardiovascular disease is the leading cause of death for individuals diagnosed with type II diabetes mellitus (DM). Changes in cardiac function, left ventricular wall thickness and fibrosis have all been described in patients and animal models of diabetes; however, the factors mediating increased matrix deposition remain unclear. The goal of this study was to evaluate whether cardiac fibroblast function is altered in a rat model of type II DM. Main methods Cardiac fibroblasts were isolated from 14 week old Zucker diabetic and lean control (LC) adult male rat hearts. Fibroblasts were examined for their ability to remodel 3-dimensional collagen matrices, their adhesion, migration and proliferation on collagen and changes in gene expression associated with collagen remodeling. Key findings Cardiac fibroblasts from diabetic animals demonstrated significantly greater ability to contract 3-dimensional collagen matrices compared to cardiac fibroblasts from LC animals. The enhanced contractile behavior was associated with an increase in diabetic fibroblast proliferation and elevated expression of α-smooth muscle actin and type I collagen, suggesting the transformation of diabetic fibroblasts into a myofibroblast phenotype. Significance Cardiac fibrosis is a common complication in diabetic cardiomyopathy which may contribute to the observed cardiac dysfunction associated with this disease. Identifying and understanding the changes in fibroblast behavior which contribute to the increased deposition of collagen and other matrix proteins may provide novel therapeutic targets for reducing the devastating effects of diabetes on the heart.