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The successful isolation and culture of Sertoli cells depend on a series of delicate processes of mechanical isolation and enzymatic digestion of the testicular tissue, taking advantage of an array of enzymes (such as DNAse, collagenase, and pancreatin) in order to digest the extracellular matrix components. The complexity of these processes may present some differences depending on the origin of the testicular sample (whole tissue or biopsy) and of the species in question. Rat and mouse Sertoli cells are obtained by a similar protocol, whereas bovine and human Sertoli cells require a more extensive mechanical and enzymatic processing.

Nuclear magnetic resonance (NMR)-based metabolomics is widely used in the research of metabolic conditions of complex biological systems under various conditions, and its use has been found in the field of male fertility. Here we describe the implementation of total and targeted NMR-based metabolomics in the research on Sertoli cell metabolism. Main principles and techniques of cell medium, cellular extracts, and intact cells are explained, as well as some classical experiments that can give complementary information on the Sertoli cell metabolism.

The purity of the primary cultures of Sertoli cells is a factor for the validation of the studies using this methodological approach. There is a probability of contamination of these cultures with other testicular cellular types, such as peritubular cells and germ cells (that represent a large percentage of the volume of the seminiferous tubules).For the evaluation of the purity of cultures, the immunocytochemistry technique (immunoperoxidase or immunofluorescence) is frequently used to label a protein marker specific for the Sertoli cells within the testicular environment. The expression of several protein markers can be used, with different particularities, being that vimentin is often used as a marker for Sertoli cells. Vimentin is expressed independently of the differentiation state of the cells and is observed around the nuclei and in the cytoplasm of Sertoli cells.

Movement of water into and out of the cell is fundamental to life. In the male reproductive tract, at the level of the seminiferous epithelium, Sertoli cells are pivotal in mediating the movement of water into the luminal compartment, a process providing a means of transport of spermatozoa into the epididymal ducts. Here, we describe a stopped-flow light-scattering methodology to easily assess the Sertoli cell osmotic water permeability. The devised methodology is well suited for studying how the Sertoli cell's permeability changes following exogenous stimuli.

Sertoli cells play a central role in spermatogenesis. They maintain the blood-testis barrier, an essential feature of seminiferous tubules which creates the proper environment for the occurrence of the spermatogenesis. However, this confinement renders germ cells almost exclusively dependent on Sertoli cells' nursing function and support. Throughout spermatogenesis, differentiating sperm cells become more specialized, and their biochemical machinery is insufficient to meet their metabolic demands. Although the needs are not the same at all differentiation stages, Sertoli cells are able to satisfy their needs. In order to maintain the seminiferous tubule energetic homeostasis, Sertoli cells react in response to several metabolic stimuli, through signaling cascades. The AMP-activated kinase, sensitive to the global energetic status; the hypoxia-inducible factors, sensitive to oxygen concentration; and the peroxisome proliferator-activated receptors, sensitive to fatty acid availability, are pathways already described in Sertoli cells. These cells' metabolism also reflects the whole-body metabolic dynamics. Metabolic diseases, including obesity and type II diabetes mellitus, induce changes that, both directly and indirectly, affect Sertoli cell function and, ultimately, (dys)function in male reproductive health. Insulin resistance, increased estrogen synthesis, vascular disease, and pubic fat accumulation are examples of metabolic-related conditions that affect male fertility potential. On the other hand, malnutrition can also induce negative effects on male sexual function. In this chapter, we review the molecular mechanisms associated with the nutritional state and male sexual (dys)function and the central role played by the Sertoli cells.