R. Molenaar

[22] Preparation of isolated leydig cells

Publisher Summary This chapter discusses relevant observations on the preparation, purification, and characterization of isolated Leydig cells and explains that different in vitro preparations of Leydig cells have been used for studies on the biochemical mechanisms involved in regulation of steroidogenesis. The chapter also explains that several alternative methods for cell isolation and characterization have been developed and the techniques have been used to isolate Leydig cells from porcine and mouse testes and Leydig cell tumors. A careful investigation and standardization of the methods appears necessary, because for Leydig cells isolated from adult rats, a large variation in the capacity for steroid production has been observed. The recovery of Leydig cells after the isolation procedure is low, and there are indications that extensive cell damage can occur during tissue dispersion. When isolated cells are used for investigations on the biochemical mechanisms involved in the regulation of steroidogenesis, it is essential to work with homogeneous preparations of viable cells and prevent various degrees of functional heterogeneity and other artifacts, which may be caused by the isolation procedure.

Regulation of Steroidogenic Activities in Leydig Cells by LH and an LHRH Agonist

Regulation of steroidogenesis in Leydig cells is the result of interactions between stimulatory and inhibitory processes regulated by protein hormones, peptides and steroid hormones. Investigations of isolated intact Leydig cells can assist in the elucidation of the individual regulating systems, provided that specific steps of the complete process can be measured, inhibited or activated. The mechanism of action of LH on Leydig cells has been studied for many years and much information has been obtained about the initial events. It is now generally accepted that LH stimulation of steroid production involves a concomitant increase in cAMP-dependent protein kinase and phosphorylation of various specific proteins. It is not clear yet if, and which phosphoproteins are important for stimulation of steroid production. Direct activation of mitochondrial cholesterol sidechain cleavage activity (CSCC) by phosphoproteins appears to be unlikely and there are indications that some specific rapidly turning over proteins regulate the CSCC enzyme. The ultimate regulation of CSCC activity by LH depends probably on a sequence of reactions which involves activation of membrane receptors, increased concentrations of second messengers, protein phosphorylation and synthesis of specific proteins. Recently, it has been shown that LHRH and analogues may directly regulate steroid production in gonadal cells and various preliminary observations indicate that the mechanism of action of gonadotrophic hormones and releasing hormones may be different (Hsueh et al., 1981). From a comparison of the effects of LH and LHRH on metabolic activities of Leydig cells, common important regulatory pathways for regulation of steroid production can be discerned. In addition, other regulatory systems may modulate the effects of LH.

REGULATION OF STEROIDOGENESIS IN ISOLATED LEYDIG CELLS

It is well-known that LH plays an important role in the (acute) regulation of steroidogenesis in Leydig cells. In the last years, it has become clear that in addition to LH, hormones such as prolactin and oestradiol, as well as an intratesticular LHRH-like factor, may also play an important role in the (long-term) regulation of the responsiveness of Leydig cells to LH. The ultimate regulation of the production of biologically active steroids in Leydig cells is the result of interactions of many stimulatory and inhibitory processes, influenced by different hormones and/or factors. Investiga- tion of isolated intact cells under defined conditions enables a dissection of individual regulatory systems. It appears, however, that the quality of isolated cells is not always optimal and that different in vitro conditions may greatly influence the steroid production by Leydig cells.