Morphology and function of human Leydig cells in vitro. Immunocytochemical and radioimmunological analyses

The aim of our study was to show whether the cells isolated from testes of patients underwent bilateral orchiectomy for prostatic cancer are able to grown in vitro, and if so, are functionally active. Immuncytochemistry was performed to show the functional status of human cultured cells. In detail, immunolocalization of luteinizing hormone receptors (LHR), mitochondria, and cytoskeletal elements was demonstrated. Moreover, radioimmunological assay was used to measure testosterone secretion by cultured Leydig cells. Using Nomarski interference contrast and fine immunofluorescence analysis the positive immunostaining for LHR was observed in almost all Leydig cells, however it was of various intensity in individual cells. Testosterone measurement revealed significant difference between testosterone secretion by hCG-stimulated and unstimulated Leydig cells (p<0.05). Moreover, testosterone levels were significantly higher in 24- and 48-hour-cultures than in those of 72 hrs (p<0.05). Morphological analysis of Leydig cells in culture revealed the presence of mononuclear and multinucleate cells. The latter cells occurred in both hCG-stimulated and unstimulated cultures. In Leydig cells labeled with a molecular marker MitoTtracker, an abundance of mitochondria and typical distribution of microtubules and microfilaments were observed irrespective of the number of nuclei within the cell, suggesting no functional differences between mono- and multinucleate human Leydig cells in vitro. Since the percentage of multinucleate cells was similar in both hCG-stimulated and unstimulated cultures (23.70% and 22.80%), respectively, the appearance of these cell population seems to be independent of hormonal stimulation.Key words: human Leydig cells, LH receptors, primary culture, hCG-stimulation, immunocytochemistry, testosterone secretion, multinucleate cells, multicolor staining.It is well established that testosterone biosynthesis depends on the existence of mature Leydig cells in the testicular interstitium. Human Leydig cells arise from mesenchymal cells or fibroblast-like precursor cells through a hormonally regulated differentiation process (Chemes, 1996). Production of testosterone in human and mammalian Leydig cells is dependent on LH stimulation in vivo and on LH/hCG stimulation in in vitro conditions; to respond to hormonal regulation the cells are equipped with functional receptors for LH (Amador and Bartke, 1987; Simpson et al., 1987; Mendis-Handagama et al.,1990; Cooke, 1996; Ramadoss et al., 2006). In man, the Δ⁵-metabolic pathway is the major pathway for the metabolism of pregnenolone to testosterone (Rommerts, 1990). According to Hammar and Petersson (1986) in human testis from young and elderly men with prostatic carcinoma also the 5-ene pathway is preferred. For optimal steroidogenic function a number of neuroendocrine and neuronal markers have been demonstrated in human Leydig cells in vivo by the group of Holstein (Middendorff et al., 1993; 1995). Moreover, production of testosterone in Leydig cells, requires the presence of functionally active enzymes acting within mitochondria and the smooth endoplasmic reticulum (Payne and O’Shaughnessy, 1996; for review see Haider, 2004).Recent studies have shown that Leydig cells become hypofunctional with age. In the rat, aged Leydig cells produce less testosterone than Leydig cells from young adult rats (Luo et al., 1996; for review Zirkin et al., 1997). A detailed characteristics of aged rat Leydig cells in vivo, including reduced testosterone biosynthesis and reduced cell volume has been described by Ewing and Zirkin (1983). Now, there is evidence from in vitro studies that reactive oxygen species can result in the inhibition of testosterone production in mouse Leydig cells by affecting steroidogenic enzymes (Stocco et al. 1993; Peltola et al., 1996; Cao et al., 2004).Considering human samples as a very rare and valuable biological material, the aim of this study was to show whether Leydig cells obtained from testes of elderly patients who underwent orchiecto-my for prostatic cancer are able to grown in vitro, and if so, are functionally active. For this purpose localization of luteinizing hormone receptors (LHR) and visualization of mitochondria and cytoskeletal elements in both hCG-stimulated and unstimulated Leydig cell cultures were performed, as well as testosterone secretion by cultured Leydig cells was measured. It is worth noting that the effect of LH and an involvement of cytoskeletal proteins in steroidogenesis of mouse Leydig cells in vitro have been demonstrated by our own (Bilinska, 1989) and mitochondria have been described as integrally involved in Leydig cell steroidogenesis (Bilinska 1994; Kotula-Balak et al., 2001).