Locally produced factors

Leydig cells in the testis are surrounded by other cells belonging either to the seminiferous tubules, such as Sertoli cells, or by cells in the interstitial tissue, such as macrophages. Many observations indicate that these neighbouring cells can potentially influence the function of Leydig cells in a paracrine fashion. FSH stimulates development of Leydig cells, probably via Sertoli cell products. Disturbances in the spermatogenic epithelium also affect Leydig cells. Moreover, conditioned media from Sertoli cells or seminiferous tubules can modify the steroidogenic activities of Leydig cells, suggesting the presence of many stimulatory and inhibitory components (reviewed bySharpe 1993; Saez 1989 and 1994;Gnessi etal. 1997). Although the existence of paracrine regulating systems can be inferred from these data, in the literature there is almost no consistency in the various reports. In most studies the results appear to depend chiefly on the species used, the techniques applied for the isolation of cells or secretion products, cell culture conditions, etc. Even when one batch of secreted Sertoli cell products was used to regulate steroid production in one standardised Leydig cell preparation, it was found that the short-term effects of the Sertoli cell products were stimulatory, whereas the long-term effects were strongly inhibitory (van Haren et al. 1995). These long-term inhibitory effects of Sertoli cell products in vitro are in sharp contrast with the long-term stimulatory effects that Sertoli cells exert on Leydig cells in vivo. In a recent study using knock out mice, it was again shown that Sertoli cells are important, but the data also illustrated that the constitutive activity of the receptor is sufficient to stimulate this process. The presence of active FSH seems not to be required (Baker etal. 2003). In a limited number of studies specific (recombinant) growth factors have been used, but this has not resolved the existing confusion. Another question is whether specific products, which are active when added to isolated Leydig cells in a chemically defined medium, are also effective in vivo when they act together with many other local products. in vitro experiments have already demonstrated that the activity of a certain compound can be inhibitory or stimulatory, depending on previous exposure or permanent exposure to other signal molecules. The action of growth factors is thus context-dependent (Sporn and Roberts 1988) or in other words, it can be a part of a cellular signalling language with individual growth factors functioning as the letters of the alphabet. In a similar fashion, as letters in the alphabet can form words with a particular meaning, a certain combination of growth factors may give a (more) useful message to the cell than the isolated growth factors. It will not be difficult to understand that this cellular language can become complex when many signal molecules are used.

There is now agreement that individual local secretion products can only be considered as potential paracrine factors if four criteria are fulfilled: 1) the molecule should regulate at least one biological activity of the target cell; 2) the molecule must be secreted in adequate quantities to guarantee a physiological response; 3) regulation of secretion of the molecule must be possible; 4) changes in the local concentration of the molecule should influence the properties of the target cell in vivo. Since it is almost impossible to fulfil all these criteria for one particular compound, this section on the physiological relevance of local regulation could be very short. However, since local regulatory systems are in general very important for specific cell function and because so much research effort has been made to understand these systems, a brief summary of the past and the present of paracrine regulation will be given before some conclusions are made.

The appreciation of local regulation and the shift from endocrine research to paracrine/autocrine research started approx. 25 years ago when it was shown that FSH could stimulate Leydig cells in hypophysectomized rats (Odell et al. 1973). A second wave occurred when LHRH or LHRH-like molecules became available and when direct effects on Leydig cells could be shown (Hsueh and Jones 1981). This period of initial great excitement was followed by a period of disappointment when it was not possible to show significant secretion of endogenous "LHRH-like" compounds by testicular cells. In the following years results became available from many studies using growth factors which are known to be produced in the testis such as IGF-1, TGFp, EGF/TGFa, FGF, PDGF, inhibin/activin, interleukin, TNFa, etc. (reviewed by Saez 1994 and Gnessi et al. 1997). In more than hundreds of publications many inhibitory and stimulatory effects of these compounds could be shown on steroidogenesis in vitro. Again, much was speculated about the biological relevance of these findings for the regulation of Leydig cells in the testis. However, when the results of all these investigations are taken together, it is still not clear how important these molecules, alone or together, are for the regulation of Leydig cells in vivo. When using information obtained from in vitro experiments, to explain Leydig cell functioning in situ, two typical aspects of Leydig cells in vivo should not be forgotten. Firstly, Leydig cells in vivo are surrounded by an interstitial fluid that will average out specific paracrine influences of neighbouring cells. Secondly, the Leydig cells are a part of a closed feedback system with the brain and the pituitary as the sensor and regulator of LH secretion. Local testicular influences that result

Pituitary

Leydig ten

Other testis

+ cells

Testosterone

Fig. 1.5 Regulation of Leydig cell steroidogenesis by LH and locally produced factors.

in activation or inhibition of steroid production will therefore be compensated by alterations in LH secretion (see Figure 1.5).

The problems connected with understanding the physiological role of paracrine factors in general will be illustrated with the possible paracrine role of IGF-1 for Leydig cell steroidogenesis (for a review of the experimental findings related to this subject, see Lin 1996) because all the known properties make this molecule a most promising paracrine regulator. IGF-1 is produced locally in many tissues and in combination with the six different IGF binding proteins, the IFG/IGF-BP system has been proposed as a super-system for fine tuning of local hormone action. Since IGF-1, IGF binding proteins and specific proteases can be produced by the target cells themselves as well as by neighbouring cells, autocrine and paracrine regulatory systems can be integrated (Collett-Solberg and Cohen 1996). Many in vitro studies have shown that this IGF-1 system can also influence Leydig cells. IGF-1 can enhance the stimulatory effects of LH/hCG on Leydig cells through specific IGF-1 receptors and IGF-1 and also some IGF binding proteins can be produced by Leydig cells themselves. One could postulate therefore that part of the stimulatory action of LH on steroid production is mediated by an external and obligatory IGF-1 loop (production of IGF-1 by the Leydig cell and immediate stimulation of the IGF-1 receptors on the Leydig cell). Other testicular cells that can secrete compounds into the interstitial fluid can amplify or inhibit this external loop by either increasing IGF-1 concentration or by decreasing IGF-1 through binding to the specific binding proteins. Thus in theory the Leydig cell appears to be surrounded by a network of regulatory molecules with IGF-1 as co-stimulator and fine tuner of LH and the binding proteins as fine-tuners of IGF-1 action. Many in vitro data support this model, but van Haren et al. 1992 could not show any effect of IGF-1 on the induction of cholesterol side chain cleavage P450 enzyme activity by LH in cultured Leydig cells. On the other hand, actions of IGF-II are obligatory for the FSH induction of aromatase in human follicles (Yan and Giudice 1999). The completely different effects of IGFs (no effect versus obligatory role) illustrate that in vitro investigations cannot answer the question on the physiological importance of the IGF system for induction of steroidogenic enzymes in gonadal cells. Transgenic animals with specific genes knocked out could shed new light on this problem. In this connection Baker etal. 1996 showed that mutant male mice with an inactive IGF-1 gene were reduced in size and were infertile. However, a close inspection showed that although the size of the testis was approx 40% of the normal and the total (not the free levels) peripheral testosterone levels were approx 20% of normal values, sperm cells of these mutant mice were able to fertilize wild type oocytes. Moreover, the infertility of the males was caused by the absence of mating behaviour. These results show that in the complete absence of the IGF-1 system indeed the normal growth of the animal is disturbed, but the data also show that the Leydig cells are still functional and that the testis, although reduced in size, can still produce active spermatozoa. It appears from this study as well as from many other studies using knock out animals, that the importance of a particular gene product (such as IGF-1) cannot be answered with a firm yes or no. It is very likely, as is the experience from many other studies with knock out animals for growth factors and regulating molecules, that the function of one defective gene can be compensated by other components of the external cellular regulatory network. It is becoming increasingly understood that physiological regulation of cell function is more than regulation of a simple linear process. Cell regulation involves many regulatory systems, and adaptive epigenetic networks and redundancy are nowwell known features of the cellular regulatory systems that operate (Strohman 1993). Although we know that a complex epigenetic regulatory network exists we are still far away from understanding this extensive signalling system (Dumont etal. 2001). Answering the question of the importance of the IGF-1 system for the regulation of steroid production in a normal organism is only possible when the amount of IGF-1 can be increased or decreased near the Leydig cells within the testis after normal development of the animal. This can be accomplished with animals in which genes in specific cell types can be manipulated conditionally (conditional knock out). Such experimental animals are now being investigated, but so far the focus has not been on Leydig cells.

Altogether, regulation of steroid production by LH not only involves complex interactions of many transcription factors for regulating gene expression in the nucleus and other regulatory pathways in the extra-nuclear compartment of the cell, but a similar level of complexity of local regulating molecules exists outside the Leydig cell. Although this extra-cellular local regulatory system is complex, it seems to be very flexible and adaptive as shown by many gene knock out experiments. In contrast to the flexibility of local regulatory systems, LH receptor activation is always required for proper cell function. This has clearly been illustrated by the abnormalities caused by activating and inactivating mutations in the LH receptor gene, as discussed previously. LH thus appears to be high in the hierarchy of regulating molecules. This is in accordance with its role as an endocrine regulator. Another argument for a subordinate role of the paracrine system is that for regulation of steroidogenesis in testicular Leydig cells there are no reports that the local regulatory network can modify the properties of the Leydig cell to such an extent that maintenance of peripheral testosterone levels requires abnormally high or low levels of LH. An imbalance between LH and testosterone levels, however, can occur when genetic defects in steroidogenic enzymes or in the LH receptor cause insufficient production of testosterone. Under these conditions the feedback system tries to compensate for this deficiency with high levels of LH. In light of the many problems that still exist in our understanding of the paracrine control of rodent Leydig cells, the mechanisms involved in paracrine regulation of human Leydig cells remain totally obscure. On the other hand, when all the information discussed above is taken together, it is not difficult to conclude that the main regulator for the steroid production of the human Leydig cells is still LH. A recent clinical study on the effect of human recombinant FSH supports this view, because it was concluded that "Sertoli cell paracrine factors do not seem to play a major physiologic role in man when LH is active" (Young etal. 2000).

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