:: Asian Journal of Andrology ::

Regulation of spermatogenesis by paracrine/autocrine testicular factors

Mahmoud Huleihel^{1, 3}, Eitan Lunenfeld^{2,
3}

¹Department of Microbiology and Immunology and BGU Cancer Research Center
²Department of Obstetrics and Gynecology, Soroka University Medical Center
³Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Asian J Androl 2004 Sep; 6: 259-268

Keywords: spermatogenesis; paracrine; autocrine; cytokine; growth factor

Abstract

Spermatogenesis is a complex process regulated by endocrine and testicular paracrine/autocrine factors. Gonadotropins are involved in the regulation of several testicular paracrine factors, mainly of the IL-1 family and testicular hormones. Testicular cytokines and growth factors (such as IL-1, IL-6, TNF, IFN-, LIF and SCF) were shown to affect both the germ cell proliferation and the Leydig and Sertoli cells functions and secretion. Cytokines and growth factors are produced by immune cells and in the interstitial and seminiferous tubular compartments by various testicular cells, including Sertoli, Leydig, peritubular cells, spermatogonia, differentiated spermatogonia and even spermatozoa. Corresponding cytokine and growth factor receptors were demonstrated on some of the testicular cells. These cytokines also control the secretion of the gonadotropins and testosterone in the testis. Under pathological conditions the levels of pro-inflammatory cytokines are increased and negatively affected spermatogenesis. Thus, the expression levels and the mechanisms involved in the regulation of testicular paracrine/autocrine factors should be considered in future therapeutic strategies for male infertility.

1 Spermatogenesis

Spermatogenesis is the process of male germ cell proliferation and differentiation [1]. It starts with a differentiating division of the spermatogonial stem cells and continues with sequential cell divisions of spermatogonia and meiosis of spermatocytes to form round spermatids [1, 2]. Spermiogenesis is the successful transformation of the round spermatids into the complex structure of the spermatozoon [2].

Spermatogonia are classified into subtypes according to the differentiation status. In rodent, such as rats and mice, the spermatogonia are grouped into type A, intermediate and type B spermatogonia. The type A spermatogonia are further subdivided into A0 (undiffe-rentiated) and A1-A4 (differentiating) spermatogonia. The As spermatogonia are generally considered to be the stem cells. It is not yet clear, however, whether other spermatogonia have stem cell activity or whether all As spermatogonia are acting as stem cells [2].

Little is known of the regulatory mechanisms involved in the spermiogenesis process. There is increasing evidence for the involvement of a number of genes, which are expressed by the haploid genome. The genes which encode protamine and the proteins that replace the histones bound to DNA are important since, like many other genes expressed in haploid cells, they are transcribed early in spermiogenesis and the resulting RNA is stored until later in this process for translation into protein [2].

2 Testicular compartments and cellular interactions

In mammals the process of spermatogenesis occurs within seminiferous tubules that release spermatozoa into the rete testis. The seminiferous tubules contain germ cells and Sertoli cells. Peritubular myoid cells surround the tubules and are in contact with the basal surface of the Sertoli cells and spermatogonia. Leydig cells are located in the interstitium of the testis between tubules. Cell-cell interactions between all four types are likely. Recent evidence suggests that the system governing spermatogenesis includes also immune cell types, which work in close concert with the testicular cells, affect each other and are controlled to some extent by each other [3]. Macrophages have been shown to reside in the interstitial compartment of the testis. These testicular macrophages are often physically associated with the Leydig cells. Conditioned medium from testicular macrophage cultures has been shown to stimulate Leydig cell steroidogenesis. Although testicular macrophages will likely have traditional bactericidal and phagocytic activities in the testis, regulatory interactions between testicular macrophages and other testicular cell types, particularly Leydig cells, may also exist [4-6]. Lymphocytes are also present in the testis [4]. The tubules, however, have been shown to be an immunologically protected tissue [3].

Stem cell spermatogonia were recognized as undifferentiated individual spermatogonia present on the basement membrane of the seminiferous tubules until type B spermatogonia divide to produce preleptotene sperma-tocytes. Around 75 % - 90 % of the cells disappear through apoptotic cell death [2]. This may indicate that interactions between cells of seminiferous tubules and interstitial tissue could be involved in the regulation of testicular cell function and spermatogenesis under physiological and pathological conditions.

3 Interactions between hormones and testicular cytokines in the regulation of spermatogenesis

The regulation of spermatogenesis involves both endocrine and paracrine mechanisms [2].

3.1 Regulatory cytokines and hormones

Interleukin (IL)-1 and IL-6 which are produced by Sertoli cells may potentially act as a physiological paracrine factor on lymphocytes and other testicular cells. These cytokines may be also needed to regulate local lymphocyte functions important for immunological protection of the tissue [3, 7]. Other leukocyte products, such as IL-1, IL-2, interferon-gamma (IFN-) and tumor necrosis factor-alpha (TNF-), were shown to regulate (induce/inhibit) Leydig cell steroidogenesis [3, 7-14] and transferrin secretion by Sertoli cells [8, 15]. It has been suggested that IFN may inhibit estradiol secretion by Sertoli cells [12]. IL-6 was shown to affect transferrin secretion by Sertoli cells [8, 15, 16]. Several aspects of testis cell biology that are integrally associated with local cellular interactions involve growth and differentiation.

There seems to be an analogy between hematopoietic and spermatogenic systems. Both systems are responsible for long-term repopulation. Furthermore, in these two systems stem cells are capable of self- renewal, maintaining the supply of lineages, and differentiation to provide a continuous yield of functional cells [3, 7]. Whilst in vitro methods have led to the identification of growth factors/cytokines and their genes in human hematopoietic stem cells, enabling the understanding of stem cell kinetics and differentiation, in vitro studies of postnatal spermatogenic stem cell populations are not yet clear. It is of particular interest to determine whether the same growth factors controlling hematopoietic stem cell cycling have physiological roles later during spermato-genesis.

The testicular interstitium is an immunologically privileged site. An immunosuppressive factor was isolated from rat testis interstitial fluid (ISF) and is thought to play a role in the immune privilege of the testis [3, 17]. An intact blood-testis barrier, functional germ cell Sertoli-cell interaction, and normal testicular steroidogenesis are essential for intact spermatogenesis. Little is known about the involvement of cytokines in spermatogenesis/spermiogenesis.

This may suggest that testicular paracrine/autocrine factors could also involved in the biomolecular process of protection of the testis as immune privileged site.

3.1.1 Paracrine/autocrine factors

Cytokines and growth factors are polypeptides produced by variety of cells of immune and non-immune origin, mainly after stimulation [18]. There is increasing evidence that a multiplicity of growth factors and cytokines are involved in local mechanisms regulating stem cell renewal by mitosis and by implication the process of meiotic cell division [2]. Cytokines have been identified as key factors in this local network [19-21].

Interleukin-1 (IL-1): IL-1 activity has been shown to be present in extracts of rat spermatozoa and in the rete testis fluid [22, 23]. The role of the very high densities of IL-1-binding sites in the epididymis is at present unclear. IL-1 activity detected in extracts of epididymal tissue and epididymal sperm may be the endogenous ligand for these binding sites. IL-1 in the epididymis may serve important biological functions related to the spermiogenic process or to the transit of sperm [24, 25]. IL-1 was found in testicular cells, including Sertoli cells, germ cells in the seminiferous tubules and Leydig cells and macrophages in the interstitial compartment of the testis [26]. IL-1 production by Sertoli cells from immature rats and mice was barely detectable [27-29]. The expression of IL-1, but not IL-1, was shown to be developmentally regulated in rat testis. It was expressed in a stage-dependent pattern during the cycle of the seminiferous epithelium. This expression in Sertoli cells was proposed to be dependent upon interaction with germ cells [23, 30, 31]. Pachytene spermatocytes and early spermatids had no effect on IL-1 production; residual bodies/cytoplasts from elongated spermatids dramatically stimulated IL-1 production of Sertoli cells [27]. Recently, we have shown that Sertoli cells produce IL-1ra, which was increased after stimulation with IL-1 and LPS [32]. Also, germ cells were shown to produce both IL-1 and IL-1ra but not IL-1 [33]. We have shown that human spermatozoa produce and secrete bioactive IL-1 [34-36]. Moreover, there is evidence confirming the involvement of IL-1 in testicular control of spermatogenesis [37]. In the interstitial compartment of the testis, IL-1 has been shown to affect immature Leydig cell proliferation and adult Leydig cell functions and to induce acute inflammation-like changes in testicular microcirculation [38-40]. IL-1 is a potent inhibitor of Leydig cell function, IL-1 blocks human CG-induced cAMP and testosterone formation as well as cytochrome p450 side-chain cleavage messenger RNA expression in Leydig cells, and this may contribute to the inhibitory effects of IL-1 on Leydig cell steroidogenesis [41]. Furthermore, within the seminiferous tubules, IL-1 is able to modulate Sertoli cell functions. IL-1 has been shown to affect various testicular functions such as stimulation of germ cells, inhibition of FSH-induced aromatase activity in immature Sertoli cells, and inhibition of the gene expression of IGF-I in Leydig cells [31, 42, 43].

IL-1 was shown to promote DNA synthesis and differentiation of, spermatogonia and preleptotene spermatocytes [31, 44]. IL-1 may play a critical role in postmeiotic germ cell development through the control of energy (glucose) metabolism [45]. IL-1 was also able to elicit a transient but significant increase in Sertoli cell sertolin expression. Sertolin expression was also shown to increase with testicular development and is likely to be associated with the onset of spermatogenesis [46].

RT-PCR analysis has shown the presence of both types of IL-1 receptor (type I/II) mRNAs in isolated rat, mouse, and human somatic testicular cells (macrophages, Leydig cells, Sertoli cells and peritubular cells). While also present in rat and mouse isolated pachytene spermatocytes and early spermatids, the only germ cell types that were found not to express IL-1RI mRNA were the elongating spermatids [47].

Leukemia inhibitory factor (LIF): LIF is a multifunctional polypeptide cytokine/growth factor, which belongs to the IL-6 family. It exists in both soluble and matrix-bound forms and displays biological activities ranging from the differentiation of myeloid leukemia cells into macrophage lineage, to affect bone metabolism, inflam-mation, neuronal development, embryogenesis and the maintenance of implantation [5, 48]. LIF is rarely observed under normal conditions. Immune stimulation was shown to induce its expression in different organs, body fluids and cells such as lung fibroblasts, epithelial cells and muscle cells and mesangial cells [48]. LIF has been shown to promote the proliferation of murine primordial germ cells (PGC) [49] and their survival by preventing their apoptosis [50]. It was suggested that LIF may be involved in the regulation of fetal stem germ cells [49-53] and Sertoli cells [54]. Peritubular cells were the main testicular cells that produce LIF. This suggests that LIF may be a paracrine regulator of both testicular compartments. In addition, hCG was found to stimulate Leydig cell LIF production [55].

Stem cell factor (SCF): In normal tissues, SCF and its receptor (c-kit) are expressed in mast cells, melanocytes, testis and in bone marrow in the progenitor (CFU-c) compartment, and in non-hematopoietic cells such as vascular endothelial cells, interstitial cells, astrocytes, renal tubules, and epithelial cells. SCF and c-kit play an important role in spermatogonial development. Mutations in the gene encoding either SCF or c-kit result in infertility due to defective migration, proliferation and survival of primordial spermatogonia. Disruption of SCF action prevents spermatogonia from undergoing mitosis[56, 57]. The presence of Sertoli cells producing membrane-bound SCF and germ cells expressing the functional c-kit receptor leads to qualitatively normal repopu-lation of the testes with developing germ cells. All differentiating types of spermatogonia were shown to express c-kit in contrast to the undifferentiated spermatogonia [56, 57].

3.1.2 Expression of receptors for paracrine/autocrine factors in the testis

Several growth factors that involved in the regulation of testicular functions were shown to be expressed only in one cell type of each testicular compartment. i.e., in seminiferous tubule (ST) or interstitial tissue (IT). In the mouse testis, TNFaR55 was reported to be expressed in Leydig and Sertoli cells but not in germ cells [58, 59]. LIF-R is expressed by Leydig and germ cells [54]. TNF R55 and LIF-R are receptors regulating differentiated functions of testicular cells [60] and LIF modulates germ cell proliferation [61]. Lactate dehydrogenase C, an enzyme of glucose metabolism, is exclusively present in postmeiotic germ cells [62]. The distribution of TGF-beta was demonstrated in human testicular cells [63]. TGF RI was detected in the adult rat testis, in Leydig cells and premeiotic germ cells. In the adult porcine model, Sertoli cells only weakly expressed TGF RI [64, 65].

These data emphasize the key role and the cellular origin of the various cytokines and their receptors in the testicular cells. These cytokines are expressed under physiological conditions. In addition, their levels are altered in pathological conditions. Thus, the levels of these autocrine/paracrine factors should be considered in male infertility.

3.1.3 Paracrine/autocrine factors and seminiferous function; Sertoli cell - germ cell interactions

Sertoli cells are considered to create a highly specialized environment in which spermatogenesis proceeds. Only the base of Sertoli and spermatogonia are in contact with the basement membrane of the tubule [2].

Sertoli cells limit the expansion of the spermatogonial population. As the number of Sertoli cells present determines testicular size, it is assumed that each Sertoli cell supports a defined number of germ cells and therefore spermatogonia [66]. Throughout its progression from spermatogonia to meiosis and spermiogenesis, a germ cell remains in intimate contact with a Sertoli cell. Many morphological and functional changes that germ cell undergo during spermatogenesis are controlled by Sertoli cells [66]. It is clear that many of the interactions between the Sertoli cell and the germ cells at each maturation stage, are of a paracrine nature [2]. These include: the maintenance of mitochondrial morphology in spermatocytes [2]; germ cells are dependent on the production of lactate by Sertoli cells [2]; the transport of iron within the intratubular environment, which is dependent on Sertoli transferrin production. In turn, the capacity of the germ cells to modulate Sertoli cell activity is well established [2]. Androgen binding protein [67], plasminogen activator [68], and FSH-dependent cAMP production by Sertoli cells have been shown to be affected by germ cells [69]. This is to emphasize that not only Sertoli cell number, but also the functional Sertoli cells are crucial for the successful development and completion of spermatogenesis [2]. The differentiated Sertoli cell line, 15P-1 cells express a series of Sertoli-specific genes and in coculture with male germ cells form multicellular complexes which support the progression of pachytene spermatocytes to the haploid state [70, 71].

Among the Sertoli cell products are binding and transport proteins [72], extracellular matrix and functional proteins [73] proteases and proteases inhibitors [74], growth factors [75, 76], and energy substrates such as lactate [77]. Lactate production in Sertoli cells has been shown to be predominantly under the control of the endocrine system including FSH, insulin [78], and IGF-I [79, 4]. Recently, it was shown that IL-1 (produced in germ cells and other testicular cells) stimulates lactate and sertolin production in Sertoli cells [45]. Also, it has been reported that IL-1 was able to inhibit the secretion of both protein-2 (CP-2) and transferrin by Sertoli cells [80], whereas IL-6, IL-2 and TNF- were able to increase transferrin secretion by Sertoli cells [8]. Neither IFN- nor bFGF, both of which are germ cell-derived cytokines, were able to influence Sertoli cell sertolin expression in vitro. Thus, cytokines could be good candidates for involvement in the local control exerted between germ and Sertoli cell activity [45, 46].

The above data may indicate that the processes of spermatogenesis in the seminiferous tubules are under the regulation of both cell types - Sertoli cells and germ cells (by physical interaction and secretion of biological factors). In addition, these interactions could be also affected by endocrine factors and paracrine factors from the interstitial compartment.

3.2 Endocrine factors

The endocrine stimulation of spermatogenesis involves both follicle stimulating hormone (FSH) and leutinizing hormone (LH); the later acting through the testosterone (T), produced by the Leydig cells in the testis. Since the germ cells do not possess receptors for FSH and testoste-rone, the hormonal signals are transduced through the Sertoli cells and peritubular cells by the production of signals that have yet to be defined [2].

In rat, FSH may modulate the number of germ cells proceeding successfully through the mitotic and meiotic phase of spermatogenesis. Also, it was suggested that FSH may play a role in stimulating mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes as well as in preventing apoptosis of pachytene spermatocytes and round spermatids [2].

It has been shown that, in the absence of T, there is a loss of round spermatids between steps 7-8 of sper-matogenesis. This loss was suggested to be due to failure of Sertoli cells to produce the adhesion molecule N-cadherin, the production of which appears to require both FSH and T. T also impacts on normal spermatogonial mitosis and the successful completion of meiosis [2]. It was also shown that the requirement of T for meiosis may be even more crucial than its requirement for spermiogenesis. Recently, it was shown that human germ cells, from testicular biopsy of men with germ cell maturation arrest, could undergo rapid trans-meiotic and post-meiotic differentiation when cultured in vitro in media supplemented with high concentrations of FSH and testosterone [81, 82]. It was suggested that the main effects of T were related to the improvement of Sertoli cell survival in culture with the inhibition of the apoptotic pathway leading to DNA fragmentation [2, 81-83].

Stimulation of Sertoli cells by FSH had no effect on IL-1 production, but it increased the levels of produced IL-1ra [30, 84]. It was shown that IL-1, but not IL-1, exert a potent effect on gonadotropin action in rat Leydig cells. By using cultured porcine Leydig cells as a model, it was shown that IL-1, in contrary to previous reports, is a potent inhibitor of LH/hCG steroidogenic action. IL-1 inhibited hCG-induced testosterone secretion [85].

FSH was shown to elicit soluble and membrane-associated plasminogen activator activities, in granulose cells. The effect is inhibited by IL-1. Also, progesterone produced by FSH-stimulated granulosa cells is regulated by IL-1. The effect of IL-1 on FSH-induced estradiol production by granulosa cells was shown to be follicle size dependent [86].

In the human osteoblast HOBIT cell model testosterone increased IL-1 protein release [87]. Testosterone was shown to inhibit IL-1 secretion from peripheral blood mononuclear cells (PBMCs) from healthy male donors [88].

The endocrine and paracrine/autocrine factors in the testis are regulated by each other. Thus, the therapeutic strategies for male infertility should consider not only the endocrine factors, but also testicular paracrine/autocrine factors.

4 Involvement of pro-inflammatory cytokines in testicular pathology

Recently, increased levels of IL-1 and TNF- were demonstrated after ischemia/reperfusion of the testis [89]. This process leads to a permanent loss of spermatogenesis [90] which was related to germ cell apoptosis [91,92]. In addition, it was suggested that TNF- is involved in experimental autoimmune orchitis (EAO) [93], and TNFR I positive germ cells during EAO were demonstrated [94]. In human pathological biopsies exhibiting inflammation, the number of testicular macrophages and their TNF levels were increased [95].

In summary, spermatogenesis is a process which is regulated by endocrine and autocrine/paracrine factors. The endocrine factors affect testicular cells from the different compartments. Autocrine and/or paracrine factors affect testicular functions and also regulate the secreted levels of endocrine factors (Figure 1). The autocrine/paracrine factors produced by the testicular cells could affect each other directly or indirectly (Figure 2). Under pathological conditions, such as systemic or testicular infection/inflammation, the levels of pro-inflammatory cytokines in the testicular tissue could be increased and thus to disrupt the balance and normal levels of these cytokines in the testis and also the levels of the secreted endocrine factors (Figure 3). This disruption may affect testicular function and thus affect male fertility.

Figure 1. Endocrine factors: gonadotrophin releasing hormone (GnRH) which released from the hypothalamus affects the releasing of leutinizing hormone (LH) and follicular stimulating hormone (FSH) from the hypophysis. LH induces Leydig cells to produce testosterone (Test) which under high levels negatively affect LH production (through the hypothalamus or hypophysis). On the other hand FSH affects Sertoli cells in the seminiferous tubules to produce various autocrine/paracrine factors such as transferrin, androgen- binding protein, cytokines and inhibin (Inhib). Under high levels, these factors (mainly inhibin) negatively affect FSH production through the hypophysis. In addition, LH and FSH could affect, directly or indirectly, the production of various paracrine/autocrine factors from testicular cells of the interstitial and seminiferous tubule cells.

Figure 2. Testicular cells from the interstitial compartment (mainly Leydig cells and macrophages) produce various paracrine/autocrine factors under physiological and pathological conditions. The functions of these cells are affected by the factors produce by these cells. In addition, these factors affect peritubular cells and also the seminiferous tubule cells (Sertoli cells, germ cells and other differentiated germ cells and also mature spermatozoa) directly or indirectly. On the other hand, seminiferous tubule cells affect each other by cell-cell interaction through the autocrine/paracrine factors produce by each cell. These cells could also affect the peritubular cell functions and also the functions of the interstitial cells (mainly Leydig cells and macrophages). Thus, autocrine/paracrine factors produced by testicular cells could affect the functions of each other and also to regulate the levels of LH and FSH released by the hypophysis.

Figure 3. Under pathological conditions, such as systemic or testicular infection/inflammation, the systemic and local (in the testis) levels of pro-inflammatory cytokines (TNF, IL-1, IL-6, IFN- and others) will be increased. The elevated levels of these cytokines will negatively affect the endocrine glands (such as hypothalamus and pituitary) and thus to decrease the hormones released by these glands (mainly GnRH, LH and FSH) which regulate the spermatogenic process. In addition, Pro-inflammatory cytokines could directly affect the cells of the testis, and thus to decrease the spermatogenesis. On the other hand, these pro-inflammatory cytokines could directly affect differentiated spermatogonia and thus to affect the spermatogenesis. High levels of pro-inflammatory cytokines could directly affect spermatozoa (in the testis, epididymis or in the semen) and thus to affect its functions or viability.

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Correspondence to: Mahmoud Huleihel, Ph.D., Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion-University of the Negev, Beer-Sheva, Israel.
Tel/Fax: +972-8-647 9959
Email: huleihel@bgumail.bgu.ac.il
Received 2003-12-03 Accepted 2004-05-17