**4. Pathophysiological meaning of Sertoli cell phagocytosis**

The sperm production and testosterone synthesis are two major functions of testis. To fulfill these functions, the testis is highly organized, considering its anatomical location, histological structure, and cellular compositions. The testis is constituted by several types of tissue-specific cells. In addition to numerous germ cells, major testicular somatic cell types, including Leydig and Sertoli cells, are crucial for spermatogenesis. Leydig cells, localizing in the interstitial spaces of the testis, synthesize testosterone essential for spermatogenesis and multiple other extratesticular target organs. Sertoli cells embrace developing germ cells and constitute the seminiferous epithelium within the seminiferous tubules where spermatogenesis occurs (**Figure 1**). Sertoli cells are the only type of somatic cells in the seminiferous epithelium and play critical roles in regulating spermatogenesis by building a niche for germ cell development, providing nutrition to germ cells, and removing AGC and RB by phagocytosis. Sertoli cell phagocytosis is the most noticeable. Several consequences of phagocytotic removal of AGC and RB by Sertoli cells have been proposed. Removal of AGC and RB provides appropriate spaces in the seminiferous epithelium for healthy spermatogenesis. AGC can release autoantigens when necrosis occurs, which may induce autoimmune responses. Therefore, timely elimination of AGC before releasing autoantigens prevents autoimmune responses. After phagocytosis of AGC and RB, Sertoli cells recycle these apoptotic components as an energy source. This energy source would be important for Sertoli cells because circulating nutrients barely reach to the seminiferous epithelium due to the BTB and lacking blood vessels.

## **4.1 Space saving**

Based on origin, phagocytes can be classified into professional or nonprofessional phagocytes, respectively [49]. The hematopoietic phagocytes belong to professional and can infiltrate into the infected sites to ingest microbes and clean up damaged cells, which is critical for the innate defense against microbial infection. However, circulating phagocytes cannot migrate into tissues separated by the BTB, where resident tissue-specific phagocytes, which are considered as nonprofessional, are essential for maintaining tissue homeostasis by phagocytic removal of apoptotic substrates. The typical example is the mammalian testis. More than one hundred million sperms are produced each day in men during their whole reproductive age. Since a large number of male germ cells develop simultaneously within the seminiferous epithelium, there is a competition for space and nutrient if all the germ cells would develop into spermatozoa. Therefore, before maturing to sperm, most developing germ cells die through apoptosis, and the remaining spermatids shed most of their cytoplasmic portions as RB. Since the number of germ cells that Sertoli cells can support for finalizing their development is limited, we can speculate that the elimination of AGC and RB by Sertoli cells is important to ensure

enough spaces for a healthy germ cell production and maintain tissue homeostasis. However, this speculation lacks experimental evidence. By contrast, there is a body of substantial evidence that the phagocytic removal of AGC and RB prevents an autoimmune response.

### **4.2 Removal of autoantigens**

Male germ cells, which are mostly developed after the establishment of central immune tolerance, express a large number of novel proteins. These new proteins of male germ cells can be recognized as "foreign antigens" by the immune system. However, male germ cells do not induce an autoimmune response in the male reproductive tracts under physiological conditions due to their special immune microenvironment. The testis is a distinct immunoprivileged organ. Immune privilege represents a special immunological status that exists in several mammalian organs, including the eye, brain, pregnant uterus, and testis, where allografts or/and xenografts can survive without evoking immune rejection [50]. The testis tolerates both alloantigens and immunogenic autoantigens [51]. Various mechanisms are involved in the maintenance of testicular immune privilege [42], in which Sertoli cells play crucial roles.

Sertoli cells modulate testicular immune privilege with different mechanisms (**Figure 5**). The BTB protects the majority of the antigenic germ cells by sequestering autoantigens behind the BTB from immune components in the interstitial spaces. The BTB is formed between adjacent Sertoli cells near the basal membrane of the seminiferous epithelium (**Figure 1**). Several cellular junctions, including tight junction, gap junction, and basal ectoplasmic specialization, are involved in the BTB formation. The BTB divides the seminiferous epithelium to the basal and adluminal compartments [52]. The BTB limits the access of immune contents residing in the interstitial spaces into the adluminal compartment and sequesters the germ cell autoantigens within the adluminal compartments. Therefore, the BTB plays an important role in maintaining immune privilege within the adluminal compartments of the seminiferous epithelium. Although the BTB sequesters the late stage of germ cells in the adluminal compartments, the early stage of germ cells, including preleptotene spermatocytes and spermatogonia that reside outside the BTB, also produces antigenic proteins [53]. Moreover, certain germ cell antigens behind the BTB can egress into the interstitial spaces, and these antigens do not induce an immune response in the testis [54]. These observations suggest that the BTB cannot completely sequester germ cell antigens and should be only partially responsible for testicular immune privilege. In fact, the interstitial spaces outside the BTB also enjoy immunoprivileged status. A dense network, including the tissue structure, local immunosuppressive milieu, and systemic immune tolerance, coordinately regulates the immunoprivileged environment in the testis [42, 55]. In addition to the BTB, Sertoli cells produce various anti-inflammatory factors that regulate the testicular immune microenvironment [56]. Sertoli cells express activin A and activin B [57]. Activin A inhibits the expression of pro-inflammatory cytokines, thereby suppressing the testicular inflammatory responses. TGF-β is also predominantly produced by Sertoli cells in the testis. As an anti-inflammatory factor, TGF-β1 protects islet β-cell grafts after co-transplantation with Sertoli cells [58]. Moreover, Sertoli cells express Fas ligand (FasL) and programmed death ligand-1 (PD-L1), two negative immunoregulatory ligands which are both involved in the maintenance of testicular immune privilege [59, 60].

Phagocytosis is a biological process that regulates immunity [61]. The phagocytic removal of AGC and RB by Sertoli cells is critical for timely elimination

**77**

**Figure 5.**

*Sertoli Cell Phagocytosis: An Essential Event for Spermatogenesis*

of autoantigens that may be released if AGC and RB are broken down. Toll-like receptors (TLRs) belong to a subfamily of pattern recognition receptors that initiate innate immune responses. Several TLRs are expressed in testicular cells and can be activated by their relative ligands [41, 62, 63]. Damaged tissues and necrotic cells may release endogenous TLR ligands, namely, damage-associated molecule patterns (DAMPs), which can induce noninfectious inflammatory response (**Figure 3**, left side). Various DAMPs, including high-mobility group box 1 (HMGB1) and several heat stock proteins (HSPs), have been recognized to activate TLR2 and TLR4 [64, 65]. Notably, HMGB1 and HSPs are abundantly expressed in male germ cells and can be released under stress conditions [66, 67]. Therefore, necrotic germ cells and RB breakdown may release endogenous TLR ligands, thus inducing inflammatory responses. Accordingly, physical trauma and chemical noxae that may damage germ cells are risk factors of chronic testicular orchitis [68]. An impaired removal of AGC leads to autoimmune orchitis [69]. The damaged male germ cells (DMGCs) induce the expression of various inflammatory mediators, including pro-inflammatory factors and chemokines, in Sertoli cells, thereby promoting leukocytes' infiltration to the testis [70]. The DMGC-induced inflammatory cytokine expression and immune cell infiltrations require TLR2 and TLR4 in Sertoli cells. Therefore, timely removal of AGC and RB by Sertoli cells is essential for maintain-

*PSc, primary spermatocyte; SSc, secondary spermatocyte; Rs, round sperm; ES, elongated sperm.*

*Role of Sertoli cells in testicular immune privilege. Sertoli cells produce various anti-inflammatory cytokines, including activin A and TGF-β, which inhibit immune response of dendritic cells (DC) and testicular macrophages (Mφ). Sertoli cells, together with germ cells, also express high level of Fas ligand (FasL) and programmed death ligand 1 (PD-L1) that can inhibit immune response by inducing apoptosis of T lymphocytes. Sg, spermatogonia;* 

ing immune homeostasis in the testis to prevent autoimmune orchitis.

*DOI: http://dx.doi.org/10.5772/intechopen.86808*

*Sertoli Cell Phagocytosis: An Essential Event for Spermatogenesis DOI: http://dx.doi.org/10.5772/intechopen.86808*

### **Figure 5.**

*Male Reproductive Health*

autoimmune response.

crucial roles.

**4.2 Removal of autoantigens**

enough spaces for a healthy germ cell production and maintain tissue homeostasis. However, this speculation lacks experimental evidence. By contrast, there is a body of substantial evidence that the phagocytic removal of AGC and RB prevents an

Male germ cells, which are mostly developed after the establishment of central immune tolerance, express a large number of novel proteins. These new proteins of male germ cells can be recognized as "foreign antigens" by the immune system. However, male germ cells do not induce an autoimmune response in the male reproductive tracts under physiological conditions due to their special immune microenvironment. The testis is a distinct immunoprivileged organ. Immune privilege represents a special immunological status that exists in several mammalian organs, including the eye, brain, pregnant uterus, and testis, where allografts or/and xenografts can survive without evoking immune rejection [50]. The testis tolerates both alloantigens and immunogenic autoantigens [51]. Various mechanisms are involved in the maintenance of testicular immune privilege [42], in which Sertoli cells play

Sertoli cells modulate testicular immune privilege with different mechanisms (**Figure 5**). The BTB protects the majority of the antigenic germ cells by sequestering autoantigens behind the BTB from immune components in the interstitial spaces. The BTB is formed between adjacent Sertoli cells near the basal membrane of the seminiferous epithelium (**Figure 1**). Several cellular junctions, including tight junction, gap junction, and basal ectoplasmic specialization, are involved in the BTB formation. The BTB divides the seminiferous epithelium to the basal and adluminal compartments [52]. The BTB limits the access of immune contents residing in the interstitial spaces into the adluminal compartment and sequesters the germ cell autoantigens within the adluminal compartments. Therefore, the BTB plays an important role in maintaining immune privilege within the adluminal compartments of the seminiferous epithelium. Although the BTB sequesters the late stage of germ cells in the adluminal compartments, the early stage of germ cells, including preleptotene spermatocytes and spermatogonia that reside outside the BTB, also produces antigenic proteins [53]. Moreover, certain germ cell antigens behind the BTB can egress into the interstitial spaces, and these antigens do not induce an immune response in the testis [54]. These observations suggest that the BTB cannot completely sequester germ cell antigens and should be only partially responsible for testicular immune privilege. In fact, the interstitial spaces outside the BTB also enjoy immunoprivileged status. A dense network, including the tissue structure, local immunosuppressive milieu, and systemic immune tolerance, coordinately regulates the immunoprivileged environment in the testis [42, 55]. In addition to the BTB, Sertoli cells produce various anti-inflammatory factors that regulate the testicular immune microenvironment [56]. Sertoli cells express activin A and activin B [57]. Activin A inhibits the expression of pro-inflammatory cytokines, thereby suppressing the testicular inflammatory responses. TGF-β is also predominantly produced by Sertoli cells in the testis. As an anti-inflammatory factor, TGF-β1 protects islet β-cell grafts after co-transplantation with Sertoli cells [58]. Moreover, Sertoli cells express Fas ligand (FasL) and programmed death ligand-1 (PD-L1), two negative immunoregulatory ligands which are both involved

in the maintenance of testicular immune privilege [59, 60].

Phagocytosis is a biological process that regulates immunity [61]. The phagocytic removal of AGC and RB by Sertoli cells is critical for timely elimination

**76**

*Role of Sertoli cells in testicular immune privilege. Sertoli cells produce various anti-inflammatory cytokines, including activin A and TGF-β, which inhibit immune response of dendritic cells (DC) and testicular macrophages (Mφ). Sertoli cells, together with germ cells, also express high level of Fas ligand (FasL) and programmed death ligand 1 (PD-L1) that can inhibit immune response by inducing apoptosis of T lymphocytes. Sg, spermatogonia; PSc, primary spermatocyte; SSc, secondary spermatocyte; Rs, round sperm; ES, elongated sperm.*

of autoantigens that may be released if AGC and RB are broken down. Toll-like receptors (TLRs) belong to a subfamily of pattern recognition receptors that initiate innate immune responses. Several TLRs are expressed in testicular cells and can be activated by their relative ligands [41, 62, 63]. Damaged tissues and necrotic cells may release endogenous TLR ligands, namely, damage-associated molecule patterns (DAMPs), which can induce noninfectious inflammatory response (**Figure 3**, left side). Various DAMPs, including high-mobility group box 1 (HMGB1) and several heat stock proteins (HSPs), have been recognized to activate TLR2 and TLR4 [64, 65]. Notably, HMGB1 and HSPs are abundantly expressed in male germ cells and can be released under stress conditions [66, 67]. Therefore, necrotic germ cells and RB breakdown may release endogenous TLR ligands, thus inducing inflammatory responses. Accordingly, physical trauma and chemical noxae that may damage germ cells are risk factors of chronic testicular orchitis [68]. An impaired removal of AGC leads to autoimmune orchitis [69]. The damaged male germ cells (DMGCs) induce the expression of various inflammatory mediators, including pro-inflammatory factors and chemokines, in Sertoli cells, thereby promoting leukocytes' infiltration to the testis [70]. The DMGC-induced inflammatory cytokine expression and immune cell infiltrations require TLR2 and TLR4 in Sertoli cells. Therefore, timely removal of AGC and RB by Sertoli cells is essential for maintaining immune homeostasis in the testis to prevent autoimmune orchitis.

### **4.3 Providing energy**

Another meaning of male germ cell death and removal of AGC and RB serves as energy sources for Sertoli cells [71]. After phagocytosis by Sertoli cells, AGC and RB fuse with lysosomes. AGC and RB are subsequently broken down and recycled as energy sources for ATP production. The most noticeable phenotype of Sertoli cells is the formation of numerous lipid droplets in the cell cytoplasm. These lipid droplets result from the breakdown of engulfed AGC and RB [13, 72]. Unlike the majority of cell types that mainly use glucose as an energy source, Sertoli cells predominantly use lipids to produce ATP [71]. The lipids from AGC and RB should be the main energy sources for Sertoli cells. Sertoli cells provide essential physical and environmental support for spermatogenesis, which are energy consumers. Corresponding to their function, Sertoli cells exhibit an active energy metabolism and produce high levels of ATP [71, 73]. While lipids and glucoses can be substrates for ATP production within cells under physiological conditions, the majority of cell types use glycogen to produce ATP, whereas lipids serve as energy storage. Only minor cell types, such as adipocytes, myocardial cells, and Sertoli cells, have been confirmed to actively utilize lipids to produce ATP. Why these cell types predominantly use lipids as energy sources remains unclear. However, the active usage of lipids by Sertoli cells is compatible with the special microenvironment in the testis. Sertoli cells are barely reached by the nutrition from the peripheral circulation due to the barriers in the basement membrane, BTB, and the absence of blood capillaries within the seminiferous epithelium. The simplest way for Sertoli cells to have enough energy to support spermatogenesis is to recycle the lipid contents of AGC and RB. Therefore, the phagocytic removal of AGC and RB by Sertoli cells is necessary for Sertoli cells to ensure their functions, which confers a novel meaning for a large number of germ cells to undergo apoptosis during spermatogenesis.
