**3.2 Role of TAM receptors in mediating Sertoli cell phagocytosis**

TAM receptors belong to a subfamily of the transmembrane receptor tyrosine kinases (**Figure 4**), which include three members, Tyro3, Axl, and Mer [22]. Gas6 is a functional common ligand of TAM receptors [23]. The TAM/Gas6 system regulates cell survival, innate immune response, and phagocytosis of apoptotic cells [24–27]. TAM receptors are involved in several pathological conditions, such as chronic inflammatory and autoimmune diseases [28, 29], viral infection [30–32], and cancer [33–35]. Notably, TAM receptors are essential for spermatogenesis and male fertility [36, 37].

The mechanisms by which the TAM/Gas6 system regulates testicular functions have been intensively investigated [38]. TAM receptors and Gas6 are abundantly expressed in Sertoli and Leydig cells [39]. All three Tyro3, Axl, and Mer receptors are expressed in Sertoli cells, whereas Leydig cells express Axl and Mer. Gas6 is uniquely expressed in Leydig cells. TAM receptors negatively regulate the expression of pro-inflammatory cytokines in both Sertoli and Leydig cells [40, 41], which might contribute to the immunoprivileged status of the testis [42]. In particular, the TAM receptors and Gas6 are essential for the phagocytic removal of AGC by Sertoli cells [43]. TAM receptors cooperatively regulate Sertoli cell phagocytosis of AGC. All three Tyro3, Axl, and Mer receptors participate in recognizing and binding AGC to Sertoli cells, whereas Mer is responsible for triggering phagocytic intracellular signaling that promotes engulfment of AGC. Any individual TAM receptors in Sertoli cells exhibit similar binding ability to AGC. However, Sertoli cells lacking all three TAM receptors remarkably decrease the binding between Sertoli cells and AGC. The TAM-mediated binding of Sertoli cells to AGC cannot be homologous

### **Figure 2.**

*SR-BI/PS-mediated phagocytosis. PS is translocated from the inner leaflet to outer leaflet of cellular membrane during apoptosis. PS is recognized by SR-BI located on the surface of phagocytes, and subsequently apoptotic cell is engulfed by phagocytosis via cytoskeletal changes.*

**73**

**Figure 3.**

*Sertoli Cell Phagocytosis: An Essential Event for Spermatogenesis*

adhesion because germ cells do not express any TAM receptors. Gas6 is required for TAM-mediated phagocytosis of AGC by Sertoli cells. The N-terminal region of Gas6 binds to PS on the surface of AGC, and the C-terminal of Gas6 is recognized by TAM receptors, allowing Gas6 to bridge the binding between Sertoli cells and AGC (**Figure 3**, right side). Gas6 also plays a role in mediating Sertoli cell phagocytosis of AGC through the activation of Mer, thus triggering intracellular phagocytic signal-

*Mechanisms and consequences of Sertoli cell phagocytosis of apoptotic germ cells (AGC) and residual bodies (RB). AGC and RB are phagocytized by Sertoli cells through two mechanisms (right side). SR-BI expressed on the Sertoli cell membrane binds with phosphatidylserine (PS) located on the surfaces of AGC and RB, thereby engulfing AGC and RB. TAM receptors mediate the phagocytosis of AGC and RB by Sertoli cells through Gas6 that bridges TAM receptors on Sertoli cell membrane and PS on the surfaces of AGC and RB. After phagocytosis, AGC and RB fuse with lysosomes and are recycled as energy sources for ATP production. If AGC and RB are not efficiently engulfed by Sertoli cells (left side), AGC and RB break down and release damageassociated molecular patterns (DAMPs). DAMPs can be recognized by toll-like receptors (TLRs) and initiate innate immune responses through TRIF and MyD88 signaling pathways. These pathways activate nuclear factor kappa B (NF-κB), mitosis antigen protein kinases (MAPKs), and interferon regulatory factors (IRFs), thereby inducing the expression of inflammatory cytokines, including TNF-α, IL-1, IL-6, INF-α, and IFN-β.*

Sertoli cells abundantly express dynamin 2, and dynamin 2 is involved in the regulation of Sertoli cell phagocytosis [12]. Dynamin 2 regulates the actin assembly in Sertoli cells during phagocytosis. A dynamin 2 inhibitor reduces Sertoli cell phagocytosis through the impairment of phagocytic cup formation. Knockdown of dynamin 2 perturbs actin polymerization and recruitment to target liposomes. The role of dynamin 2 in regulating Sertoli cell phagocytosis requires the interaction between dynamin and amphiphysin 1 [44]. Dynamin 2 and amphiphysin 1

ing that modulates the cytoskeleton of Sertoli cells for engulfing AGC.

**3.3 Other molecules regulating Sertoli cell phagocytosis**

*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 3.**

*Male Reproductive Health*

male fertility [36, 37].

which phagocytes engulf apoptotic cells (**Figure 2**). The interaction of PS and SR-BI induces cytoskeletal changes that form phagocytic cup, thereby resulting in the engulfment of apoptotic cells. As shown in **Figure 3** (right side), SR-BI is expressed in Sertoli cells, and PS is exposed on the surfaces of AGC and RB [19–21]. Several in vitro studies provide evidence that Sertoli cells engulf AGC and RB through the interaction of SR-BI and PS. The phagocytosis of AGC and RB by Sertoli cells can be inhibited by the presence of annexin V that specifically binds to PS on the surfaces of AGC and RB [3, 21]. Moreover, an antibody against SR-BI disables the phagocytosis of AGC by Sertoli cells [19]. The SR-BI/PS-mediated phagocytosis of AGC and RB by Sertoli cells is confirmed in vivo, in which injection of anti-SR-BI antibody and annexin V into the seminiferous tubules increases the number of AGC [3]. Therefore, both in vitro and in vivo studies confirm that Sertoli cells recognize

TAM receptors belong to a subfamily of the transmembrane receptor tyrosine kinases (**Figure 4**), which include three members, Tyro3, Axl, and Mer [22]. Gas6 is a functional common ligand of TAM receptors [23]. The TAM/Gas6 system regulates cell survival, innate immune response, and phagocytosis of apoptotic cells [24–27]. TAM receptors are involved in several pathological conditions, such as chronic inflammatory and autoimmune diseases [28, 29], viral infection [30–32], and cancer [33–35]. Notably, TAM receptors are essential for spermatogenesis and

The mechanisms by which the TAM/Gas6 system regulates testicular functions have been intensively investigated [38]. TAM receptors and Gas6 are abundantly expressed in Sertoli and Leydig cells [39]. All three Tyro3, Axl, and Mer receptors are expressed in Sertoli cells, whereas Leydig cells express Axl and Mer. Gas6 is uniquely expressed in Leydig cells. TAM receptors negatively regulate the expression of pro-inflammatory cytokines in both Sertoli and Leydig cells [40, 41], which might contribute to the immunoprivileged status of the testis [42]. In particular, the TAM receptors and Gas6 are essential for the phagocytic removal of AGC by Sertoli cells [43]. TAM receptors cooperatively regulate Sertoli cell phagocytosis of AGC. All three Tyro3, Axl, and Mer receptors participate in recognizing and binding AGC to Sertoli cells, whereas Mer is responsible for triggering phagocytic intracellular signaling that promotes engulfment of AGC. Any individual TAM receptors in Sertoli cells exhibit similar binding ability to AGC. However, Sertoli cells lacking all three TAM receptors remarkably decrease the binding between Sertoli cells and AGC. The TAM-mediated binding of Sertoli cells to AGC cannot be homologous

*SR-BI/PS-mediated phagocytosis. PS is translocated from the inner leaflet to outer leaflet of cellular membrane during apoptosis. PS is recognized by SR-BI located on the surface of phagocytes, and subsequently apoptotic cell* 

and engulf AGC and RB in the SR-BI/PS-dependent fusion.

**3.2 Role of TAM receptors in mediating Sertoli cell phagocytosis**

**72**

**Figure 2.**

*is engulfed by phagocytosis via cytoskeletal changes.*

*Mechanisms and consequences of Sertoli cell phagocytosis of apoptotic germ cells (AGC) and residual bodies (RB). AGC and RB are phagocytized by Sertoli cells through two mechanisms (right side). SR-BI expressed on the Sertoli cell membrane binds with phosphatidylserine (PS) located on the surfaces of AGC and RB, thereby engulfing AGC and RB. TAM receptors mediate the phagocytosis of AGC and RB by Sertoli cells through Gas6 that bridges TAM receptors on Sertoli cell membrane and PS on the surfaces of AGC and RB. After phagocytosis, AGC and RB fuse with lysosomes and are recycled as energy sources for ATP production. If AGC and RB are not efficiently engulfed by Sertoli cells (left side), AGC and RB break down and release damageassociated molecular patterns (DAMPs). DAMPs can be recognized by toll-like receptors (TLRs) and initiate innate immune responses through TRIF and MyD88 signaling pathways. These pathways activate nuclear factor kappa B (NF-κB), mitosis antigen protein kinases (MAPKs), and interferon regulatory factors (IRFs), thereby inducing the expression of inflammatory cytokines, including TNF-α, IL-1, IL-6, INF-α, and IFN-β.*

adhesion because germ cells do not express any TAM receptors. Gas6 is required for TAM-mediated phagocytosis of AGC by Sertoli cells. The N-terminal region of Gas6 binds to PS on the surface of AGC, and the C-terminal of Gas6 is recognized by TAM receptors, allowing Gas6 to bridge the binding between Sertoli cells and AGC (**Figure 3**, right side). Gas6 also plays a role in mediating Sertoli cell phagocytosis of AGC through the activation of Mer, thus triggering intracellular phagocytic signaling that modulates the cytoskeleton of Sertoli cells for engulfing AGC.

### **3.3 Other molecules regulating Sertoli cell phagocytosis**

Sertoli cells abundantly express dynamin 2, and dynamin 2 is involved in the regulation of Sertoli cell phagocytosis [12]. Dynamin 2 regulates the actin assembly in Sertoli cells during phagocytosis. A dynamin 2 inhibitor reduces Sertoli cell phagocytosis through the impairment of phagocytic cup formation. Knockdown of dynamin 2 perturbs actin polymerization and recruitment to target liposomes. The role of dynamin 2 in regulating Sertoli cell phagocytosis requires the interaction between dynamin and amphiphysin 1 [44]. Dynamin 2 and amphiphysin 1

### **Figure 4.**

*TAM receptors and Gas6 system. TAM receptors belong to transmembrane proteins. The extracellular N-terminal region of TAM receptors binds to C-terminal domain of Gas6. The binding of Gas6 to TAM receptors results in the activation of intracellular tyrosine kinase (TK) domain of TAM receptors, thereby promoting cell survival and phagocytosis of apoptotic cells and inhibiting innate immunity.*

can be specifically bound and simultaneously accumulated at ruffles of phagocytic cups. The interaction of dynamin 2 and amphiphysin 1 depends on the PS exposure on AGC.

Dimeric transferrin inhibits phagocytosis of RB by Sertoli cells in an autocrine manner [45]. Transferrin is a glycoprotein that transports iron and is highly expressed in Sertoli cells. Iron is essential for the inhibitory effect of transferrin on Sertoli cell phagocytosis. Transferrin can be physiologically secreted by Sertoli cells and inhibits the phagocytic removal of RB in autocrine manner.

ELMO1 is an evolutionarily conserved engulfment protein that mediates the internalization of apoptotic cells. However, ELMO1-deficient mice are viable and largely normal except for evident testicular pathology [46]. The seminiferous epithelium is disrupted, and AGC number is increased in the testis of ELMO1-deficient mice, therefore reducing sperm output. ELMO1 mediates the phagocytic removal of AGC by Sertoli cells. The engulfment receptors BAL1 and RAC1 (upstream and downstream of ELMO1, respectively) are involved in ELMO1-mediated Sertoli cell phagocytosis of AGC.

Noncoding miRNA regulates Sertoli cell phagocytosis. An early study showed that Dicer, a key enzyme that processes miRNA precursors into its functional form, is required for Sertoli cell function [47]. Dicer knockout mice are fetal lethal. The conditional Dicer knockout in Sertoli cells remarkably increases AGC numbers and leads to primary infertility, suggesting that miRNAs are involved in Sertoli

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and lacking blood vessels.

**4.1 Space saving**

*Sertoli Cell Phagocytosis: An Essential Event for Spermatogenesis*

the expression of miR-471-5p and its target proteins.

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

cell function and spermatogenesis in mice. Whether phagocytic ability of Sertoli cells is impaired by Dicer mutation remains unclear. However, the miR-471-5p has been recently identified to regulate phagocytosis of AGC by Sertoli cells [48]. The overexpression of miR-471-5p in Sertoli cells increases AGC number due to a defective phagocytic ability of Sertoli cells in transgenic mice. The role of miR-471-5p in regulating Sertoli cell phagocytosis requires its interaction with the autophagy protein LC3. Interestingly, androgen favors Sertoli cell phagocytosis by regulating

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

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

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

*Male Reproductive Health*

can be specifically bound and simultaneously accumulated at ruffles of phagocytic cups. The interaction of dynamin 2 and amphiphysin 1 depends on the PS exposure

Dimeric transferrin inhibits phagocytosis of RB by Sertoli cells in an autocrine

ELMO1 is an evolutionarily conserved engulfment protein that mediates the internalization of apoptotic cells. However, ELMO1-deficient mice are viable and largely normal except for evident testicular pathology [46]. The seminiferous epithelium is disrupted, and AGC number is increased in the testis of ELMO1-deficient mice, therefore reducing sperm output. ELMO1 mediates the phagocytic removal of AGC by Sertoli cells. The engulfment receptors BAL1 and RAC1 (upstream and downstream of ELMO1, respectively) are involved in ELMO1-mediated Sertoli cell

Noncoding miRNA regulates Sertoli cell phagocytosis. An early study showed that Dicer, a key enzyme that processes miRNA precursors into its functional form, is required for Sertoli cell function [47]. Dicer knockout mice are fetal lethal. The conditional Dicer knockout in Sertoli cells remarkably increases AGC numbers and leads to primary infertility, suggesting that miRNAs are involved in Sertoli

manner [45]. Transferrin is a glycoprotein that transports iron and is highly expressed in Sertoli cells. Iron is essential for the inhibitory effect of transferrin on Sertoli cell phagocytosis. Transferrin can be physiologically secreted by Sertoli cells

*TAM receptors and Gas6 system. TAM receptors belong to transmembrane proteins. The extracellular N-terminal region of TAM receptors binds to C-terminal domain of Gas6. The binding of Gas6 to TAM receptors results in the activation of intracellular tyrosine kinase (TK) domain of TAM receptors, thereby promoting cell survival and phagocytosis of apoptotic cells and inhibiting innate immunity.*

and inhibits the phagocytic removal of RB in autocrine manner.

**74**

on AGC.

**Figure 4.**

phagocytosis of AGC.

cell function and spermatogenesis in mice. Whether phagocytic ability of Sertoli cells is impaired by Dicer mutation remains unclear. However, the miR-471-5p has been recently identified to regulate phagocytosis of AGC by Sertoli cells [48]. The overexpression of miR-471-5p in Sertoli cells increases AGC number due to a defective phagocytic ability of Sertoli cells in transgenic mice. The role of miR-471-5p in regulating Sertoli cell phagocytosis requires its interaction with the autophagy protein LC3. Interestingly, androgen favors Sertoli cell phagocytosis by regulating the expression of miR-471-5p and its target proteins.
