**2. Germ cell apoptosis and Sertoli cell phagocytosis**

The mammalian testis consists of two distinct compartments: the seminiferous tubules and the interstitial spaces among the tubules (**Figure 1**). The two major functions of the testis include spermatogenesis, occurring within the seminiferous epithelium, and steroidogenesis by Leydig cells that are in the interstitial spaces.

### **Figure 1.**

*Human testicular schematic of histological structure and cellular constituents. The testis consists of two separate regions, namely, seminiferous tubules and interstitial spaces (left panels). The seminiferous tubules are composed of multiple layers of peritubular myoid cells (PMC) that constitute a tubular wall and Sertoli cells embracing different stages of male germ cells to form the seminiferous epithelium where spermatogenesis is fulfilled (right upper panel). The seminiferous epithelium is divided into two compartments, namely, the basal compartments and adluminal compartments, by the BTB that is formed by various junctions (right low panel) between two adjacent Sertoli cells, near the basal side. Different stages of developing germ cells, including spermatogonia (Sg), primary spermatocytes (PSc), secondary spermatocytes (SSc), round spermatids (RS), and elongated spermatids (ES), are localized on the seminiferous epithelium from the basal compartments to adluminal compartments. The interstitial spaces are composed of various cell types, the majority of which are Leydig cells (LC), but also macrophages (Mφ), as well as minor dendritic cells (DC) and T lymphocytes (T).*

**71**

**3.1 Role of SR-BI/PS system**

*Sertoli Cell Phagocytosis: An Essential Event for Spermatogenesis*

The seminiferous tubules possess a special microenvironment essential for spermatogenesis, which is composed of columnar Sertoli cells tightly encompassing developing germ cells. The blood-testis barrier (BTB) that is formed by two adjacent Sertoli cells near the basal side of the tubules is critical for maintaining the tissue homeostasis and immune microenvironment for normal germ cell development. During spermatogenesis, more than 75% germ cells have been estimated to undergo apoptosis [5, 6]. Apoptosis can occur at any stage of germ cells. Male germ cell survival and apoptosis are highly regulated by endocrine hormones [7]. In particular, follicle-stimulating hormone (FSH) produced by the pituitary and testosterone synthesized in Leydig cells is essential for healthy spermatogenesis. Low level of FSH increases germ cell apoptosis. The administration of testosterone in vivo inhibits germ cell apoptosis. Both FSH and testosterone could not act on germ cells because these cells do not express the receptors of two hormones. By contrast, FSH and testosterone can regulate the functions of Sertoli cells that express the hormonal receptors. Therefore, FSH and testosterone indirectly regulate germ cell apoptosis via Sertoli cells. The cascade of caspase activation is involved in the initiation of germ cell apoptosis [8]. Caspase 2 activation initiates the caspase cascade, in which

Like other apoptotic cells, the translocation of phosphatidylserine (PS) to the surface of the cellular membrane is a characterization of male germ cell apoptosis [9]. The PS on the surface of apoptotic cells can be recognized by SR-BI and engulfed by phagocytes. At the final stage of germ cell development, most of the cytoplasm portions of spermatozoa are shed as RB before spermatozoa release to the lumen of the seminiferous tubules. However, immunohistochemical staining only detects a limited number of AGC. The RB are also rarely observed by histological analysis. These phenomena are assumed due to the rapid removal of apoptotic cells and RB through phagocytosis, a common way for engulfing apoptotic cells [1]. In accordance with this speculation, the inhibition of Sertoli cell phagocytosis in vivo

greatly increases AGC numbers within the seminiferous tubules [3].

Phagocytosis of AGC and RB by Sertoli cells can be assessed by various approaches [10]. Confocal and transmission electron microscopy are reliable approaches that can distinguish apoptotic components ingested by Sertoli cells. However, these expensive and time-consuming approaches are not suitable for routine tests. Several simplified protocols to indirectly measure Sertoli cell phagocytosis have been reported [11–13]. These protocols require further optimization to avoid data misinterpretation. Lipid droplets are cyclically formed in the cytoplasm of Sertoli cells, which coincides with the spermatogenic cycle [14]. Therefore, it has been proposed that lipid droplets in Sertoli cells might result from the degradation of apoptotic components, including RB and AGC. An in vitro study confirmed that phagocytosis of AGC by Sertoli cells resulted in lipid droplet formation in Sertoli cells, which was used for evaluation of phagocytosis of AGC by Sertoli cells [13].

**3. Mechanisms underlying phagocytosis of AGC and RB by Sertoli cells**

and apoptotic cells [15–17]. PS is a type of phospholipid that is located on the inner leaflet of the plasma membrane bilayer of healthy cells [18]. However, PS translocates to the outer leaflet of the cellular membrane during cell apoptosis and is exposed on the surface of apoptotic cells [9]. PS on the apoptotic cell surface can be recognized and bound by SR-BI on phagocytes, which is a key mechanism by

SR-BI is a receptor for high-density lipoprotein and can bind to acidic liposomes

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

BAX is involved in the cleavage of caspases.

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

*Male Reproductive Health*

on phagocytes and phosphatidylserine (PS) exposed on apoptotic cell surfaces is a universal mechanism by which phagocytes engulf apoptotic cells [1]. This mechanism is also involved in the regulation of the phagocytosis of AGC and RB by Sertoli cells [2]. Tyro3, Axl, and Mer (TAM) tyrosine kinase receptors and their functional common ligand, growth arrest specific gene 6 (Gas6), are also essential for optimal phagocytosis of AGC by Sertoli cells. Several other genes that regulate Sertoli cell phagocytosis of AGC have been recognized. The mechanisms underlying phago-

Impairment of Sertoli cell phagocytosis is associated with pathogenesis and dysfunction of the testis, thus impairing male fertility. The inhibition of Sertoli cell phagocytic ability disrupts spermatogenesis [3]. Gene mutation that impairs Sertoli cell phagocytosis may lead to autoimmune orchitis and male infertility [4]. The pathogenic

The mammalian testis consists of two distinct compartments: the seminiferous tubules and the interstitial spaces among the tubules (**Figure 1**). The two major functions of the testis include spermatogenesis, occurring within the seminiferous epithelium, and steroidogenesis by Leydig cells that are in the interstitial spaces.

cytic removal of AGC by Sertoli cells are the main focus of this article.

conditions due to impaired Sertoli cell phagocytosis are mentioned in the text.

*Human testicular schematic of histological structure and cellular constituents. The testis consists of two separate regions, namely, seminiferous tubules and interstitial spaces (left panels). The seminiferous tubules are composed of multiple layers of peritubular myoid cells (PMC) that constitute a tubular wall and Sertoli cells embracing different stages of male germ cells to form the seminiferous epithelium where spermatogenesis is fulfilled (right upper panel). The seminiferous epithelium is divided into two compartments, namely, the basal compartments and adluminal compartments, by the BTB that is formed by various junctions (right low panel) between two adjacent Sertoli cells, near the basal side. Different stages of developing germ cells, including spermatogonia (Sg), primary spermatocytes (PSc), secondary spermatocytes (SSc), round spermatids (RS), and elongated spermatids (ES), are localized on the seminiferous epithelium from the basal compartments to adluminal compartments. The interstitial spaces are composed of various cell types, the majority of which are Leydig cells (LC), but also macrophages (Mφ), as well as minor dendritic cells (DC) and T lymphocytes (T).*

**2. Germ cell apoptosis and Sertoli cell phagocytosis**

**70**

**Figure 1.**

The seminiferous tubules possess a special microenvironment essential for spermatogenesis, which is composed of columnar Sertoli cells tightly encompassing developing germ cells. The blood-testis barrier (BTB) that is formed by two adjacent Sertoli cells near the basal side of the tubules is critical for maintaining the tissue homeostasis and immune microenvironment for normal germ cell development. During spermatogenesis, more than 75% germ cells have been estimated to undergo apoptosis [5, 6]. Apoptosis can occur at any stage of germ cells. Male germ cell survival and apoptosis are highly regulated by endocrine hormones [7]. In particular, follicle-stimulating hormone (FSH) produced by the pituitary and testosterone synthesized in Leydig cells is essential for healthy spermatogenesis. Low level of FSH increases germ cell apoptosis. The administration of testosterone in vivo inhibits germ cell apoptosis. Both FSH and testosterone could not act on germ cells because these cells do not express the receptors of two hormones. By contrast, FSH and testosterone can regulate the functions of Sertoli cells that express the hormonal receptors. Therefore, FSH and testosterone indirectly regulate germ cell apoptosis via Sertoli cells. The cascade of caspase activation is involved in the initiation of germ cell apoptosis [8]. Caspase 2 activation initiates the caspase cascade, in which BAX is involved in the cleavage of caspases.

Like other apoptotic cells, the translocation of phosphatidylserine (PS) to the surface of the cellular membrane is a characterization of male germ cell apoptosis [9]. The PS on the surface of apoptotic cells can be recognized by SR-BI and engulfed by phagocytes. At the final stage of germ cell development, most of the cytoplasm portions of spermatozoa are shed as RB before spermatozoa release to the lumen of the seminiferous tubules. However, immunohistochemical staining only detects a limited number of AGC. The RB are also rarely observed by histological analysis. These phenomena are assumed due to the rapid removal of apoptotic cells and RB through phagocytosis, a common way for engulfing apoptotic cells [1]. In accordance with this speculation, the inhibition of Sertoli cell phagocytosis in vivo greatly increases AGC numbers within the seminiferous tubules [3].

Phagocytosis of AGC and RB by Sertoli cells can be assessed by various approaches [10]. Confocal and transmission electron microscopy are reliable approaches that can distinguish apoptotic components ingested by Sertoli cells. However, these expensive and time-consuming approaches are not suitable for routine tests. Several simplified protocols to indirectly measure Sertoli cell phagocytosis have been reported [11–13]. These protocols require further optimization to avoid data misinterpretation. Lipid droplets are cyclically formed in the cytoplasm of Sertoli cells, which coincides with the spermatogenic cycle [14]. Therefore, it has been proposed that lipid droplets in Sertoli cells might result from the degradation of apoptotic components, including RB and AGC. An in vitro study confirmed that phagocytosis of AGC by Sertoli cells resulted in lipid droplet formation in Sertoli cells, which was used for evaluation of phagocytosis of AGC by Sertoli cells [13].

## **3. Mechanisms underlying phagocytosis of AGC and RB by Sertoli cells**

### **3.1 Role of SR-BI/PS system**

SR-BI is a receptor for high-density lipoprotein and can bind to acidic liposomes and apoptotic cells [15–17]. PS is a type of phospholipid that is located on the inner leaflet of the plasma membrane bilayer of healthy cells [18]. However, PS translocates to the outer leaflet of the cellular membrane during cell apoptosis and is exposed on the surface of apoptotic cells [9]. PS on the apoptotic cell surface can be recognized and bound by SR-BI on phagocytes, which is a key mechanism by

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