The transport of cholesterol in Sertoli cells
A large proportion of nutrients, such as lipids, used for spermatogenesis are provided by supporting Sertoli cells. Sertoli cells have the ability to synthesize cholesterol using acetate in vitro (19), however, it has been demonstrated that this source of cholesterol is insufficient for steroidogenesis in vivo. As the amount of cholesterol required for spermatogenesis far exceeds the capacity of Sertoli cells biosynthesis, specialized cholesterol transporters such as SR-BI facilitate the uptake of cholesterol from the blood (20). Research has revealed that, at least in rodents, HDL serves as the primary source of cholesterol for Sertoli cells (21), which may be further supported by the observations that the basal membrane segregating seminiferous tubules from blood capillaries blocks the entry of LDL but permits HDL entry (8,21). In rats, Sertoli cells obtain cholesterol from HDL primarily through apolipoprotein E-dependent pathways (22). In addition, Sertoli cells may also obtain cholesterol from phagocytosed apoptotic germ cells and lipid-rich remnant recycling (22). To eliminate toxicity, surplus cholesterol undergoes esterification and storage in lipid droplets, which is instrumental in the maintenance of cholesterol equilibrium. Lipid droplets are highly mobile and dynamic structures, and are reported to interact with cell junctions and organelles via transient or stable surface exchange, ensuring the effective synthesis, uptake, usage and elimination of cholesterol (23). Furthermore, Sertoli cells also maintain cholesterol homeostasis through reverse cholesterol transport (RCT). RCT involves various procedures that enable the efflux of excess cholesterol to HDL and promote the transport of cholesterol from non-hepatic peripheral tissue back to the liver via the plasma to maintain cholesterol homeostasis, which primarily relies on the ABCA1 cholesterol transporters (3). The excessive accumulation of cholesterol esters (CE) in Sertoli cells leads to disruption of cholesterol homeostasis, ultimately resulting in complete subfertility or infertility. This phenomenon was demonstrated in various knockout mouse models that are deficient in functional nuclear receptors, such as retinoid X receptor (RXR) (24,25), and also in ABCA1 knockout mice (3). As ABCA1 is an established liver X receptor (LXR) target gene, in double knockout mice (LXR α/β−/−), the deficiency of ABCA1 decreases the cholesterol efflux from Sertoli cells and results in the accumulation of CE (3,24). Therefore, Sertoli cells modulate every step of cholesterol metabolism, including cholesterol uptake, efflux, storage and recycling, which indicates that Sertoli cells should be a focus in the interpretation of spermatogenesis. Fig. 1 is a graphic illustration of the transport of cholesterol in Sertoli cells.
The role of cholesterol metabolism in spermatogenesis
The role of cholesterol in sperm production and male fertility is well established. The process of spermatogenesis consists of a sequence of differentiation and proliferative phases, which are further divided into spermatogenic, meiotic and mitotic stages. Each stage involves different cell types, including spermatids, spermatocytes and spermatogonia (26,27). Additionally, increases in lipid droplets are observed throughout spermatogenesis (28), demonstrating an intimate association between lipid metabolism alterations and fertility during spermatogenesis. Steroidogenesis also requires large amounts of cholesterol (29), while in the seminiferous tubules, cholesterol is required for the differentiation of germinal cells to spermatozoids (gametogenesis/spermatogenesis) (30). Considerable evidence also indicates that, in males and females, cholesterol is required for the development of fertility and gametes. In male mice, the absence of the 24-dehydrocholesterol reductase gene, which encodes a cholesterol biosynthetic enzyme, induces infertility (31). Therefore, cholesterol is required for the mass production of germ cells during spermatogenesis. The plasma membranes of sperm are also heavily loaded with cholesterol when sperm are released from the seminiferous epithelium. Previously, experiments that determined the role of cholesterol de novo synthesis identified that the incorporation rate of 14C acetate into cholesterol was increased during the development of pachytene, leptotene and zygotene stages (32). This indicates that de novo synthesis of cholesterol is increased in these germ cells, which was associated with increased diameter and surface area of germ cells. In late pachynema, the rate of cholesterol synthesis tends to decrease and remains low throughout the following phases of spermatogenesis, including sperm maturation. In contrast to cholesterol, the incorporation capacity of acetate into dolichol was enhanced in round spermatids and late pachytene spermatocytes, and later diminished and remained low in mature sperm cells (32). These observations indicate that the preliminary phases of cholesterol synthesis in round spermatids and pachytene spermatocytes may precede dolichol synthesis, which acts as a critical constituent in the production of membrane glycoproteins. However, the detailed role of cholesterol metabolism within germ cells has not been identified clearly, which is primarily due to the complex connections between germ cells and the supporting Sertoli cells.
Proteins responsible for the regulation of cholesterol metabolism in Sertoli cells
ABCA1
ABCA1, a member of the ABC transporter superfamily, transfers phospholipids and cholesterol out of the peripheral cells to lipid-free apolipoprotein A1, which results in the formation of pre-βHDL and is the rate-limiting step in the process of HDL biosynthesis (33,34). Genetic mutations in ABCA1 lead to the absence of plasma HDL, the accumulation of CE in various body tissues and an increased incidence of atherosclerosis (AS), which are collectively termed Tangier disease (35). By contrast, ABCA1 overexpression in mice enhances plasma HDL levels and prevents the development AS (36,37). However, research has demonstrated that, although ABCA1 in macrophages has an important role in preventing atherosclerosis, it has limited influence on HDL levels in the plasma. In addition, in mice, liver ABCA1 serves as a predominant factor to determine plasma HDL levels (38,39). ABCA1 is highly expressed in the testes (40,41), indicating that ABCA1 may also be involved in the regulation of testicular lipid transport, which is largely segregated from the peripheral circulation (42). Selva et al (3) observed that, in the seminiferous tubule, Sertoli cells account for the majority of the ABCA1 expression. Consistent with the high ABCA1 expression levels, Sertoli cells that express ABCA1 exhibit substantial cholesterol efflux to lipid-free apolipoprotein A1. Conversely, ABCA1-deficient Sertoli cells exhibited decreased cholesterol efflux and accumulation of oil red O-positive lipid droplets, effects that were reversed by ABCA1 restoration, demonstrating that the cholesterol efflux from cultured Sertoli cells is mediated by ABCA1. Fig. 2 is a schematic representation of the regulation of cholesterol content in Sertoli cells. Importantly, in apolipoprotein A1−/− mice, Sertoli cells do not exhibit excess lipids, indicating that deficiency of plasma HDL is insufficient to lead to lipid accumulation in Sertoli cells and that ABCA1 has a direct role in modulating Sertoli cell lipid efflux (43). Furthermore, ABCA1−/− mice exhibit reduced testosterone levels and fewer spermatozoa compared with wild-type (WT) mice, indicating that ABCA1 deficiency compromises the Sertoli cell functions and male fertility, but does not lead to complete sterility (3). A deeper understanding of the roles of ABCA1 in lipid transport in spermatogenesis requires further investigation.
SR-BI
SR-BI, which belongs to the SR superfamily, is an important integral membrane glycoprotein on the cell membrane. Structurally, SR-BI and CD36 share 30% homology in amino acid sequence. Functionally, it is a physiological receptor for HDL that facilitates the uptake of HDL-CE, a process that is termed RCT (44). Unlike LDL receptors, which internalize whole lipoprotein particles, SR-BI selectively uptakes cholesterol from HDL into cells. In the testes, the highest expression of SR-BI is observed in steroidogenic Leydig cells (45,46), and the remaining expression occurs in Sertoli cells (46). Previously, Shiratsuchi et al (47) demonstrated that the activity of phosphatidylserine (PS)-mediated phagocytosis of spermatogenic cells by Sertoli cells was enhanced by SR-BI upregulation and inhibited by an anti-SR-BI antibody, indicating that SR-BI acts as a PS receptor, enabling Sertoli cells to recognize and phagocytose spermatogenic cells. Furthermore, Rigotti et al (45) demonstrated that in the testes, a large proportion of lipoprotein-derived cholesterol used for steroidogenesis is acquired through SR-BI (45). Akpovi et al (20) observed that, in mink testes, the overexpression of SR-BI is associated with increased esterified cholesterol levels in Sertoli cells. In addition, Casado et al (48) reported that hormone-sensitive lipase knockout mice exhibited upregulated SR-BI expression, increased phosphorylated (p)-extracellular signal-regulated kinase, p-Akt and p-SRC proto-oncogene levels, and lipid accumulation in Sertoli cells, indicating that SR-BI may be involved in the uptake of CE for spermatogenesis and steroidogenesis in the testes.
LXRα/β
LXRα/β have important roles in the maintenance of cellular cholesterol homeostasis. The tissue distribution of these two LXR isoforms is different. LXRβ is expressed ubiquitously throughout the body, while LXRα is predominantly distributed in the kidney, liver and intestine (49,50). Oxysterols, which are the oxidized derivatives of cholesterol, are the endogenous ligands for LXRs. In addition, T0901317 and GW3965 are synthetic specific ligands. Upon activation, LXRs form heterodimers with RXRs, which are capable of activating the transcription of genes involved in cholesterol efflux. Previously established LXR target genes include ABCA1/G1/G5/G8 (51–53), apolipoprotein A1, apolipoprotein E (54,55) and SR-BI (56). Robertson et al (25) demonstrated that from as early as 2.5 months, cholesterol began to accumulate in the Sertoli cells of LXRβ−/− mice. At around 10 months, although Sertoli cells structure remained comparatively intact and spermatocytes and spermatogonia were still detectable, large and numerous droplets, in addition to the absence of mature germ cells, were observed. By 20 months, lipid accumulation was highest and few cell types were observed. Genetic data from whole testis indicated that the LXRβ was the predominant transcript in the testis (25). Furthermore, Annicotte et al (57) reported that LXRβ was present specifically in the Sertoli cells and seminiferous tubules as early as 16.5 days post-conception in embryos, which further highlights the importance of LXRβ in these cells. Furthermore, results also indicated that LXRα cannot be detected with in situ hybridization (57), and that LXRα−/− mice did not exhibit lipid accumulation and exhibited almost undisturbed spermatogenesis, indicating that the presence of LXRβ is enough to maintain cholesterol homeostasis in the testis. More importantly, in the LXRα−/−/β−/− mouse, the testicular phenotype is more destructive compared with that observed in the LXRβ−/− mouse. Furthermore, it was also demonstrated that WT animals that were fed a diet mixed with the synthetic LXR agonist T0901317 exhibited increased levels of the LXR target genes sterol regulatory element-binding protein-1c (SREBP-1c) and ABCG1 in their testes (53,58), which was supported by results in MSC-1 cells treated with T0901317 where ABCA1 levels were increased (51). LXR deficiency may also cause dysfunction in these regulatory elements in Sertoli cells. However, the accurate molecular mechanisms underlying the regulation of cholesterol homeostasis by LXR in Sertoli cells and male fertility require further investigation.
RXR α/β/γ
RXRα/β/γ belongs to the vertebrate nuclear receptor superfamily (59). In rats, RXRα/β transcripts are distributed widely throughout the body, whereas RXRγ is only expressed in certain types of tissues. It has been demonstrated that RXRβ is necessary for normal spermatogenesis in rats, and RXRβ−/− males exhibit abnormalities in spermiogenesis and spermiation. The biological functions of Sertoli cells may be severely affected by abnormal RXRβ expression, which is supported by various observations. In the testes of WT mice, only Sertoli cells express RXRβ. In addition, in RXRβ−/−mice, Sertoli cell abnormalities precede the presence of abnormal spermatids by at least a week. In 29-day-old mutants, prior to the completion of the first spermatogenic cycle, lipid droplets were observed in Sertoli cells (49). Therefore, the earliest lipid accumulation detected in Sertoli cells cannot due to the phagocytosis of spermatids remnants. Furthermore, in the testes of retinoic acid receptor (RAR)α−/− mice and not RARβ−/−mice, although sperm release was blocked during phagocytosis of retained spermatids, lipid droplets in Sertoli cells were not observed (60), indicating that lipid accumulation cannot be attributed to the impaired spermiation. Combined, the results indicate that the lipid deposition observed in the Sertoli cells of RXRβ−/− mice reflects a complex metabolic disorder. As Sertoli cells are critical for spermatid maturation, the obstacles of spermiation and spermiogenesis in RXRβ−/− mutant testes may due to compromised function of Sertoli cells. Furthermore, as RXRβ may serve as a heterodimeric partner for peroxisome proliferator-activated receptor (PPAR)β, which is expressed abundantly in Sertoli cells (61), compromised PPAR function may also contribute to the lipid deposition in RXRβ−/− mutant Sertoli cells (62). However, the role of RXR in the lipid metabolism of Sertoli cells requires further investigation.
FSH and insulin
FSH, which is produced and secreted by the anterior pituitary gland, is a crucial reproduction factor in mammals. In females, FSH facilitates the maturation of follicles and the production of estrogen, while in males it enhances Sertoli cell proliferation in the immature testis and mature spermatogenesis (63,64). The levels of FSH are regulated by hormones released by gonadotropin through estrogen feedback and the hypothalamic pituitary gonadal axis (65). Guma et al (66) demonstrated that FSH and insulin promoted the lipid metabolism of Sertoli cells by augmenting acetate incorporation into Sertoli cell lipids. These stimulatory activities of FSH and insulin do not involve protein synthesis, indicating a potential effect via modulation of enzyme activity or glucose transport in Sertoli cells. In addition, treatment with insulin, but not FSH, also enhanced the ATP citrate lyase activity, indicating the biological relevance of insulin on the fatty acid synthesis of Sertoli cells. Oliveira et al (67) demonstrated that insulin signaling is indispensable for Sertoli cell lipid metabolism, as the absence of insulin transforms Sertoli cell metabolism from glycolysis to the Krebs cycle, which inhibits the development of germ cells.
Sex sterols and PPAR α/β/δ/γ
Sex steroids are also involved in the modulation of cholesterol metabolism in Sertoli cells. The effects of 5α-dihydrotestosterone on Sertoli cell cholesterol metabolism resembled those of insulin, but were less obvious. By contrast, 17β-estradiol (E2) promotes increases in acetate concentration by amplifying the transcript levels of acetyl-CoA hydrolase, therefore contributing to the production of by-products that are essential for the maintenance of cholesterol synthesis in Sertoli cells (68). In mature Sertoli cells, 18-carbon polyunsaturated fatty acids (PUFAs) are efficiently converted into 22- and 24-carbon PUFAs with the assistance of certain metabolic enzymes, including fatty acid elongases, and Δ5 and Δ6 desaturases (69). However, hormonal dysregulation may disrupt PUFA synthesis, as Sertoli cells treated with testosterone exhibited reduced activities of the Δ5 and Δ6 desaturases, therefore repressing the steps involving Δ5 and Δ6 desaturases (70). These effects are important as inhibition of Δ5 and Δ6 desaturases activity is likely to restrict the incorporative process of long chain PUFAs into sperm membranes, which may ultimately decrease sperm membrane flexibility and fluidity. PUFAs possess several double bonds in their backbone, which, with an increased number of lipids, improves the fluidity and flexibility of sperm membranes (71).
In addition, lipid oxidation in Sertoli cells is also regulated by PPARα/β/δ/γ, which serve as sensors and derivatives of fatty acids and thus influence the lipid and cholesterol metabolic pathways. Regueira et al (72) demonstrated that the activation of PPARα and PPARβ/δ augmented the expression of the cholesterol transporter SR-BI in Sertoli cells, therefore promoting the selective uptake of CE. Furthermore, PPAR activation enhanced acetyl-CoA carboxylase phosphorylation, resulting in reduced enzyme activity, which promotes the incorporation of fatty acyl-CoA into the mitochondria for further oxidation, and increased long and medium chain dehydrogenase enzyme and L-carnitine palmitoyl transferase 1 mRNA levels (72). These results indicate that PPARα and PPARβ/δ are indispensable for cholesterol metabolism and lipid oxidation in Sertoli cells, while the upstream factors regulated by the PPAR system remain uncertain.