Sertoli cells

Sertoli cells were first identified in the 19th century by Enrico Sertoli, who in his original article described a type of somatic cell with an irregular large shape and speculated that these cells were involved in the formation of viable spermatozoa in spermatogenesis (Oliveira and Alves, Reference Oliveira and Alves2015; Griswold, Reference Griswold2018). Sertoli cells are polarized epithelial cells that extend from the base of the seminiferous epithelium to the lumen of the seminiferous tubule. This means that the basal part of the Sertoli cell is attached to the membrane part of the seminiferous tubule (Berger and Nitta-Oda, Reference Berger and Nitta-Oda2018). Sertoli cells expand to surround germ cells and provide a support network such that most of the surfaces of the Sertoli cells are in contact with germ cells (Crisóstomo et al., Reference Crisóstomo, Alves, Gorga, Sousa, Riera, Galardo, Meroni, Oliveira, Alves and Oliveira2018). Therefore, an accurate understanding of the morphology of Sertoli cells gives us a clear view of their role in spermatogenesis (Tindall et al., Reference Tindall, Rowley, Murthy, Lipshultz and Chang1985). Tarulli et al. (Reference Tarulli, Stanton and Meachem2012) likened spermatogenesis to a symphony in which germ cells occur in regulated waves. Based on this analogy, the population of Sertoli cells that surround germ cells can be considered as a concert hall that provides the environment to maximize sound dynamics. Sperm could be considered the output sound, and their quality depends on Sertoli cells, seen as the size of the concert hall, and Sertoli cell maturity, as the quality of the concert hall.

Morphological research on Sertoli cells has progressed through three phases. First, routine light microscopy (LM) examination continued until the 1960s. The invention of the transmission electron microscope (TEM) led to the second phase of research, which lasted until 2000 and this microscope was very useful for taking high-quality images of Sertoli cell membranes and organelles. The last phase of research, immunohistochemistry and immunofluorescence, began before 2000, but gradually became the main tool for three-dimensional imaging of interactions between Sertoli cells and germ cells, and for localization of specific testicular proteins. Main immunohistochemical nuclear markers for Sertoli cells commonly used for morphological studies include SRY-box containing gene 9 (SOX9), androgen receptor (AR), Wilms tumour protein-1 (WT1), GATA-binding protein 1 (GATA1), GATA4 and cyclin-dependent kinase inhibitor 1B (p27kip1) (França et al., Reference França, Hess, Dufour, Hofmann and Griswold2016).

In mammals, Sertoli cells have been observed as different sizes, with an individual volume of c. 2000–7000 µm². Sertoli cell volume density relative to total seminiferous tubular epithelium is also highly variable, ranging from approximately 15% in mice to 40% in humans. These cells also have an irregularly shaped cytoplasm that varies from species to species (Oliveira and Alves, Reference Oliveira and Alves2015). The first important step in the development of the testis from other glands, resulting in fetal masculinity, is the differentiation of Sertoli cells and Leydig cells. In mammals, during puberty, Sertoli cells undergo profound modifications in their morphology and function, and are morphologically and biochemically different from other undifferentiated cells. The formation of the male reproductive system and the degeneration of the female reproductive system depend on this (Baker and O’Shaughnessy, Reference Baker and O’Shaughnessy2001; Oliveira and Alves, Reference Oliveira and Alves2015).

Electron micrographs have shown that Sertoli cells have a clear and irregular cytoplasm. Sertoli cells contain many organelles such as several mitochondria that explain their high metabolic activity (Wang et al., Reference Wang, Wen, Yuan, Sun, Niu and He2016). The shape of the mitochondria varies depending on species but most mitochondria are spherical or elongated, but can also be cup shaped or doughnut shaped (Gautam et al., Reference Gautam, Rao, Gothwal, Garg and Bhattacharya2019).

In most species, Sertoli cells contain large, irregular nucleoli that, depending on a person’s age, can deform during the seminiferous epithelium cycle. The nucleus is usually found near the basement membrane but in some species the nucleus can be found in the higher parts of the epithelium near the lumen. The nuclear envelope contains indentations due to the accumulation of vimentin and intermediate filaments. These indentations indicate the maturation of Sertoli cells and are not present during growth or in patients with fertility disorders. In addition, pores and fissures are seen whose density depends on the stage of the spermatogenesis cycle (Oliveira and Alves, Reference Oliveira and Alves2015; França et al., Reference França, Hess, Dufour, Hofmann and Griswold2016). Adjacent Sertoli cells attach to each other with tight junctions that create a continuous lock between adjacent Sertoli cells and separates the basal (containing spermatogonia and preleptotene spermatocytes) from the adluminal compartments of the seminiferous epithelium (containing advanced germ cells), also creating the BTB or Sertoli cell barrier. This name is misleading because this barrier is between two intratubular compartments not between blood and testicle tissues (Ritzén, Reference Ritzén1983; Shi et al., Reference Shi, Li, Ren, Xie, Yin and Mo2018). This junctional structure has a porosity of c. 1000 Daltons, which means that anything with a higher molecular weight cannot pass from the outside to the inside of the lumen. The movement of germ cells through the BTB involves the reconstruction of these cell junctions at different stages of the seminiferous epithelial cycle. However, this barrier is stable and it is never completely disassembled or assembled. It is dynamic between these two states. This means that, as the germ cells cross the BTB toward the lumen, the junction in front of the germ cells separates, while the previous cells regroup. This protect germ cells in the adluminal compartments from release of foreign matter and from the immune system (Chojnacka et al., Reference Chojnacka, Zarzycka and Mruk2016; Shi et al., Reference Shi, Li, Ren, Xie, Yin and Mo2018) (Figure 1).

Figure 1. Immunohistochemistry staining of Sertoli cells in mice. Blue 4′,6-diamidino-2-phenylindole (DAPI) staining of all cells within the seminiferous tubule (A1, B1). Sertoli cell staining with Sox9 (A2) and red vimentin (B2). Merged images of DAPI and Sox9 (A3) and vimentin (B3) (scale bars, 50 μm) (Azizi et al., Reference Azizi, Niazi Tabar, Mohammadi and Skutella2021).

At the beginning of meiosis, germ cells outside the BTB become dependent on Sertoli cells as soon as they cross it, in order to survive and continue the process of spermatogenesis. Sertoli cells provide the factors needed for germ cell metabolism and development, therefore are also called nurse cells, which are vital for the normal development of germ cells (Siemann et al., Reference Siemann, Strange, Maharaj, Shi and Verma2017; Crisóstomo et al., Reference Crisóstomo, Alves, Gorga, Sousa, Riera, Galardo, Meroni, Oliveira, Alves and Oliveira2018).

Throughout the Sertoli cell cytoplasm are lysosomes, multivascular bodies (MVB) and lipid droplets that indicate the phagocytic activity of these cells and their frequency depends on the stage of spermatogenesis (Gautam et al., Reference Gautam, Rao, Gothwal, Garg and Bhattacharya2019). Sertoli cells phagocytose germ cells that have degenerated during spermatogenesis In addition, the cytoplasm remaining after mature spermatozoa are released can be digested by Sertoli cells and phagocytic lysosomes. Autophagy is also active in Sertoli cells and is a lysosomal pathway that removes defective organelles and proteins in the cell. Autophagy functions in cell viability or with apoptosis in cell death (Chen et al., Reference Chen, Zhou, Wang, Qian and Han2013; Yefimova et al., Reference Yefimova, Messaddeq, Harnois, Meunier, Clarhaut, Noblanc, Weickert, Cantereau, Philippe, Bourmeyster and Benzakour2013).

Seminiferous tubule adluminal fluid is needed to carry sperm and for spermatogenesis; this fluid is made by Sertoli cells and its composition differs from plasma and testicular interstitial fluid in testicular cells. One of the main differences is that the adluminal fluid of seminiferous tubule has a higher potassium content but a lower calcium content than found in plasma (Vogl et al., Reference Vogl, Lyon, Adams, Piva and Nassour2018) (Figure 2).

Figure 2. Fluidigm real-time RT-PCR. Highly significant expression of SOX9 (A) and vimentin (B) in Sertoli cells (SCs) compared with germ cells (GCs) (*P < 0.05) (Azizi et al., Reference Azizi, Niazi Tabar and Mohammadi2020).

The main regulators of Sertoli cells are hormones, which include follicle-stimulating hormone (FSH), sex steroid hormones, and thyroid hormones (TH). Sertoli cells express the FSH receptor and men with smaller testicles have not expressed the FSH receptor, so their spermatogenesis has been impaired. FSH is secreted from the anterior pituitary gland in response to gonadotropin-releasing hormone (GnRH), which is released from the hypothalamus (Figure 3). This hormone stimulates Sertoli cells to secrete androgen binding protein (ABP) (Petersen and Söder, Reference Petersen and Söder2006).

Figure 3. The hypothalamus secretes a hormone called gonadotropin-releasing hormone (GnRH), which triggers the release of FSH and LH hormones from the pituitary gland. FSH stimulates Sertoli cells to secrete factors such as transferrin and lactate. It also stimulates the secretion of inhibin B from Sertoli cells, which results in negative feedback of FSH secretion. LH stimulates testosterone secretion by acting on Leydig cells, and directly affects the process of spermatogenesis (Gurung and Jialal, Reference Gurung and Jialal2020).

FSH also causes the release of both inhibin B and activin from Sertoli cells that create negative and positive feedbacks for FSH secretion. FSH also inhibits testosterone (T) secretion from Leydig cells by stimulating the production of estradiol (E2) from Sertoli cells (Steinberger et al., Reference Steinberger, Heindel, Lindsey, Elkington, Sanborn and Steinberger1975). In addition, FSH and insulin increase the lipid metabolism of Sertoli cells by increasing acetate compounds in Sertoli cell lipids (Guma et al., Reference Guma, Wagner, Martini and Bernard1997).

Unlike germ cells, Sertoli cells also express androgen receptors. Androgen is needed to start and continue the process of spermatogenesis, and if its receptor is not expressed in Sertoli cells, spermatogenesis stops at the spermatocyte or primary spermatid stage. The presence of this hormone is also essential for Sertoli cell maturation and therefore formation of the BTB (Walker, Reference Walker2003). Thyroid hormone receptors (TRs) are also expressed on Sertoli cells, and T3 is likely to bind directly to these receptors. Consequently, TRs can mediate the possible role of THs in sperm production. This receptor is at its highest concentration at the postnatal and pre-pubertal periods in Sertoli cells and stimulates their differentiation and maturation (Singh et al., Reference Singh, Hamada and Agarwal2011).

Effects of COVID-19 on Sertoli cells

In December 2019 in Wuhan, China, a new type of coronavirus was discovered in patients who had pneumonia-type symptoms, this virus was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and this new coronavirus disease was officially named by the WHO as coronavirus disease 2019 (COVID-19), which is currently a significant constraint to public health (Shen et al., Reference Shen, Xiao, Aierken, Yue, Wu, Liao and Hua2020; Zhou et al., Reference Zhou, Niu, Jiang, Zhang, Zheng, Wang, Zhu, Gao, Huang, Wang and Sun2020). By November 2020, based on WHO data, approximately 60,000,000 people were reported to have had COVID-19 disease. Data from China also showed that older people, especially those with serious underlying diseases, are more at risk of dying than younger people. In total, 80% of deaths have occurred in adults over 60, and only 1% of deaths have occurred in people under 19 years of age (CDC COVID-19 Response Team, 2020). Although many COVID-19 patients are asymptomatic, some patients develop pneumonia and 10% of patients require intensive care unit (ICU) care and mechanical ventilation. Symptoms of the virus vary from patient to patient but are usually accompanied by fever and dry cough, shortness of breath, lethargy, and muscle and bone pain. Less common symptoms include sore throat, diarrhoea, nausea, dizziness, haemoptysis and chest pain, and progression to pneumonia usually occurs 1–2 weeks after the initial symptoms (Ragab et al., Reference Ragab, Salah Eldin, Taeimah, Khattab and Salem2020).

The SARS-CoV-2 pathogen is a positive single-stranded large RNA virus (Younis et al., Reference Younis, Abassi and Skorecki2020), which belongs to the Sarbecovirus subgenus, and the Betacoronavirus genus (Lu et al., Reference Lu, Zhao, Li, Niu, Yang, Wu, Wang, Song, Huang, Zhu, Bi, Ma, Zhan, Wang, Hu, Zhou, Hu, Zhou, Zhao, Chen, Meng, Wang, Lin, Yuan, Xie, Ma, Liu, Wang, Xu, Holmes, Gao, Wu, Chen, Shi and Tan2020). Virus sequencing has shown that SARS-CoV-2 has many similarities with the original SARS-CoV, and has 76.5% identity with SARS-CoV in its amino acid sequence (Wang and Xu, Reference Wang and Xu2020; Younis et al., Reference Younis, Abassi and Skorecki2020). This suggests that SARS-CoV and SARS-CoV-2 proteins have almost identical three-dimensional structures in the receptor-binding domain (Younis et al., Reference Younis, Abassi and Skorecki2020). Therefore, SARS-CoV-2 probably uses the SARS-CoV receptor, namely angiotensin-converting enzyme (ACE2), to enter the host cells (Shen et al., Reference Shen, Xiao, Aierken, Yue, Wu, Liao and Hua2020). The reason for the rapid spread of SARS-CoV2 is that it has a higher affinity for binding to the ACE2 receptor than SARS-CoV (Ou et al., Reference Ou, Liu, Lei, Li, Mi, Ren, Guo, Guo, Chen, Hu, Xiang, Mu, Chen, Chen, Hu, Jin, Wang and Qian2020). COVID-19 has four main structural proteins, which include N (nucleocapsid), E (envelope), S (spike) and M (membrane) (Figure 4). COVID-19 binds to ACE2 through its receptor-binding domain (RBD), which is present in its S protein (Chen et al., Reference Chen, Liu and Guo2020). The spike is a homotrimer glycoprotein that binds to the integral membrane type I in ACE2, then pH-dependent endocytosis occurs. The acidic pH of lysosomes and endosomes activates the enzymes cathepsin B and cathepsin L and breaks down the S glycoprotein into two subunits, S1 and S2. Subunit S1 is used for binding and S2 for integration with the host cell membrane (Figure 5). Host cells also bind to neighbouring cells to form syncytia by expressing spike protein on their surface. Creating syncytia not only disrupts the function of the organ involved but also allows the virus to spread further and escape the immune system (Hikmet et al., Reference Hikmet, Méar, Edvinsson, Micke, Uhlén and Lindskog2020; Kearney, Reference Kearney2020; Walls et al., Reference Walls, Park, Tortorici, Wall, McGuire and Veesler2020). In general, structural and functional studies have shown that SARS-CoV-2 uses the SARS-CoV receptor ACE2 to enter and infect cells (Hoffmann et al., Reference Hoffmann, Kleine-Weber, Schroeder, Krüger, Herrler, Erichsen, Schiergens, Herrler, Wu, Nitsche, Müller, Drosten and Pöhlmann2020), so ACE2 analysis is necessary to determine the route of infection.

Figure 4. Coronavirus structure and features. COVID-19 is an enveloped single-stranded RNA virus composed of envelope (E), nucleocapsid (N), spike (S) and membrane (M) proteins. Two long sequences of SARS-CoV-2 RNA encode special proteins such as an RNA-dependent RNA polymerase that is crucial for viral spread during infection. Other parts of the RNA sequence represent structural proteins including envelope, spike and nucleocapsid (Chen et al., Reference Chen, Liu and Guo2020).

Figure 5. Coronavirus replication in host cells. The S protein in SARS-CoV-2 allows the virus to bind to the ACE2 receptor on the surface of the host cell. Once the virus enters the host cell, viral RNA is released, and then the virus uses the host cell’s ribosome as a protein-making machine to translate its genes. One of the translated proteins is an RNA-dependent RNA polymerase that is not found in eukaryotic cells and which the virus needs it for its RNA replication. Some replicated RNAs are re-translated by the host ribosome to produce the structural proteins of the virus. These then move to the rough endoplasmic reticulum and from there to the Golgi apparatus. In the Golgi apparatus, these proteins undergo modifications and combine with replicated RNAs. After packaging, a new virus is created that exits the cell by exocytosis (Millet and Whittaker, Reference Millet and Whittaker2018).

Angiotensin-converting enzyme2 (ACE2) is a carboxypeptidase enzyme that contains zinc in its structure. ACE2 is a single-pass type I membrane protein that was identified in 2000 as an angiotensin-converting enzyme (ACE) homologue (Turner, Reference Turner, Unger, Steckelings and Souza dos Santos2015; Hikmet et al., Reference Hikmet, Méar, Edvinsson, Micke, Uhlén and Lindskog2020). This protein negatively regulates the renin–angiotensin system (RAS), which is a hormonal system that is involved in regulating multiple functions of tissues and organs. It is particularly critical in maintaining blood pressure homeostasis, electrolyte balance and inflammatory responses. ACE2 performance in this system is the opposite and moderator of ACE action. ACE catalyzes angiotensin I (ANG I) to angiotensin II (ANG II) during the RAS, and results in narrowing of the arteries. By contrast, ACE2 is responsible for the generation of angiotensin 1-7 (ANG 1-7) from ANG II; this results in dilation of the arteries (Karnik et al., Reference Karnik, Unal, Kemp, Tirupula, Eguchi, Vanderheyden and Thomas2015; Jia, Reference Jia2016).

Based on previous reports, there are no gender differences in ACE2 activity between the lungs and hearts of normal and healthy mice but, in male kidneys, ACE2 is more active than in female kidneys. Therefore, ACE2 expression patterns may differ in male and female gonads (Liu et al., Reference Liu, Chen, Tang, Zhang, Chen, Yan, Yuan, Yang, Kong, Yan and Qiao2020).

Previous studies have also shown that male patients infected with SARS-CoV-2 experience widespread destruction of germ cells, have few or no spermatozoon in the seminiferous tubules, and testes basement membrane begins to thicken. This indicated that there are many SARS-CoV-2 receptors, ACE2, in the testes (Shen et al., Reference Shen, Xiao, Aierken, Yue, Wu, Liao and Hua2020; Wang and Xu, Reference Wang and Xu2020).

Based on single-cell RNA sequencing (scRNA-seq) analysis of testicular tissue, ACE2-expressing cells are found in almost all cell types in testis. Analyses of ACE2 RNA expression profiles at single-cell resolution in adult human testicles have shown that spermatogonial stem cells (SSC), leptotene spermatocytes type1 (L1), leptotene spermatocytes type 3 (L3), zygotene spermatocytes (Z), diplotene spermatocytes (D), spermatocyte 7 (SPC7), spermatid stage 1 (S1) and all three types of testicular somatic cells, namely Sertoli cells, Leydig cells, and myoid cells, are susceptible to SARS-CoV-2 infection. Previous experiments have also shown that more than 90% of Sertoli cells express ACE2, suggesting that testicular somatic cells, especially Sertoli cells, may be more susceptible to SARS-CoV-2 infection than germ cells (Babushkina et al., Reference Babushkina, Belokopytova, Grachev, Meko and Vaganov2017; Fan et al., Reference Fan, Li, Ding and Lu2020; Liu et al., Reference Liu, Chen, Tang, Zhang, Chen, Yan, Yuan, Yang, Kong, Yan and Qiao2020). Somatic cells are a main component of the testicular microenvironment and play an important role in maintaining SSC and performing spermatogenesis. In particular, Sertoli cells are in direct contact with spermatogenic cells to control differentiation. Therefore, the dysfunction of these cells disrupts testicular function (Chen and Liu, Reference Chen and Liu2015; Shen et al., Reference Shen, Xiao, Aierken, Yue, Wu, Liao and Hua2020).