What are Sertoli cells? Historical, methodological, and functional aspects

Sertoli cells are somatic cells that are in close contact with germ cells in the mammalian testes. They have multiple functions and fulfill key roles for the development and proper maturation of spermatogenic cells into functional spermatozoa. One of their most important properties is to release trophic factors and supply nutrients to germ cells. But they are also involved in the regulation of the immune system in testis, and provide an immunologically privileged environment for developing germ cells. Because they are so essential for reproductive cells, their alterations can have detrimental consequences for fertility. Many environmental factors and exposures such as high caloric diet, toxins, and pollutants are thought to compromise Sertoli cells physiology. This review describes the discovery of Sertoli cells and the methods used for their study, summarizes their major properties and functions, and describes their dysfunctions in pathologies, particularly associated with environmental stressors.


INTRODUCTION
Sertoli cells are somatic cells that constitute the main structural component of seminiferous tubules in testes, where germ cells reside. They have important "nursing" functions for spermatogenic cells and provide nutrients necessary for testis development and spermatogenesis. 1 Sertoli cells also provide immunological protection to germ cells, and prevent novel antigens arising during spermatogenesis to evoke immune autoreactivity. 2 Sertoli cell functions are essential for the survival of germ cells and fertility, which are key functions for species based on staining and microscopy examination, documented how they provide structural support to seminiferous tubules across different stages of spermatogenesis. 6 Studies by Viktor von Ebner late 19th century established their name as "Sertoli" cells, in honor of E. Sertoli as their discoverer. 7 Back then, V. von Ebner and E. Sertoli were still debating about whether Sertoli cells are precursors of spermatogenic cells or not. This thought was widely spread in the community, but opposed by E. Sertoli himself. 5 The idea that Sertoli cells form a syncytium was also put forward by V. von Ebner and others, 8 but not supported by E. Sertoli, who described them as individual cells. In both cases, E. Sertoli's hypotheses were proven to be right long after his death in 1910. Follow-up studies using electron microscopy, a method that became available mid 20th century, visualized cell membrane and cell-cell junctions in testis, and revealed the tight and highly specialized interactions between Sertoli and germ cells. 9 In the 1990s, Lonnie Russel put forward a three-dimensional model of Sertoli cells, highlighting their complex structure with numerous ramifications of the plasma membrane creating a large surface area for interactions. 10 In parallel, testicular cell culture systems were developed 11 and allowed to obtain enriched cultures of Sertoli cells. 12 When combined with immunohistochemistry and molecular methods, these techniques made it easier to study Sertoli cells and gave rise to important new knowledge about their functions.

STUDYING SERTOLI CELLS-A METHODOLOGICAL OVERVIEW
Methods have been developed to isolate and enrich Sertoli cells from testicular tissue, by distinguishing them from other testicular cells while keeping them functionally intact. primarily identified by the distinct appearance of their nucleus, which is rather big and euchromatic with visible nucleoli 14 and is located close to the basement membrane. 10 Their fibroblast-like appearance due to their multiple cytoplasmic extensions, particularly developed in culture, was also used. 15 With the invention of labeled antibodies to detect cell-specific markers, identifying Sertoli cells became easier. One of the most common markers of Sertoli cells is SRY-box-containing gene 9 (Sox9), a transcription factor necessary for the development of the testicular chord. 16 In testes, Sox9 is expressed exclusively in Sertoli cells and its expression is age-dependent. Sox9 peaks prenatally, then decreases during development and is low in adulthood. Sertoli cells express other markers at specific development stages. For example, they express anti-Mullerian hormone (Amh), a marker that appears at the time of sex determination in early embryogenesis. Mature Sertoli cells also have GATA-binding protein 1 (Gata1), whose level oscillates with spermatogenesis cycles in seminiferous tubules. 4 Also, the Sertoli cell marker follicle-stimulating hormone receptor (FSHr) oscillates with spermatogenesis cycles, and is responsible for the induction of many Sertoli-specific genes. 17

How to isolate Sertoli cells
The first methods to isolate Sertoli cells were based on enrichment from immature rat testes around the initiation of puberty at postnatal day 18-20, followed by purification in culture. 12 To collect Sertoli cells, testes are usually subjected to successive steps of enzymatic digestion to remove interstitial and peritubular cells, followed by fractionation of the remaining seminiferous tubules into single cells. Initial protocols used digestion with trypsin and DNAse followed by collagenase. 12,20 Then, hyaluronidase was used to better separate Sertoli cells from germ cells. 23 Other protocols also used digestion in collagenase first, and then in trypsin and hyaluronidase, or glass beads (for canine cells). 24 After enzymatic digestion, the resulting cell suspension of seminiferous tubules can be plated in culture dishes, where Sertoli cells grow and reach an enrichment of up to 95%.
Several strategies can be used to remove contaminating germ cells in vitro after enzymatic digestion. Initial protocols used high incubation temperature to detach germ cells from the dish, so they can be washed away. 20 Later, hypotonic shock treatment of cultures was used to eliminate germ cells, while Sertoli cells remain largely untouched. 25 Plating the cell suspension on DSA-lectin-coated dishes was also proposed, which allows Sertoli cells to attach to lectin, while germ cells remain free-floating and can easily be removed by washing. 26 These methods have been further improved using different enzymatic digestion and culture techniques, and were adapted to different species. 27,28 Recent methods also addressed the issue of contamination by other somatic cells such as peritubular myoid cells, which can attach to culture plates.
Nowadays, serum-free media are often used to inhibit the growth of contaminating cells, while not affecting the more frugal Sertoli cells. 28 While initial enrichment methods were efficient for immature testes, they were not suited to obtain mature Sertoli cells even after Methods to enrich adult rodent Sertoli cells not requiring any culture have been developed. One of them is based on a three-step enzymatic digestion with collagenase, trypsin, and then hyaluronidase followed by hypotonic shock. With several washing steps, many dying germ cells can be removed to achieve around 80% purity of Sertoli cells. 24 The use of FACS further improved the quality of Sertoli cells preparations. But unlike for immune cells, only few FACS protocols could be established for Sertoli cells as commonly used markers such as vimentin, Sox9, and Gata4 are not surface markers but intracellular. Surface markers such as FSH receptor have been proposed to sort adult Sertoli cells. 33 But their use required to culture the cells for several days to allow them to recover from enzymatic digestion, which may lead to cleavage or internalization of surface markers. 34,35 The introduction of transgenic technology helped solve many of these issues.

Cell lines as model systems
As it is difficult to isolate pure fractions and sufficient number of Sertoli cells from testes, Sertoli cells have been immortalized to facilitate their use in vitro. TM4, 39  SSCs differentiation into haploid stages in vitro. 43 Sertoli cell lines have been instrumental to study Sertoli cell functions, with recent studies addressing the effects of androgen signaling, 44 the action of specific miRNAs 45 and nuclear receptors. 46 They have also been used to study the consequences of environmental toxins in relation to infertility. [47][48][49] Although Sertoli cell lines are useful study models, their distinct features and differences with primary cells and cells in vivo can complicate conclusions and require validation.

MAJOR ROLES AND FUNCTIONS OF SERTOLI CELLS
Sertoli cells have multiple functions, particularly for developmental guidance, immunological protection, and trophic support of germ cells.

4.1
Guiding spermatogenesis Sertoli  The first entry into the spermatogenic cycle is initiated by a surge in retinoic acid (RA) during puberty. 66 Thereafter, RA supports spermatogenic cycles by pulses. 67  Thus, depending on the stage, Sertoli cells react more or less sensitively to external regulators such as FSH. 69 Sertoli cell sensitivity dictates the cyclic expression of GDNF and FGFs, which are increased upon FSH stimulation. 70 Conversely, when RA receptors are highly expressed in Sertoli cells, they react more to RA than to FSH, which activates the secretion of differentiation factors such as bone morphogenetic protein 4 (Bmp4), but inhibits renewal factors. 66  The BTB is induced and maintained upon gonadotropin and androgen signaling, which regulate the expression of its components in Sertoli cells. 82 Inhibition of these hormones is commonly used to interfere with BTB formation or permeability and study the role of BTB in spermatogenesis. 83,84 Spermatogenesis is severely impacted upon BTB dysfunction as exemplified by studies on SCARKO (Sertoli cell androgen receptor knock out) mice, which lack androgen receptor (AR) in Sertoli cells. 83 Due to the lack of AR signaling in Sertoli cells, the expression of BTB components is decreased in these mice, resulting in higher permeability of the BTB. Affected mice develop auto-antibodies against spermatid-specific proteins, and leukocytes infiltrate the seminiferous tubules, indicating a failed immune privilege in the testis.
However, a direct disruption of BTB components, such as claudin, does not lead to the production of auto-antibodies, nor an infiltration of immune cells into testes. 84,85 This indicates that rather the regulatory effect of AR signaling in Sertoli cells provides immunological protection for differentiating germ cells than the BTB components alone.
Pioneer work by Knud Sand showed that foreign organs can be transplanted into rodent testes, without being rejected by the immune system. 86 Later, it was found that Sertoli cells are responsible for enabling such a strong immune privilege, by influencing both the innate and adaptive immune responses. Sertoli cells can target natural killer cells 87 and inhibit complement factors of the innate immune response, empeding complement-mediated cell lysis. 88 Moreover, Sertoli cells can reduce the adaptive immune response by reducing lymphocyte proliferation through interleukin-2 inhibition. 89

Trophic support of germ cells and waste management
Due to the BTB, passage of blood factors to the adluminal compartment is tightly controlled and restricted. This allows remarkable protection of spermatocytes and spermatids, but makes them highly dependent on Sertoli cells to provide nutrients from the blood. The main energy source for adluminal spermatogenic cells is lactate, which needs to be produced by Sertoli cells in high amounts. 93,94 Sertoli cells use most of their imported glucose for lactate production through glycolysis. 94 Glucose is imported via glucose transporters (GLUTs) from the blood stream and converted to pyruvate. A majority of pyruvate is then converted to lactate and shipped to the lumen of seminiferous tubules via monocarboxylate transporters. Germ cells, which also express these transporters, can then take up the released lactate. 95 This process provides sufficient energy for adluminal germ cells, but leaves little energy from carbohydrate pathways for Sertoli cells themselves. Sertoli cells gain their energy through the oxidation of lipids instead, which is their main source of energy. 96 They receive their necessary lipids by recycling germ cell waste that accumulates during development. If the environment plays a role in the origin of testicular pathologies, it is important to better understand the role of Sertoli cells in sensing and propagating information about the environment. This may help address the growing rate of infertility, which affects around 48.5 million couples worldwide, 106 and also the possible long-term consequences for germ cells.

CHALLENGES
The lack of studies on the role of Sertoli cells in fertility may be due to the difficulties in identifying molecular and functional changes in Sertoli cells. Sertoli cells are rare cells in adult testes 13 and have mostly been studied in vitro. 28 113 Such staining may be transferable to other species, and therefore be less restrictive than transgenic lines which are mostly in mice. While improving enrichment strategies, one also has to consider that Sertoli cells go through various "stages of seminiferous tubules" (modes A and B illustrated in Figure 2). 37  adulthood. The difficulty is that many adult Sertoli cell markers are also expressed in other cells in the body. 116 An alternative method to achieve Sertoli cell-specific alterations in adult animals is to use viral vectors. A few studies already used AAVs and lentiviral vectors to specifically transduce Sertoli cells in vivo. 117,118 It may be possible in the future to treat infertility or allow reversible contraception by developing specific drugs targeting the testicular environment. The tight BTB and the presence of drug transporters in testis, which actively remove drugs from the tissue, 119

CONCLUSIONS
While the nursing functions, immunological properties, and structural role of Sertoli cells have been well studied, more research on their epigenome and their involvement in testicular pathologies is needed.
With the development of new model systems and targeted approaches to manipulate Sertoli cells and their testicular environment, we aim to better understand their responses to environmental factors and their