**1. Introduction**

The testis is a reproductive gland that is part of the internal structures of the male reproductive tract (MRT) and is involved in spermatogenesis and steroidogenesis. Each testis is composed of a tangle of tubes, the seminiferous ducts. These ducts are formed by Sertoli cells (SCs) and the germinal epithelium, which is responsible for ensuring protection and nutrition to accurate spermatogenesis. Leydig cells (LCs) are found in the testis interstitium, adjacent to the seminiferous tubules. LCs promote steroidogenesis through the secretion of male sex hormones, especially testosterone, responsible for the development of male genital organs and secondary sexual characters [1,2].

The testis is considered an immune-privileged organ [3]. This is essential to ensure the immunogenic germ cell protection against immune system activation during spermatogenesis. This is mainly provided by the combination of a local immunosuppressive environment and systemic immune tolerance [4–6]. It has long been assumed that the blood–testis barrier (BTB) constitutes the main mechanism of the immune-privileged status of the testis [7]. In addition to BTB and anatomical impairment of external cells' and molecules' entrance to testis, SCs also provide anti-inflammatory mediator secretion aiming to maintain the tolerogenic microenvironment [8]. However, many local immune modulators, including macrophages, dendritic cells (DCs), natural killer cells (NKs), mast cells and T-lymphocytes, contribute to the intercommunication among testicular components [9–12].

The testis is commonly exposed to pathogens derived from blood, trauma, or through the genitourinary tract. To protect itself against all these pathogens, the testis also needs the ability to overpower immune privilege. This is achieved by inducing local innate immune responses [3]. Even counting this frontline protection, some pathogens have an immune scape mechanism that leads to infection and persistence in the MRT. Reproductive tract infections (RTI) can be caused by bacterial, parasitic, and viral pathogens [13]. RTI promoted by viral infections are notorious, as shown by the World Health Organization (WHO) in 2006, which estimated that 500 million people live with genital herpes, 300 million women have human papillomavirus (HPV), and approximately 240 million people suffer from chronic hepatitis B [14]. In 2016, the WHO also estimated that over 17 million people are living with HIV on antiretroviral therapy. However, the number of HIV-positive cases is increasing worldwide [15].

Some diseases can persist a long time in human semen. Ebola [16], Zika virus (ZIKV) [17], HIV [18], and 27 other types of viruses that contaminate humans have been found in semen and testis for differing periods [19]. Despite the knowledge that various types of viruses can be found in semen, their sexual transmission capacity is still poorly understood. Some of these are not considered sexually transmitted diseases because this route is not the main form of contagion. However, ZIKV has already been confirmed by the WHO to have sexual transmission (World Health Organization, 2016) and considered to be the first arbovirus reported to be associated with sexual transmission [20,21]. Due to this fact, attention is being turned to the possibility that other arboviruses may be present in the MRT. Compared to ZIKV, the literature regarding this effect is scarce, and the available data suggests that arbovirus sexual transmission is a relevant point of concern. The presence of ZIKV in the male genital tract and its ability of sexual transmission leads to unanswered questions such as (1) has the ZIKV a tropism for any specific cell in the male reproductive system?, (2) what features may favor the ZIKV persistence in testicles when compared to other arboviruses?, (3) can the spermatozoa harbor the virus?, (4) how long does the virus remain viable in the male genital tract?, (5) how can the prolonged presence of ZIKV in the male genital tract cause infertility?, (6) is this ZIKV-induced testicular damage reversible? Based on these questions, it is clear the importance of continuing to investigate the role of ZIKV in the male reproductive system. In addition, a vaccine against ZIKV may be the best way to protect the population from infection, and control the disease and its consequences. The vaccination should protect against future and possible damage to the male genital tract, avoiding fertility-related problems. Therefore, in this review, we will address recent findings of ZIKV infections in the MRT, focusing on cellular mechanisms, immune and physiological responses, and the ability to other arboviruses to remain in the testicle.

#### **2. Male Reproductive Tract (MRT) and Cellular Composition of Testis**

The MRT is composed of sexual organs that play a major role in the male germ cells (or sperm) production. It has mainly consisted of a pair of testicles that are specialized for androgen hormones and gamete production, an intromittent organ that is responsible for depositing sperm on the female reproductive tract and finally a couple of sexual accessories ducts and glands vital for sperm maturation, nutrition, and storage [22,23]. The different cell types that compound these tissues of MRT maintain crosstalk that allows the production of viable sperm in the testis (Figure 1). Once the homeostasis of the system is broken, this process is impaired and the fertility capacity is altered [24].

The testis is composed of interstitial LCs located between blood vessels and the seminiferous tubules, where sperm is produced [25]. LCs secrete androgens that participate in conjunction with pituitary hormones (gonadotropin) in germ cell development [26]. On the other hand, seminiferous tubules include the germ cells, which give rise to spermatozoa through a series of differentiation steps and the somatic SCs [23]. Somatic SCs are essential not just for testes formation but are one of the major conductors of gametogenesis [27]. The immunological infiltrate in the interstitial compartment of the normal testis, especially resident macrophages, is also important to directly influence testicular microenvironment [28].

**Figure 1.** Cellular crosstalk during normal spermatogenesis. Pituitary hormones follicle-stimulating hormone (FSH) and Luteinizing Hormone (LH) have an important role in spermatogenesis. FSH leads to Sertoli cell proliferation stimulating the release of inhibin. LH triggers the production of the testosterone by Leydig cells, which can stimulate the release of metabolic and growth factors by Sertoli cells and indirectly trigger spermatogenesis in germ cells. Metabolic factors, such as lactate and growth factors, can directly drive the spermatogenesis in germ cells. Oppositely, inhibin produced by Sertoli cells can inhibit FSH release by pituitary gland acting as a negative feedback regulation.

The seminiferous tubules present an anatomical barrier that impairs the blood-derived factor input to the testicular microenvironment without any regulation [29]. BTB is the main factor responsible for regulating the paracellular transit of molecules. The BTB is the result of tightly cellular junctions of adjacent SCs in addition to epithelial and myeloid cell interaction [27]. The presence of this barrier creates separated compartments and protects against immunological infiltrate that could lead to testicular inflammation [30]. The unbalanced inflammatory response can disrupt BTB integrity, causing non-specific entry of harmful molecules that impair sperm cell maturation. Nevertheless, cytokine release is a regulatory factor during spermatogenesis in controlled levels [31]. It is important to emphasize that the transit of immune cells is not fully blocked once leukocytes have been reported in normal testicular surroundings, especially close to spermatozoa. Macrophages are the most abundant immunological cells that reside in seminiferous tubules environment and present an important role of immune-surveillance of the germ cell development process.

In the testis, macrophage characterization demonstrated novel functions associated with germ cell development, androgen hormone production, and maintenance of a homeostatic microenvironment [28]. Studies have shown that there are two distinct macrophages populations in testicular surroundings: the CD163− newly arrived macrophages and CD163+ resident testicular macrophages. The CD163+ macrophages are polarized to the type 2 macrophage (M2) profile that constantly secretes anti-inflammatory molecules, such as interleukin-10, in the seminiferous tubules acting as a protective component against sperm cell damage [32]. On the other hand, newly arrived CD163− macrophages are related to the inflammation maintained in the seminiferous tubules. These cells secrete higher levels

of pro-inflammatory cytokines, such as interleukin-1β and tumoral necrosis factor-<sup>α</sup>, and present a higher expression of nitric oxide synthase (iNOS), demonstrating a pro-inflammatory profile, a key characteristic of type 1 macrophage (M1) [32]. The communication of these cells with LCs, SCs, and germ cells seems to be important in the development process that leads to sperm production. Macrophages are being called as true sentinels of testis function [28]. In addition, Matusali and colleagues have found evidence of ZIKV infection of the testicular CD163+ resident macrophages [33]. ZIKV-induced cell death of CD163+ resident macrophages could also contribute to the inflammation in testis.

In addition to macrophages, other immunological cells are found in testicular surroundings. DC are antigen-presenting cells found in testicular interstitial spaces and represent a minor population of leukocytes in the testis. DCs induce activation and differentiation of lymphocytes in response to allo-antigens and minimize autoimmune response by tolerating T-cells to auto-antigens under physiological conditions [12]. Other immune cells, including NKs, T-cells, and CD4+CD25+ regulatory T-cells (Tregs), are also found [10]. Besides, mast cells are present in a grea<sup>t</sup> number regarding immune cell populations in the testis during puberty [34]. However, the functions of these cells in the maintenance of testicular immune-privileged sites remain unclear [35].

The process of male mature gamete production is called spermatogenesis and consists of the intense proliferation and subsequent differentiation of spermatogonial stem cells to spermatozoa [25]. The crosstalk between constituent cells of the testis is essential in this process [36,37], once the energy source of gametes during differentiation depends on the lactate that is provided by SC. On the other hand, glucose capitation depends on androgen hormone signalization provided by LC, as well as pituitary hormones, insulin sensibility, and paracrine communication [36,37]. This is one of the central reasons that explains why altering testicular cell metabolism impairs the production of viable sperm [38]. Important factors as epigenetics (including miRNA regulatory activity), growth factors, and cytokine release also influence the process in the quantity and quality of the sperm [39,40].

Spermatogenesis starts in puberty, long after the perinatal self-tolerance process. For this reason, sperm cells contain a new repertory of proteins that present a grea<sup>t</sup> potential of activating an immune response, leading to autoimmunity [3]. Studies have shown that activation of T-lymphocytes and the production of specific antibodies against sperm cells are related to the infertility process. It was also reported that the production of intense pro-inflammatory cytokines is related to loss of BTB integrity and loss of viable sperm, leading to infertility [30,41]. Avoiding this massive activation, the testis presents a unique tolerogenic microenvironment, making the organ immune-privileged, and protecting mature gametes against the immune-cell-induced death and inflammation.

The immune-privileged microenvironment is essential for the viability of sperm cells and maintenance of testis function while, at the same time, serving as a site for the persistence of infections due to the tolerogenic surrounding. Microorganisms coming from blood or urogenital infections enter into a testicular environment and disrupt tissue homeostasis, leading to activation of local immune system [3]. This process triggers testicular inflammation and may alter tissue metabolism, signalization, cellular function, and leading to impaired spermatogenesis and spermiogenesis [42,43]. Many pathogens have been shown to cause male infertility by many mechanisms, induced inflammation being the key for most of them.
