Anatomically, male reproductive tract is constituted by testis, epididymis, vas deferens, urethral adnexal glands (ampullae, vesicular glands, prostate, and bulbourethral gland) and the penis (48). The testis are paired organs located outside the abdominal cavity inside the scrotum. The testicular surface is covered by the tunica albuginea, with a thickness of 1 to 2 mm, composed of collagen fibers and containing the blood vessels (testicular artery and testicular vein) that are visible on the testicular surface, in which each species present a characteristic pattern (49, 50). Regarding muscle constitution, the testis is covered by the smooth muscle dartos tunic and suspended by the cremaster muscle, which contribute to testicular thermoregulation, moving the gonads away or closer to the inguinal-abdominal area (51). This mechanism is related to testicular temperature maintenance, which is essential for spermatogenesis to occur normally. In species that the testis are inside the scrotum, the temperature of the gonads must be between 4° and 7° C below body temperature (51, 52).

More externally, the testis are composed of a fibrous capsule called the tunica albuginea; more internally, there are the septa and the mediastinum, which make up the connective tissue (53). The tunica albuginea gives off the septa that spreads into the testicle. As the septa enters the testis, the testicular parenchyma divides into pyramidal lobes (54). These septa tend towards the central region forming the mediastinum of the testis, and their location can vary from axial to displaced towards the epididymis (55) ( Figure 1 ).

Histologically, the testicular tissue has several cell types, which can be divided in germ cells lineage, from gonocytes to mature spermatozoids, support, and immune cells (56). The extracellular matrix is an association of structural and functional components that provide not only a structural network for cells but also a biochemical and biomechanical signaling that is able to impact on cell physiology directly (57, 58). The testicular ECM is formed by several components as collagen fibers, elastic fibers, glycosaminoglycans and proteoglycans (56) ( Table 1 ).

Functionally, testicular ECM is organized into a specialized structure called the blood-testis barrier (BTB), formed mainly by type IV collagen, laminin, heparan sulfate proteoglycans, entactin and fibronectin (64). This barrier, along with the tight junctions of Sertoli cell membranes, restricts the flow of molecules from the bloodstream, selecting which components will come into the testicular parenchyma (65, 66).

In mammals, the testis consist of two compartments: the interstitial and seminiferous tubules compartments (67). The interstitial one contains nerves, blood, and lymphatic vessels (67). Its main cell type is the Leydig cells, that are responsible for testosterone synthesis. Other important cell types presented in this region are peritubular, endothelial, smooth muscle, perivascular cells, and testicular macrophages (68). Regarding the seminiferous tubules, several cell layers constitute them. In the periphery, there are Sertoli cells and spermatogonium. Inside the tubules, there are spermatic cells lineage, which includes spermatocyte I, spermatids and more centrally, spermatozoids (69).

The testicular microenvironment is essential for the spermatogenesis process to occur normally, providing favorable conditions for anchorage, cell growth, nutrient diffusion, and mechanical support necessary for tissue homeostasis (56). The complex structure of the testis directly acts on sperm maturation and the male hormones production (70). Sertoli cells are the main cell type responsible for the production and secretion of metal ion binding proteins, lipids, proteases, protease inhibitors, hormones, and growth factors. This secretion products act on germ cells, tissue remodeling, spermatid release, basement membrane formation and intercellular junctions (65). Another cell type essential for spermatogenesis is the Leydig cell, which acts directly on Sertoli cells and sperm development (65). Hormones such as testosterone and follicle-stimulating hormone (FSH) act on Sertoli cells gene expression, regulating their activity according to the spermatogenesis cycles (71).

Another important structure that is connected to the testicles is the epididymis, which is divided into caput, body, and cauda, situated longitudinally in the caudal portion of the testis (72). The caput of the epididymis is in the upper portion, while the cauda is located in the lower portion of the testis. The epididymis consists of a long tube that is coiled. In the caput the sperm maturation phase occurs, and in the body and cauda, sperm motility occurs (73, 74).

The development of male gametes is a complex differentiation process that takes place in the testis and produces sperm (75, 76). Spermatogenesis begins with the proliferation and differentiation of diploid spermatogonial stem cells, followed by meiosis of spermatocytes that form round spermatids (77). In mammals, the spermatogenesis process is composed of three distinct phases: the mitotic or spermatogonial phase, in which the gonocytes or stem spermatogonia undergo mitotic divisions until the formation of primary spermatocytes; meiotic or spermatocyte phase, stage in which spermatocytes undergo reduction divisions that result in the formation of spermatids; and the spermiogenesis or differentiation phase, the period in which the morphological and functional changes of spermatids occur until the moment of their release into the lumen of the seminiferous tubule, where they become spermatozoa (38, 78, 79). The stages of differentiation of primordial germ cells into mature sperm are schematized in Figure 2 .

During embryonic development, primordial germ cells migrate from the yolk sac region to the undifferentiated gonads (80). After reaching the forming gonad, primordial cells begin the process of division before forming gonocytes. In males, these gonocytes undergo differentiation before puberty to form A0 spermatogonia, from which other germ cells originate (81). During the first phase of spermatogenesis, the proliferation and differentiation of spermatogonia occurs (75). The types of undifferentiated and differentiated spermatogonia vary according to the species (82).

In mice and rats, spermatogonia are divided into three types: A, Intermediate and B. Type A spermatogonia are subdivided, according to morphological criteria, into A isolated (Ai), A paired (Ap), A aligned (Aal), A1, A2, A3 and A4 (83). This classification varies according to the degree of differentiation, with Ai spermatogonia being the least differentiated and A4 the most differentiated within the type A subdivision. (84). Ap and Aal spermatogonia are called proliferative spermatogonia, A1 to A4 spermatogonia as in differentiation, Intermediate and B spermatogonia as differentiated (85). In humans, spermatogonia divide into dark type (A_dark), pale type (A_pale) and type B. Type A_dark spermatogonia reproduce through mitosis that generate both A_dark and A_pale spermatogonia (81). During adult life, undifferentiated A_pale spermatogonia divide, giving rise to B spermatogonia. A_dark spermatogonia are quiescent reserve cells, as they show low proliferative capacity throughout spermatogenic activity, while A_pale spermatogonia are in continuous proliferation (86). Although both types of spermatogonia (A_dark and A_pale) are in the basement membrane, they differ morphologically, with A_dark being small, round, or slightly ovoid, while A_pale are larger, oval or nearly round (87).

The period of spermatogenesis varies according to the species. In cattle, for example, the entire spermatogenesis process lasts an average of 61 days, divided into three phases (33). The initial phase, known as spermatocytogenesis, is the process by which germ cells undergo mitotic divisions, and after the first division there are germ stem cells (type A spermatogonia) and primary spermatocytes (from type B spermatogonia). To give rise to primary spermatocytes, the mitotic division of A1 spermatogonia into differentiated cells called A2, A3, intermediate, B1 and B2 spermatogonia must occur (88). During the second phase, there is a reduction in the number of chromosomes, originating a haploid cell, carrying out the recombination and segregation of the genetic material. In this way, the primary spermatocytes resulting from the first phase begin DNA replication, later entering the first meiotic division to produce spermatocytes. Secondary, such spermatocytes rapidly enter the second meiotic division resulting in rounded haploid spermatids (89, 90).

During the third phase, called spermiogenesis, no more cell division phases occur, so during this phase, spermatids suffer morphological changes, differentiating into spermatozoa (91). These changes include the formation and development of the acrosome and flagellum, chromatin condensation, remodeling, and elongation of the nucleus. Besides that, there is a cytoplasm removal, which occurs before the spermatid release during the spermiation, a phase characterized for the spermatozoa releasing into the seminiferous tubules lumen (92, 93). However, these spermatozoa do not have the ability to fertilize the oocyte, and it is necessary for these spermatozoa to pass into the straight tubules, reach the rete testis and reach the epididymis, where they start the maturation process (94, 95). Thus, in bulls the mitotic division phase lasts 21 days, the meiotic division phase 23 days and spermiogenesis 17 days, resulting in 61 days of spermatogenesis (88), and in humans and rats, the spermatogenesis process lasts 74 and 35 days, respectively (96, 97).

Spermatogenesis is also regulated by endocrine factors depending on the activity of the hypothalamic-pituitary-testicular axis, whereby Gonadotropin Releasing Hormone (GnRH) stimulates the anterior pituitary to release Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH), that will produce stimuli to produce gonadal steroids and for the development of germ cells (98, 99). LH hormone receptors (LHR) stimulate the process of steroidogenesis and act during the development and maintenance of spermatogenesis (100), their expression takes place in Leydig cells and are essential for fertility in mammals (68).

During spermatogenesis, three hormones are essential for the process to occur without modification, namely testosterone, FSH and LH. The lack of any of these hormones cause germ cell apoptosis, and when administered, these hormones suppress apoptosis (101). Therefore, germ cells require the presence of these hormones for their survival. When there is a lack of testosterone, round spermatids do not complete the transition phase to elongated spermatids, as there is a loss of spermatids binding to Sertoli cells (102). FSH acts indirectly through Sertoli cells, being associated with early stages of spermatogenesis, especially during spermatocytogenesis and meiosis (103). On the other hand, testosterone is present in the later stages of spermatogenesis, such as in the spermatid differentiation stage and potentiating the effect of FSH (104).

In cattle, Leydig cells acquire the ability to respond to the LH stimulus at puberty (105), which begin to produce increasing amounts of testosterone, exerting control over Sertoli cell differentiation and, consequently, cell growth (101). Therefore, any interruption or alteration that occurs during the spermatogenesis process can generate changes in the pattern of cell development, affecting the reproductive capacity of animals (106). Testicular hormone regulation is summarized in Table 2 .

Testicular tissue cryopreservation is one of the alternatives to preserve human and animal fertility. Protection of male fertility can be performed by several techniques such as progenitor cells cryopreservation, testicular stem cells cryopreservation (spermatogonic stem cells or SSCs) or cryopreservation of testicular tissue fragments (107). The freezing of semen is the standard and most used technique for the preservation of male fertility in men and animals, being routinely performed in clinics and farms (108, 109). However, this technique is unfeasible when dealing with juvenile and prepubertal individuals, whose gonads have not yet started to produce sperm, or in adults in which mature sperm is not produced due to the occurrence of pathological and genetic disorders (110).

In such cases, testicular tissue cryopreservation is one of the viable alternatives (111, 112). The testis has a high number of germ cells, especially spermatogenic cells, which can offer an unlimited number of male gametes, when properly cultivated and preserved (33). This technique is used as treatment for several types of cancers, which mainly affect the spermatogenesis niche, and induce the death of spermatogonial stem cells (SSCs), reducing the sperm count in men (82). In addition to that, aiming to seek endangered species conservation, several studies have been carried out using this technique to try to preserve male reproductive tissue samples to further uses (32).

Studies have demonstrated that methods of collection and preservation of testis from sexually immature individuals, and from adult animals, alive or postmortem (113, 114). After immediate tissue recovery or cryopreservation, the fragments can be cultured in vivo or in vitro to obtain viable sperm (115). That is, the cryopreservation of testicular tissue fragments is used to preserve the fertility of prematurely dead animals, as well as those undergoing treatments that cause infertility, such as cancer treatment. The spermatogenic cells, present in the testicular fragments, can resume their functions in vitro after thawing, the genetic resources of high-value animals and the preservation of endangered species (116, 117).

Studies using both animal and human tissues generally advocate a DMSO-based cryopreservation medium for immature tissue cryopreservation and a glycerol-based medium for mature testicular tissues (35, 112, 118). Testicular tissue cryopreservation can be performed using techniques such as slow freezing, fast freezing, and vitrification; however, protocols for using these techniques are still being tested in different species (112, 119, 120).

Slow freezing is performed using a machine that gradually reduces the temperature until the frozen state of the testicular fragments is reached. The fragments are initially exposed to an equilibrium solution containing cryoprotective agent (CPAs) at 4° for 10 to 15 minutes, after which they are transferred to cryogenic flasks and stored in liquid nitrogen (121). This method allows the tissue to be less exposed to the deleterious effects of CPAs, however there is a high possibility of crystal formation during the process, which may invalidate the use of tissue after thawing (122). It is mostly used in immature human tissues and is associated with the survival of spermatogonia (35).

Vitrification is a technique widely used in the cryopreservation of female gonadal tissue, and in the research of this technique for the cryopreservation of testicular tissue, the technique used was solid surface vitrification. This technique consists of exposing the fragments to a vitrification solution, after which the fragments are placed in a metal cube above the liquid nitrogen, which allows the tissue to be cooled in an ultra-rapid way and, after freezing, they are stored in cryotubes and maintained in liquid nitrogen (123). Among the cryopreservation techniques, the most common methods are slow freezing and vitrification. Vitrification has the lowest operating cost and is easy to perform, in addition to avoiding crystallization in a more effective way than the other techniques due to the ultra-fast cooling (124).

During the freezing and thawing processes of the material, the loss of spermatogonia is inevitable, and to improve cell survival, cryoinjury caused by the formation of intracellular ice crystals must be avoided through the addition of cryoprotective agent and the control of freezing and thawing rates. Testicular cryopreservation is an economical and efficient method to preserve genetic material; however, its techniques are still being tested and improved, so there are several approaches that involve numerous positive and negative points to be improved. The procedure involves invasive surgery, therefore, it is extremely important to select patients, so that those who undergo testicular tissue cryopreservation are more likely to benefit from future applications, in addition, patients who need gonadotoxic therapy are at an additional risk of bleeding and infection, particularly those with hematologic disorders such as leukemia or aplastic anemia (118, 125–127).

Sperm cryopreservation is an effective method used in the management and preservation of fertility of animals and humans through assisted reproduction techniques (ART) (128, 129). This technique is based on the freezing of sperm to maintain its viability and functionality, and when performed correctly, it allows long-term freezing, as it results in the arrest of cellular metabolism that prevents cellular aging, maintaining viability and fertilization potential, an essential part for ART (130). The preservation of spermatozoa by freezing has the first record in 1776, but it was only in 1949 that the cryopreservation technique had its scientific progress with the discovery of the cryoprotective properties of glycerol (131), this advance being a point of departure within the field of fertility preservation (132). From this advance, there were significant improvements in the cryopreservation of semen of several species (133) with the creation of sperm cryobanks that took place during the 1960s for bovine species and in the 1970s for humans, the constitution of genetic resource banks began (134).

There are several conventional methods available for cryopreservation of human and animal semen: slow, fast and ultra-rapid freezing (known as kinetic vitrification) (135). The slow freezing method consists of progressive cooling divided into two or three stages over a period of 2 to 4 hours (136). In the first stage, sperm collected by ejaculation or other techniques is kept at room temperature for 10 minutes (137). In the second step, there is a gradual cooling of the samples from a temperature of 20°C to 5°C with a cooling rate of 0.5-1°C/min. After reaching a temperature of 5°C the samples are cooled again from 5°C to -80°C at a rate of 1-10°C/min, and finally, in the third step, the samples are frozen in liquid nitrogen (138). However, slow freezing leads to the formation of ice crystals resulting in high concentrations of electrolytes inside the cell, causing physicochemical damage to spermatozoa (139).

The rapid freezing method is based on the direct contact of samples with liquid nitrogen vapor for at least 10 minutes, in this method the sperm are mixed with cryoprotective agent and placed in cryotubes that will be exposed to nitrogen vapors. After the vapor exposure phase; the samples are immersed in liquid nitrogen (140, 141). The addition of CPAs to the samples seeks to minimize osmotic damage and prevent intracellular and extracellular ice crystals from forming, however, CPAs have cytotoxic characteristics (142).

Vitrification is the process of solidifying a liquid substance at extremely high freezing rates, transforming the liquid sample into an amorphous solid state (143). This freezing process prevents the formation of ice crystals (144). In addition, unlike the slow and fast methods, during vitrification, CPAs use is eliminated, as this method is cryoprotectant free (145). When used as a cryopreservation method, vitrification is commonly used for oocytes and embryos (146), since sperm vitrification is still a challenge due to the greater osmotic fragility of sperm when compared to other reproductive tissues (147).

Sperm cryopreservation is a tool for fertility preservation, sought by men who wish to start ARTs, in some cases being the only opportunity for couples to have children in the future (148). In addition, it is considered before starting any medical procedure that may affect male fertility, as in the case of non-malignant and malignant diseases, where it is necessary to submit the patient to chemotherapy, local radiology or radical testicular surgery (138). This technique is also recommended for men who have had a vasectomy. More recently, transgender patients, who chose to save their gametes for later use (149), have used cryopreservation. When used in animal species, cryopreservation is used for artificial insemination by making use of frozen and thawed sperm to improve rates of genetic improvement (150). It is considered a valuable tool, as it allows the preservation of genetic material from endangered species, through the storage of these gametes in cryobanks (5, 151).

Despite the high success rates in fertilization using cryopreserved semen, the technique has limitations such as intracellular and extracellular ice formation, osmotic and oxidative stress and toxicity from the use of cryoprotectants. These factors are responsible for cellular damage in the cryopreservation and thawing process (93, 152). Damage occurs to a greater degree during thawing, causing an imbalance in reactive oxygen species that directly affects cell metabolism and signaling, as well as DNA integrity and plasma membrane function and integrity (153). Recent studies point out that non-coding RNA (ncRNA), chromatin remodeling, DNA methylation and post-translational histone modifications are among the epigenetic factors involved in gene expression that are affected by cryopreservation and the thawing process (21). Sperm motility is the morphological parameter most affected by cryopreservation and thawing, caused by mitochondrial damage, sperm tail deformities and sperm membrane alterations (140).

Germ cell transplantation is an innovative technique that began to be used in 1994. The technique consists of isolating spermatogonial stem cells (SSCs) from a donor animal of interest and transplanting these cells into the testis of the recipient animal. After transplantation, the transplanted germ cells will continue their development and form mature and fertile sperm bearing the genetic characteristics of the donor animal (154). In recent years, this technique has been applied to mammalian species, in order to understand the processes of spermatogenesis and the biological characteristics of stem cells (155). In addition to these applications, the technique has a high potential for use in research related to biotechnology, genetically modified animals, and preservation of genetic material from endangered species or animals of high economic interest (38).

The use of this technique demonstrated for the first time that germ cells could be transferred between species and between animals of the same species (65). When microinjection of donor germ cells is performed in the seminiferous tubules of infertile recipients, a part of the donor germ cells moves to the periphery of the seminiferous tubules, moving through the Sertoli cell junctions. Due to the absence of spermatogenesis in recipient males, donated germ cells can penetrate the epithelial layer of Sertoli cells and reach the basal lamina (84). Considering the importance of understanding the steps involved during the in vitro spermatogenesis process and seeking to establish a favorable environment for the development and maturation of SSCs, further studies are still needed to prove the efficiency of SSCT in animals of the same species and between animals of different species.

Testicular tissue grafting is a technique that has been studied to restore fertility. When compared to other restoration methods, grafting has several advantages such as the SSCs remaining within their microenvironment (avoiding the need for isolation and cell expansion in vitro) and providing an in vivo environment for the complete proliferation, differentiation, and maturation of germ cells (156). The complete process of spermatogenesis by the testicular tissue graft technique, that is, by allografts and xenografts, showed promising results in non-human species such as mice (157), hamster (158, 159); rabbit (160); bovine (161); rhesus monkey (162); horse (163); cat (164); dog (119) and buffalo (165).

In 2019, the first female offspring were born using sperm from grafts from prepubertal rhesus macaques (32). Despite the great advance in non-human species, in humans, xenotransplantation of immature testicular tissue (ITT) with spermatogonial cells was not able to carry out complete spermatogenesis, not producing sperm (166). Autologous grafts in humans have not yet been reported, therefore, data on testicular tissue autotransplantation in animal species provide important knowledge for the future application of this technique in human fertility (167).

As with all reproductive technologies, testicular grafting also has disadvantages, for patients diagnosed with neoplasms, autograft presents the risk of reintroduction of residual malignant cells present in cryopreserved ITT fragments (168). Therefore, continuous research to overcome the limitations of testicular grafts and provide information for application in humans is essential so that in the future this technique provides chances of success for patients to have biological children (169).

Conventional treatments for of male infertility and subfertility are still not effective related to problems associated to spermatogenesis disorders (170). Several testicular cell culture systems have been studied to mimetize the testicular microenvironment and restore fertility. An emerging field is reproductive tissue engineering (RTE), which is based on the same principles applied for vital organs as the heart, lungs, kidneys, liver, and skin (171–173). However, RTE must, in addition to reestablishing tissue integrity, generate a suitable microenvironment for the development of germ cells (174). Male reproductive tissues are under daily endocrine stimulation, with the rise and fall of testosterone, present a complex microarchitecture that changes due to intense hormonal flow (175). Aiming to mimetize these elements and testicular ECM, several biotechnological tools have been used to reconstruct testicular tissue and carry out the process of spermatogenesis in vitro (173) ( Figure 3 ).

The understanding of the mechanisms involved during reproductive system development and how reproductive diseases occur has been of great value to develop in vitro models of reproductive tissues (41, 176). The advance of bioengineering has allowed the study of the male reproductive system in an innovative way by introducing new biomaterials that simulate organs and tissues that make up this system (177), by making bioprinted models (178–180). The microfluids (181) and biogels (58) production also may assist on cell development. The insertion of three-dimensional cell culture methods (182), maintaining physical and biochemical contact between cells and the tissue architecture brought the in vitro reality even closer to in vivo.

Studies report that cell-cell interaction in 3D culture systems influences the regulation and maturation of germ cells (183–186). 3D cultures favor the bidirectional communication between spermatozoa and the somatic cells that surround them, which are required for a proper testicular functioning and development (1, 187).

One of the limitations of in vitro spermatogenesis biotechnologies is to mimitize the testicular tissue microenvironment in vitro, as the interaction of gametes with the extracellular environment provides the necessary conditions to remain viable (188, 189). In search of alternatives to reproduce physiological conditions in vivo in the laboratory environment, tissue engineering approaches have been used to produce biomaterials that can reconstruct the structure of testicular tissue or assist in the process of spermatogenesis in vitro (190). Natural polymers such as collagen, fibrin and alginate have been explored because they are biomaterials with known biocompatibility and biodegradability (191). Synthetic polymers have also been studied to recompose the structural substrate for the maintenance of spermatogenesis, such as polyethylene glycol (PEG) and poly (epsilon-caprolactone) (PCL) (180, 192). Such materials can be chemically altered to adapt to certain conditions, however, as they are of synthetic origin, there are limitations in more accurately mimicking the extracellular environment (188).

Therefore, several synthetic and natural materials have been developed as extracellular matrix scaffolds, biogels, biodegradable polymers of gelatin, fibrin, collagen, hyaluronic acid, and poly (lactic-co-glycolic acid) that are widely used in tissue engineering (174, 193, 194).