Transplantation of spermatogonial stem cells in stallions

Spermatogenesis is a stem cell-based process in which functional mature spermatozoa are progressively produced by division and differentiation within the seminiferous tubules that compose the testicular tissue. This process is accomplished through three distinct developmental stages, namely spermatocytogenesis, meiosis, and spermiogenesis [25,26]. In stallions, spermatogenesis is typically completed in 57.4 days [27]. The first stage, i.e., spermatocytogenesis, which takes 19.4 days, is initiated by the development of spermatogonia. SSCs are the undifferentiated, immature germ cells adjacent to the basement membrane of the seminiferous tubules and are capable of self-renewal and differentiation for continuous production of mature sperm throughout the adult life [25]. A previous study showed that A₁, A₂, A₃, A₄, and B are the differentiating spermatogonial populations in mice and pigs [28], while in cattle and sheep, A₁, A₂, A₃, B₁, and B₂ were reported to be the differentiating spermatogonial populations [28]. Recently, eight different subtypes of spermatogonia, namely A_s, A_pr, A_al, A₁, A₂, A₃, B₁, and B₂, have been reported, and A_s, A_pr, A_al spermatogonia are considered SSCs [29]. The two major functions of spermatogonia are duplication by mitosis (A₀) to maintain a progenitor population for the spermatogenic lineage and production of daughter cells by mitosis to generate primary spermatocytes that differentiate into spermatozoa [30]. In stallions, the A₁ spermatogonia are destined to produce primary spermatocytes. Once they are committed to this developmental line, they divide by mitosis in five stages to form 1 (A₁), 2 (A_1.2), 4 (A_1.4), 8 (A_1.8), and eventually 16 potential A₂ spermatogonia. The spermatogonia are connected by intercellular bridges to maintain their developmental lineage. Subsequently, the A₂ spermatogonia divide by mitosis, first into two A₃ spermatogonia, then into two B₁ spermatogonia, and finally into two B₂ differentiated spermatogonia [31]. Eventually, the B₂ spermatogonia divide into two primary spermatocytes that undergo the first meiotic division, which constititutes the second stage of spermatogenesis. In this stage, which also takes 19.4 days for completion, the secondary spermatocyte is formed by division of the primary spermatocyte into two diploid secondary spermatocytes by mitotic division and exchange of genetic material. Two haploid spermatids are then produced from each secondary spermatocyte; this process takes 0.7 days. Spermiogenesis is the final stage of spermatogenesis and is completed in 18.6 days [32]. During this process, spermatids undergo dramatic changes; their nuclei transform, they lose their cytoplasm, and develop tails, thus getting ready to differentiate into mature spermatozoa [33].

The technique of germ cell isolation from testicular tissue is widely used to collect pure individual germ cells. The two-enzyme digestion method is usually applied for isolation in several species, including mouse [34], goat [35], cow [36], buffalo [37], rhesus macaque [38], and pig [39]. This helped in the establishment of a germ cell culture system and determination of various factors affecting spermatogenesis [40]. In addition to germ cell evaluation, it is used to prepare donor germ cells for transplantation into the testis of recipient animals, such as goat [13], boar [41], dog [20], and sheep [42]. The collection of pure germ cells, including SSCs, from the testicular tissue using the two-enzyme digestion technique is essential for successful transplantation because stem cells in donor germ cells are the only cell type that can completely regenerate spermatogenesis in the seminiferous tubules of the recipient testis after transplantation [43]. However, like other adult stem cells, SSCs are present in very small numbers in the testes, and as the testes mature, the number of SSCs decreases. For example, there are only approximately 35,000 SSCs per testis in adult mice, and the percentage of the total number of germ cells in the testis is 0.03% [44]. In neonatal pigs, approximately 7% of the gonocytes were present only in the seminiferous tubules [45]. Thus obtaining large numbers of progenitor cells and other undifferentiated spermatogonia using the isolation technique is difficult [46]. Recently, we evaluated the efficiency of the two-enzyme digestion technique for isolation of the testicular tissue in stallions and demonstrated its effectiveness in isolating high-purity germ cells from the testes [47]. Additionally, specific molecular markers for SSCs, including other germ cells, in the testes of stallions have been developed. Antibodies, such as UTF-1 [48], DAZL [49], PGP9.5 [50], C-kit [51], ACRBP [52], VASA [53], and Lin28 [54], can be used to identify specific germ cells at each developmental stage of spermatogenesis in vitro. In particular, UTF-1 is a specific putative marker for undifferentiated SSCs in stallions [48], and we found that 6.43% of germs cells per 10 g of testicular tissue in prepubertal stallions were positive for UTF-1 [47]. The immature testes are typically used to isolate SSCs for transplantation because they contain a greater number of gonocytes or undifferentiated SSCs than do the mature testes. Nevertheless, this method does not significantly increase the number of isolated SSCs. Hence developing other strategies to enrich the concentration of SSCs to improve the efficiency of germ cell transplantation in stallions is necessary. As an alternative to the use of immature testes, long-term in vitro culture of SSCs is widely known to elevate the number of stem cells [55,56]. Several key growth factors, such as GDNF, GFRa1, bFGF, CSF1, LIF, EGF, and IGF1, which contribute to self-renewal and proliferation of SSCs in vitro have been identified [57,58]. However, the nature of the in vitro culture environment and efficacy of these growth factors on the expansion of SSC numbers in stallions remains to be elucidated. Thus, the next challenge would be to establish an in vitro culture environment to proliferate or differentiate SSCs for transplantation into stallions.

Preparation of the ideal recipient is a key issue in improving the success rate of SSCT. The fundamental purpose of this step is to suppress all the endogenous germ cells while maintaining the full structure and function of the somatic cells in the seminiferous tubules of the recipient’s testes [59]. This provides the space for development of stem cell niches, in which donor-derived spermatogenesis can be re-established in the basement membrane of the seminiferous tubules in the recipient’s testes after transplantation. Thus, several techniques to deplete endogenous germ cells, such as local glycerol injections [60,61], local irradiation [62,63], heat treatment [64,65], systemic busulfan injections [6,14,66], and the use of genetically defective animals with congenital deletion of endogenous spermatogenesis, have been developed [65]. However, no studies have demonstrated the efficacy of these techniques in depleting endogenous germ cells in stallions. In our previous study, we found that intra-testicular injection of 70% glycerol suppressed some germ cells in the seminiferous tubules in stallions. However, it did not completely deplete endogenous germ cells for transplantation [67]. As an alternative, the chemotherapeutic agent, namely busulfan is widely used to deplete endogenous germ cells to prepare recipient mouse models for transplantation. However, the efficacy of this treatment varies in large animals; moreover, knowledge regarding it is limited owing to the differences in physiological structure, composition, and size between species [18]. Additionally, busulfan is systemically toxic; it causes bone marrow suppression and inhibits hematopoiesis, which occasionally leads to lethal effects. Therefore, the species-specific sublethal dose of busulfan required to deplete endogenous germ cells should be determined. A recent study showed that multiple injections of low concentrations of busulfan can effectively deplete endogenous germ cells without any side effects in stallions [66]. However, some problems with transplantation persist. Although treatment with busulfan is more effective than that with glycerol, it cannot completely deplete germ cells, and recovery of the endogenous germ cells is inevitable. This eventually disrupts the re-establishment of transplanted donor SSCs. Therefore, along with depletion of endogenous germ cells of the recipient, determining the optimal time period for regeneration of donor-derived spermatogenesis is important for successful transplantation in stallions.

Three methods are typically employed to ensure sufficient engraftment of donor SSCs into the lumen of seminiferous tubules of the recipient’s testes. In mice, donor SSCs have been successfully transferred into the seminiferous tubules of the recipient’s testes via efferent duct injection [6]. The efferent ducts are directly connected to the rete tubules and epididymis of the testes. The seminiferous tubules converge to form the rete tubules, which carry mature sperm from the seminiferous tubules to the efferent ducts [68]. Thus, efferent duct injection is effective in transferring donor SSCs into the seminiferous tubules of the recipient’s testes. However, this transplantation technique, which is commonly used in rodents, cannot be applied to all animals because of differences in anatomical structures, greater resistance of the lamina propria, and large volume-to-surface ratio [18]. In mammals, the highly coiled, tightly packed seminiferous tubules are lined with individual compartments consisting of inwardly extending tunica albuginea [46]. Therefore, the potential rise in intratubular pressure during transplantation may rupture the seminiferous tubules and membrane of the testes [68]. In bovine animals, donor SSCs are directly injected into the seminiferous tubules of the recipient’s testes; however, this method was found to be difficult and inefficient because of the highly convoluted tubular structure of the bovine testes. The rete testis has a distinct structure and color and can be easily distinguished from the surrounding tissues in the testicles, thus allowing more practical access to the seminiferous tubules [7]. Hence a large volume of germ cell suspension can be injected into the rete testis, thereby improving the efficiency of cell transfer and success rate of transplantation. However, the structural location of the rete testis varies between species. Unlike the rete testis of rodents, such as mice, rats, and hamsters, which is located closer to the subcapsular area of the testis, that of most other species, such as cats, dogs, bulls, boars, and monkeys, is located deep in the center of the testis [4, 68, 69]. Hence, transplantation of SSCs through the rete testis is more difficult and less feasible in large animals than it is in rodent models. To overcome this problem, a method to inject donor SSCs into the rete testis of large animals under ultrasound guidance was developed. However, a previous study showed that the rete testis cannot be identified in stallions using sonography [23]. Therefore, instead of injection into the rete testis, 5 mL of iodixanol-contrasted fluorescein solution was injected into the parenchyma below the epididymis, which helped identify contrast in the vessels extending toward the head of the epididymis. Thus, this previous finding indicated that the testicular injection method may be applicable for SSCT in stallions. Hence, we transplanted SSCs directly into the testicular parenchyma of stallions without identifying the rete testis by sonography; however, we found no donor-derived sperm in the recipient semen after transplantation. This approache revealed an additional issue that the recipient pose and anesthesia should be considered for the successful transplantation. Because since the recipient horse was standing without anesthesia while in performing transplantation, even if the needles were properly inserted into the testicular parenchyma, the needle position and germ cell migration might be interrupted by continuous movement of the recipient. However, the general anesthesia in horses carries a mortality rate 1 in 10 due to they have heavy muscles and organs, which do not function while standing, as well as low blood oxygen levels, which can also cause problems. Moreover, the expertise in anesthesia, appropriate drugs, and sepcific equipments, along with a suitable environment, are required. Thus, the testicular injection of donor SSCs without considering the pose and anesthesia of the recipient is not an optimal transplantation techniques for producing donor-derived sperm [24]. Therefore, alternative approach must be considered to obtain donor sperm without transplanting germ cells into the testes of recipient stallions. Several techniques, such as in vitro single germ cell culture [70], testicular tissue culture [71,72], xenograft [73], and xenotransplantation [74,75], have been introduced to produce donor sperm without transplanting SSC in recipient testis. However, their efficacy in stallions has not yet been confirmed. Overall, the current experimental approaches used to produce donor SSC-derived sperm in stallions are inefficient, and clarity regarding the alternative methods is lacking. Additionally, factors such as enrichment of donor SSCs and preparation of germ cells with mitotically active Sertoli cells must be considered to ensure successful transplantation. Finally, alternative approaches to obtain donor sperm without transplanting germ cells into the testes of recipient stallions are required.