Introduction

The spermatogonial stem cells (SSCs) are the adult stem cell pool capable of self-renewal and differentiation and regulates the sustained production of sperm. They form a niche in the seminiferous tubules, and the fate of the cells for self-renewal, differentiation into sperm, or apoptosis is determined by the signals of the testicular microenvironment [1]. SSCs in the testicular microenvironment are regulated by juxtaposed cells, autocrine, and paracrine factors essential for maintaining the stem cell reservoir [2]. In mice, A-single(As) spermatogonia are single cells that divide and produce inter-connected A-paired (Apr) cells, which further divide in turn to produce four A-aligned (Aln) cells. A-alinged cells again divide to produce chains of eight cells and 16 cells [3]. These are collectively grouped as undifferentiated A-type spermatogonia, expressing most of SSCs markers and possessing stem cell properties.

Improving productivity through augmenting reproduction is the prime focus of the livestock sector. Production potential has been accelerated in livestock with the upcoming assisted reproductive technology (ART) over the period of time. Artificial insemination (AI) is the most successful biotechnological tool that facilitated faster dissemination of the male germplasm using streamlined management of breeding bull [4]. SSC technology is a promising area for faster dissemination of superior male germplasm and also facilitates the production of transgenic animals with high productive and reproductive traits [5,6,7]. SSC-mediated transgenic animal production has the advantage of permanent modification of the germline. SSC transplantation and cryopreservation methods for the restoration of fertility and in vitro sperm production attained varying degrees of success. To establish germ cell transplantation in agriculturally important animals, identification of factors produced in testicular SSCs niche is essential. The establishment of a reliable and robust culture system for SSCs maintenance is of utmost important [8]. The factors regulating SSC self-renewal and exponential growth with inherent stemness remain elusive. A long-term culture system that supports, expands and maintains SSCs from livestock is yet to be developed [9].

Present scenario of SSC technology in farm animals

SSC research has various applications in livestock such as mining factors regulating male fertility, SSC transplantation and faster dissemination of superior germplasm as an alternative to AI, understanding the process and the pathways associated with SSC self-renewal and differentiation and preservation of the genetic material of valuable male [5, 10] and endangered species [11] (Fig. 1). Production of good-quality sperm from infertile animals is possible through transplantation of SSCs from superior fertile bulls [6]. In vitro spermatogenesis though successful in rodents [12] and fishes [13], still has not gained momentum in livestock and humans. However, with the help of advanced bioengineering technology, 3D scaffolds are developed that support the complete differentiation of SSCs into sperm [14]. Germ cells transplanted from donor to a recipient testis resulted in donor-derived spermatogenesis and fertile sperm production in rodents, monkeys, and livestock [15, 16], and offspring with donor germplasm was produced through natural breeding in rodents and livestock [15, 17]. Hence, SSC technology has great potential for the production of superior beef and dairy animals through natural breeding. In humans, SSC technology is advocated for prepubertal cancer patients that enable to restore the fertility at a later phase once the treatments are over. Research is being undertaken for testicular tissue grafting and xenografting, testicular tissue organ culture, and de novo testicular morphogenesis in rodents and humans [18,19,20] for facilitating the production of donor-derived spermatogenesis and restoration of fertility, but to a less extent in livestock. Gene editing in mouse SSCs using CRISPR-Cas9 technology corrected male infertility due to genetic defects and sex chromosome-linked dominant genetic diseases [21]. Such applications indicate the importance of SSC-based fertility-related research in the future, and attempts are carried out in livestock recently [15]. The researches on the isolation, purification, characterization, and culture of SSCs in livestock are advancing in many livestock species such as cattle [17], buffalo [22, 23], goat [24], sheep [25], and pig [26]. However, identification of specific SSC markers, long-term SSC culture and cryopreservation, and SSC transplantation procedures need refinement for successful application in livestock [15, 27, 28].

Fig. 1
figure 1

The various applications of SSCs culture are, a propagation of superior germplasm through transplantation of SSCs from the high fertile to the low fertile animals, b transgenic animal production, c in vitro differentiation of SSCs into the functional sperm, d characterization of SSCs in vitro to understand the physiology of SSCs, unveiling the possibility to predict fertility and to study the genetic and epigenetic changes associated with long-term culture, e understanding the spermatogenesis process by unraveling the mechanism of SSCs self-renewal and differentiation, f cryopreservation of valuable male germplasm

Full size image

SSC isolation

The testis consists of various types of cells such as Sertoli cells, Leydig cells, different stages of differentiating germ cells, and the SSCs. Undifferentiated SSC pool and cell kinetics are identified using prevailing models such as As, Apr and Aal spermatogonia, based on SSC clones in rodents [3]; and Adark and Apale spermatogonia in humans, based on nuclear morphology and hematoxylin staining [29]. In livestock, only limited studies on SSC pool and self-renewal dynamics were conducted as compared to rodents and humans. The subset of SSCs is rare in germ cells population in the testis, as 0.03% in mice [30], 4% in monkey [31], 22% in humans [32], and 0.2 to 0.3% in bovine [33]. The population of SSCs in the testis of other livestock species such as sheep, goats, pigs, and buffaloes is not documented. However, we could isolate 7.33% promyelocytic leukemia zinc finger  (PLZF +)cells from prepubertal sheep testis using enzymatic method [34].

SSCs were isolated from the testis using mechanical and enzymatic digestion methods with high viability in bovine, caprine, porcine, and bubaline [28]. An enzymatic method using collagenase IV, trypsin, DNase I, and hyaluronidase is commonly adopted for the dissociation of livestock testicular cells. Since a single enzyme is not sufficient to isolate effectively SSCs, two-step, three-step, or sequential enzymatic digestion with different enzymes have been carried out [24].

In the mechanical isolation method, tunica albuginea and visible connective tissues are removed, and seminiferous tubules are mechanically dissociated using scissors and forceps. Seminiferous tubular cells are dissociated by repeated vigorous pipetting and passing through a 10-ml hypodermic syringe fitted with a 22G needle. The cells are then filtered through 70- and 40-micron nylon meshes [35]. Enzymatic digestion of testicular tissues yields a higher number of SSCs than mechanical dissociation [36]. The STA-PUT method of SSC isolation combines the enzymatic method with velocity gradient separation [37]. This method yields a higher number of SSCs from the testis as compared to fluorescence-activated cell sorting (FACS) and is also less expensive than a cell sorter or an elutriator methods of isolation [37]. STA-PUT is an assembly of specialized glass apparatus, wherein SSCs are separated by a linear BSA gradient and sedimentation velocity at unit gravity. SSCs get separated based on the size and mass of the cells [38]. The advantages of STA-PUT method are low cost as it only involves the assembly of specialized glassware but yields SSCs with increased purity and doesn’t require specific biomarker [39]. SSCs from mice [40, 41], humans [39], bovine [42], and porcine [43] are isolated using STA-PUT method. A combination of the STA-PUT and magnetic-activated cell sorting (MACS) can be used for isolating the subpopulation of SSCs expressing a particular marker [44].

SSC characterization

Characterization of isolated, cultured, or transplanted SSCs is performed using the cell morphology, biochemical assays, or molecular markers. The morphological characterization of SSCs based on cell shape, size, nuclear to cytoplasm ratio, and chromatin condensation status has been carried out in mice [45], humans [46], bovine, porcine, caprine, and ovine [28, 47,48,49]. Undifferentiated spermatogonia contain euchromatin while differentiated spermatogonia have heterochromatin in mice [45]. Alkaline phosphatase activity is markedly high in undifferentiated spermatogonia as compared to differentiated cells and other testicular cells in humans [50], rodents [51], porcine [52], bovine [53], caprine [54], ovine [34], and bubaline [55]. Though morphological and biochemical methods are used for the initial characterization of SSCs, it cannot be confirmatory in nature. Hence, various biomolecules have been established as markers, which are expressed on the cell surface, cytoplasm, and nucleus (Fig. 2, Supplementary table 1). SSC markers have been identified in cattle [PLZF, Dolichos biflorus agglutinin (DBA), protein gene product 9.5 (PGP 9.5), Thy-1 cell surface antigen (THY1) and GDNF family receptor alpha 1(GFRα1)] [56,57,58]), buffalo (PLZF, PGP 9.5 and THY1) [22], goat [(α6 integrin (ITGA6), PLZF, β1 integrin (ITGB1), Octamer binding transcription factor 4 (OCT-4) and THY1)] [24, 59], sheep [(PLZF, PGP 9.5 and Cadherin 1(CDH1)], and pig (PLZF, DBA, PGP 9.5 and GFRα1) [26, 60]. Even though independent studies have reported these SSC markers in livestock, species-specific markers to differentiate between SSCs and other cell types are still lacking [28]. ITGA6 was reported to be a nonreliable SSC marker when compared to GFRα1 in bovine [58, 61]. PLZF and GFRα1 showed a similar pattern of expression in domestic cat SSCs [62]. Inhibitor of DNA binding 4 (ID4) is an established SSC marker in rodents [63] and humans [64], but not well characterized in livestock. Undifferentiated embryonic cell transcription factor 1 (UTF1), an SSC marker reported in rodents, humans, and monkeys [65, 66], is also conserved as a marker in stallion [67] and pig [68] but has not been documented in other species. It is also to be noted that SSC lineage-wise markers have been established in rodents and humans [69], but not in livestock. The identification of species-specific SSC markers is critical for enrichment and in vitro culture [70]. In addition, identification of SSC surface marker is necessary for isolation of viable cells through FACS or MACS method. Apart from molecular markers, retrospective studies such as tracing the SSC efficiency by teratoma formation and transplantation capability are performed as functional assays [71].

Fig. 2
figure 2

Spermatogonial stem cell markers are identified on the cell surface, cytoplasm, and nucleus. Cell surface markers such as THY1, ITGA6, ITGB1, GFRα1, CD9, PLD6, IGFBP3, SSEA1, CD14, CDH1, CD209, and GFR125; cytoplasmic markers such as UCHL1, OCT4, KLF4, UTF1, FOXO1 and PLZF; and nuclear markers such as FOXO1, OCT4, KLF4, UTF1, and PLZF are pictorially represented on the SSC

Full size image

SSC enrichment/purification

Since the SSC population is very less in the testicular cells isolate, several methods of enrichment procedures have been tried to obtain sufficient cell numbers for culture work. Some of the techniques utilized are MACS, FACS, differential plating, selection with extracellular matrix (ECM), and velocity sedimentation or density gradient centrifugation [58, 72, 73]. The combination of enrichment techniques also significantly augment the purity of spermatogonia [74]. The selection of enrichment method is crucial for successful culture and downstream applications of SSCs.

Magnetic-activated cell sorting

MACS uses the immune magnetic method for the separation of the interested cells population based on their surface markers. The knowledge of the surface marker and its expression in the cells are needed to obtain a sufficient specific cell population for culture and other down-stream applications. In humans, MACS has been efficiently used for isolation of SSCs utilizing markers such as G protein-coupled receptor 125, (GPR 125), CD49f, and THY1 [19]. THY1 (CD90) is used as a marker to isolate SSCs from cattle, pigs, and goats [75,76,77]. THY1 is a reliable surface marker for goat [77, 78], pig [75], and cattle [76]. However, THY1 is not specific to humans SSCs, and the sorting efficiency using FACS also varied between humans and porcine [79]. GFRα1 is used for the isolation of pig SSCs [26]. Surface marker, ITGA6 in humans [73], yielded 3-fold, while DBA-FITC in bovine yielded 4.6-fold [74] and c-kit in rodents yielded 4–7-fold [80] enrichment of SSCs through MACS. This approach is a high-throughput method involving less operational cost and time when compared to FACS. MACS microbeads do not alter the structure, function, and viability and will not interfere with the culture characteristics of sorted cells. However, the purity of cells for certain cell type is not adequate in MACS when compared to FACS due to the sorting of cells based only on cell affinity [81].

Fluorescent activated cell sorting

FACS is a more specific and efficient method employed for the purification of SSCs based on multiple parameters such as cell size, density, viability, and numerous SSC markers. Hence, SSCs can be sorted with high accuracy from the mixture of cells isolate by employing FACS technique [81]. Further, sorting using various surface markers enables to enrich the subset of stem cell population from the whole testicular cell isolates. The surface markers, ITGA6, THY1, GFRα1, and DBA are targeted for SSC separation in rodents and livestock. The enrichment efficiency is high using FACS (12-fold) when compared to MACS (3.3-fold) [73]. In mice, selection of cells based on FACS employing light-scattering properties and expression of the cell surface markers ITGA6, ITGAV, and the c-kit receptor resulted in 166-fold enrichment of SSCs [82]. In bovine, sorting by FACS increases in DBA-positive SSCs from 12.3% in the input sample to 50.5% in the sorted fraction [74]. When coupled with molecular marker screening and the stem cell transplant assay to validate sorted fractions, FACS becomes a powerful tool for dissecting the molecular phenotype of SSCs [73]. Identification of a greater number of specific surface markers in livestock enables to enrich SSCs more efficiently using FACS.

Differential plating

Different extracellular matrix substrates are used for the enrichment of SSCs. Laminin is used for the positive selection [70], while lectin and gelatin are used for the negative selection of SSCs [34]. Laminin is secreted by Sertoli cells as an adhesion molecule and contributes to the basement membrane. Laminin binds to ITGα6/β1 receptors on the spermatogonial cell surface, and this property is used for purifying the SSCs by differential plating. SSCs are plated on such matrixes for improving self-renewal and proliferation for a long time [34, 83, 84]. The enrichment using laminin yielded a higher percentage of SSCs when compared to gelatin. Ficoll gradient centrifugation in combination with laminin differential plating further improved the purity to 64% in the testis of cats [85]. Datura stramonium agglutinin (DSA), a lectin that especially binds to β(1-4) linked oligomers of N-acetyl-D-glucosamine in Sertoli cells is used for the isolation and purification of Sertoli cells [86]. The testicular cells isolated are enriched over DSA lectin (5 μg/mL for 1 h), yielded 2.8-fold (VASA+) enriched SSCs [87]. The overnight incubation in flasks coated with 20 μg/ml DSA results in a 3.6-fold increase in bovine typeA spermatogonia in the nonadherent fraction [74]. Where as in goat DSA- lectin enriches the SSC population up to 11.23% in goat [88]. In addition, combining Percoll density gradient centrifugation with DSA enrichment, the purity of SSC increased up to 60% in buffalo [22]. Gelatin is obtained from the matrix component and favors the attachment of somatic cells, whereas the nonadherent cells are enriched with SSCs [26, 72, 83, 89]. The floating cells from a gelatin-coated plate after 1 day of incubation relatively enriched mouse male germline stem cells for long-term proliferation in mice. In most of the studies, the concentration of gelatin used for differential plating is 0.2% [26, 72, 83], and enrichment efficiency improved on increasing the incubation duration of differential plating.

Combined enrichment methods

A multiparameter selection was adopted to improve the purification efficiency. Isolation of SSCs using multiple parameters such as intracellular complexity along with marker selection α6-integrin+, c-kit -, and α v-integrin -(CD51-) enriched SSCs up to 152- to 166-fold in FACS [82]. A combined enrichment method employing extracellular matrix, laminin, and gelatin effectively purified undifferentiated cells to 2.7-fold from monkey testis [90]. The purity of the SSCs after the combination of Percoll, laminin and gelatin was the highest with 90% [91], while gelatin alone yielded moderate purity of 73.7% [92] and 55% [22]. Centrifugation with 17% Nycodenz (new non-ionic iodinated gradient medium) followed by differential plating with fibronectin and poly-D-lysine coating enriched the purity of piglet gonocytes to >90%. Overall, the variations in the purity levels observed between studies are attributed to the developmental stage of the testis, differences in purification protocols, and selection of the markers for the enrichment of the cells [93].

SSC culture in livestock: nascent stage

The development of a cell culture medium capable of maintaining stemness and proliferation is a prerequisite for the establishment of SSCs technology. Even though SSCs have an important role in animal reproduction, the factors that regulate self-renewal, proliferation, and stem cell fate are least understood. There is a gradual decrease in the proliferation of putative SSCs during subculture over a period of time, as differentiation and apoptosis dominate the cellular events [28]. So research is focusing on the supplementation of appropriate additives, growth factors, matrix substrates, and serum-free supplements in the culture media favoring SSC self-renewal and proliferation in vitro [28]. As the culture conditions are unique for each species, recent studies are oriented towards developing the protocols and components for a particular species [27].

Short-term culture

Short-term SSC culture have been reported in caprine (7 days) [94], sheep (14 days) [59], and bovine (11 days to 3 months) [56, 57, 95, 96]. In livestock, SSC culture system was optimized by the addition of culture media with additives and other growth factors [24, 56, 97, 98]. In these studies, serum was used as an important component in the culture medium for survival and self-renewal of culture cells. Undefined factors in serum might induce cell differentiation [18] and at higher concentrations had detrimental effects on SSCs expansion in culture [87]. Hence, defined culture media without serum and feeder have been developed recently to overcome this problem [99].

Long-term culture

Long-term culture systems are essential for propagating SSCs at an exponential rate for downstream biotechnological applications. Long-term SSCs cultures have been developed in rodents, for maintaining the stemness over 2 years with the capability to undergo spermatogenesis after transplantation into the testis [18, 89, 100,101,102]. In livestock, though few long-term SSC culture has been carried out [89, 92, 95], the culture conditions are not optimized when compared to the rodent models [9]. Few studies have reported a long-term culture system that supports continuous proliferation of bovine gonocytes in vitro; however, the culture duration is less from 1 [92] to 3 months [95]. The evidences of epigenetic stability of cell line, transplantation efficiency,and production of donor-derived offspring are lacking in livestock [89] due to the gap in knowledge for the requirement of the culture media for the maintenance and proliferation of SSCs in domestic animals [9, 27, 103, 104].

Culture on feeder system

Sertoli cells are mainly used as a feeder system for SSC culture. Feeder cells facilitate the cell’s attachment and provide paracrine support for the survival of the cells. SSCs co-cultured using Sertoli cell monolayer as feeder layer increased the self-renewal process in bull [56] and buck [24]. Due to the interactions between SSCs and Sertoli cells, the undifferentiated state of SSCs is maintained [56, 105]. These colonies are also able to regenerate new colonies upon subculturing [106]. The other feeder layer generally used are Sandos inbred mice (SIM) embryo-derived thioguanine-and ouabain-resistant, mouse embryonic fibroblast and laminin-coated plates. The SIM embryo feeder layer is efficient for in vitro propagation of bovine testicular germ cells when compared to mouse embryonic fibroblast, bovine Sertoli cells, and laminin-coated plates [96]. The addition of growth factors like glial cell line-derived neurotrophic factor (GDNF) enhanced the self-renewal of the SSCs [56, 105]. Serum supplemented at lower concentration (1%) in culture medium maintained stemness for a week, while at higher concentrations, it had detrimental effects on SSC expansion [87].

Feeder and serum-free culture

To develop a more efficient and long-term SSC culture medium, several feeder- and serum-free culture systems have been tested and found superior to the conventional culture [107]. Feeder and serum-free culture media with additives like lipid-rich albumin (Albumax), fetuin (blood proteins), and knock-out serum replacement improved the culture medium and maintained the stemness of SSCs in culture for 5 months in mice [107]. The enriched undifferentiated bovine spermatogonia maintained on bovine fetal fibroblast-conditioned medium sustained stemness for 1 month [92]. Such culture systems avoid unknown variable factors in the media and prevent the growth of contaminating somatic cells [103].

Additives in SSCs

The survival, stemness, and proliferation of cells in vitro depend on the growth factors, hormones, vitamins, and other additives supplied in the medium [108]. The media such as DMEM, MEMα, and DMEM/F12 are most widely used for the culture of SSCs in domestic animals. Stem cell-specific media like StemPro-34 and knockout serum used for SSC culture in rodents [107] are proved to support SSCs from livestock species [103]. Apart from the basal media, growth factors such as GDNF, epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), colony-stimulating factor (CSF), vascular endothelial growth factor (VEGF), and insulin-like growth factor 1 (IGF1) are supplied in the SSC culture [108,109,110,111]. Other additives with unique function for cell survival and division are hormones such as FSH [33], insulin [51], beta-estradiol, and progesterone [112]. The energy substances such as D-(1)-glucose, pyruvic acid, DL lactic acid and transferrin, vitamins including ascorbic acid, and amino acids such as minimum essential medium amino acids and L glutamine and buffer Hepes [57], and polyamines like putrescine for cell proliferation are also added. The reducing agents such as sodium selenite and 2-mercaptoethanol are incorporated in the media for preventing the effect of oxygen-free radicals. Further, the culture medium contains antibiotic-antimycotic for preventing microbial contamination. Interestingly, suppression of LH levels in the culture media promotes SSC self-renewal by reducing the expression of WNT5A, and such knowledge can be considered while deciding the composition of the culture media [113].

Growth factor as supplements

Growth factors play a crucial role in regulating the fate of SSCs in the testicular microenvironment. Several factors including GDNF, LIF, EGF, and FGF2 in the culture medium have been reported to positively influence the expansion and stemness of the SSCs [89, 114]. VEGF enhanced SSC proliferation in bovine [115] and mice [110]. Growth factors promote the self-renewal, proliferation, and survival of SSCs through autocrine regulation. The combinations of growth factors appropriate for the culture medium are different for the rodents and bovines [27] and hence need to be studied for a particular species for establishing long-term culture.

Glial cell line-derived neurotrophic factor

GDNF was identified as the major growth factor to maintain stemness and self-renewal of SSCs [56, 105]. The dose of GDNF used for SSC culture ranges from 1 to 100 ng/mL [26, 35, 56, 92]. GDNF knockout mice had depleted spermatogonia, while overexpression of GDNF produced clumps of undifferentiated spermatogonia. Gonocytes proliferate to form clusters of cells in the presence of GDNF and LIF [116]. Supplimentation of these growth factors culture medium resulted in SSC proliferation and long-term culture of SSCs in rodents.

GDNF signals act through GFRα-1 and RET tyrosine kinases in various cell types. GFRα-1 and RET are expressed in gonocytes, SSCs, and differentiated spermatogonia. GDNF produced by Sertoli cells has receptors on the SSCs, acts in paracrine signaling pathways, and mediates SSCs self-renewal [117]. The signaling pathways regulated by growth factor GDNF mainly include Src signaling and Ras signaling pathways. Four Src family kinases are connected in SSC proliferation through RET activation namely Src, Yes, Lyn, and Fyn. Among these, Src and Yes sensitizes the primary SSCs to respond to GDNF. Further, Src activates a PI3K/Akt signaling pathway which in turn leads to N-myc expression and promotes SSC proliferation [117,118,119]. GDNF also activates the Ras/ERK1/2 pathways that result in phosphorylation and activation of transcription factors such as Creb-1, Atf-1, and Crem-1 for the proliferation of SSCs [117]. GDNF and FGF2, in the testicular microenvironment, have been shown to directly control the expression of transcription factors namely Id4, Etv5, and Bcl6b to stimulate self-renewal in SSCs. There is also an intrinsic mechanism that regulates the expression of those factors inhibiting/suppressing the differentiating process [120]. Knockout studies revealed that long noncoding RNAs such as, lncRNA033862 regulate SSC fate, as the lncRNA regulates Gfra1 expression levels by interacting with Gfra1 chromatin [121]. Increased GDNF signaling led to phosphorylation of AKT3 but not of AKT1 or AKT2, in undifferentiated spermatogonia suggesting that AKT3 functions in SSC self-renewal or progenitor cell expansion [88].

In rodents, the long- and short-term culture of SSCs with GDNF are established either alone or in combination with other growth factors or additives [88]. In buffalo, 7–10 [122] and 15 days [123] of SSC culture were carried out. The undifferentiated spermatogonial colonies were maintained in culture for 15 days using the combination of GDNF, EGF, and FGF2 than with the same concentrations of GDNF alone or GDNF plus either EGF or FGF2 [123]. The highest numbers of spermatogonia were obtained in cultures when a combination of growth factors like GDNF, LIF, EGF, and FGF2 was used in the culture medium [57, 96]. FSH hormone was found to stimulate GDNF production by Sertoli cells [124]. The cocktail of growth factors GDNF and FGF2 maintained SSC self-renewal and are used in long-term cultures in rodents [117]; however, no such beneficial effects upon addition of FGF to SSCs culture system in livestock are reported [109].

Epidermal growth factor

EGF has been used in the SSC culture media [57]. The receptors for EGF have intrinsic tyrosine kinase activity, and such receptors are present in SSCs [125]. The EGF and EGFR can promote the overexpression of cyclo-oxygenase 2, which can improve the mitotic activity in SSCs [126]. EGF activates cSrc/STAT pathway of SSCs in vitro and enhances the proliferation of SSCs [127]. The effect of the addition of EGF to the culture medium either as a single growth factor [128], or in combination with other growth factors [59] and additives [129], has been studied with success. Various doses of EGF ranging from 0.1 to 20ng/ml as a single factor or with other factors have been reported to improve stemness and proliferation of the stem cells [128,129,130]. EGF had a positive effect on PLZF expression levels in porcine SSCs [109]. A nonadherent culture system containing growth factors EGF along with GDNF and FGF2 favored in vitro short-term culture in mice and dogs [131].

Fibroblast growth factor

In rodents, the combinations of bFGF with other growth factors, GDNF and EGF, are required for the long-term culture of germ cells [18, 132]. The concentrations of FGF used in livestock are ranging from 5ng/ml in goats [94] to 10 ng/ml in bulls [133] and porcine [109]. However, studies reported the possibility of the negative effect of FGF on porcine and bovine SSC stemness unlike rodents [27, 109]. In bovines, the addition of FGF2 to the culture system enhanced somatic cell proliferation and induced differentiation of the gonocytes [27], whereas in pigs, FGF decreased the expression of PLZF in the culture system [109], although the exact difference in the mode of action is least understood. FGF can induce the production of GDNF and GFRα1 in neural cells, and these act in autocrine pathways to decrease apoptotic signaling [134] and such mechanisms may determine the fate of the cultured cells [18].

Insulin-like growth factor 1

Insulin-like growth factor-1 (IGF-1) secreted from Leydig and Sertoli cells impart stemness to the spermatogonial cells. The blockage of IGF-1 pathway resulted in reduced alkaline phosphatase activity [51]. IGF1R signaling is essential for the proliferation of SSCs by promoting the G2/M progression of the cell cycle [135]. The molecular mechanism, i.e., the cross talk between IGF1/IGF1R and SDF1/CXXR4 signaling has been elucidated to induce self-renewal of SSCs under the hypoxic microenvironment in mice [136]. Such studies open up a new window for manipulations of the SSC culture system. IGF1 (100 ng/mL) in culture media increased the numbers and diameters of SSCs colonies on day 16 and also improved cryosurvivability in bovine [137]. In combination with GDNF and bFGF, IGF1 improves in vitro proliferation of goat SSCs, while preventing the uncontrolled proliferation of somatic cells [138]. Further, IGF-1 promotes spermatogenesis by increasing the round and elongated spermatids and decreasing the apoptosis of germ cells in the culture of mouse testicular fragments [139].

Leukemia inhibitory factor

LIF in combination with other growth factors such as GDNF, EGF, and FGF supplemented in SSC culture systems [57, 59, 109, 138] enhanced the self-renewal capacity. LIF is known to maintain the pluripotency of SSCs [140]. In humans, the combination of bFGF (1 ng/mL) and LIF (1500 unit/mL) produced the largest diameter of SSC colonies [141]. SSCs co-cultured with Sertoli cells with the addition of LIF enhanced the alkaline phosphatase activity and the expression of SSC specific genes [142].

Advanced culture conditions

Recent studies provided insights for modulating the bioenergetics of SSCs culture. Apart from the use of additives for improvising the SSCs culture conditions, optimization of the oxygen tension and metabolic pathways may improve the SSC proliferation in vitro and transplantation efficiency in vivo. SSC culture conditions with reduced oxygen tension maintained stemness for a long term is a progressive step for maintaining stemness [92]. The exact role of oxygen on SSCs remains unknown; few studies established that hypoxia by modulating the expression levels of Hif genes and reactive oxygen species promoting the self-renewal of SSCs [143]. Optimizing the SSC culture conditions that favor glycolytic activity and modulating similar culture environments is essential for maintaining functional integrity [102]. For instance, omission of lipid from SSCs culture enhances glycolysis pathway, which helps in proliferation of SSCs [102]. The addition of 6-bromoindirubin-3′-oxime, an inhibitor of glycogen synthase kinase-3α, enhanced the proliferation of SSC in long-term culture of SSCs in bovine [95]. Though the doubling time for SSCs in culture is relatively slow, the SSCs can multiply 1:2 to 1:4 every 7 days once the culture micro milieu is optimal. Hence, the time required to generate sufficient SSCs needs careful planning [144]. Advanced systems including a three-dimensional (3D) culture system that mimics the testicular architecture in vitro and provide ECM support for the proliferation are also beneficial. Three-dimensional materials such as nanofiber matrices [145] and soft agar culture system [146] are also used for the culture of the SSCs. ECM from homologous species provides the best bioenvironment for SSCs culture in vitro [147].

SSC cryopreservation and transplantation

Successful cryopreservation and transplantation of SSCs are essential procedures that need to be standardized in livestock for wider application of SSC technology and research has been focused in this direction in rodents [148, 149]. The extent of application of SSCs depends on the success of long-term storage of cultured cells by employing the cryopreservation technique. During cryopreservation, cooling and thawing process induce damage to cells and tissue which directly affect their viability [150]. The selection of appropriate cryoprotectant and the cryopreservation protocol is necessary for preserving the cell structural and functional integrity. The permeating cryoprotectants are small molecules less than 100 dalton including DMSO, ethylene glycol, glycerol, glucose, propylene glycol, diethyl glycol, proline, etc. which can penetrate the plasma membrane and prevent excessive dehydration of cells during freezing [151]. The non-permeating cryoprotectants are large in size and can protect the cells from cryoinjury are usually of polymers, polyvinylpyrrolidone, polyethylene glycol, trehalose, sucrose, etc. [152, 153]. Further, the time taken for freezing the cells from room temperature also affects the viability of SSCs. In this direction, cryopreservation of SSCs using cryoprotectants by employing slow freezing with rapid thawing protocol has been successfully tested [148, 154]. Appropriate selection of cryopreservation protocol for SSC storage should reduce DNA fragmentation, mitochondrial damage, oxidative stress, and osmotic stress during cryopreservation [155, 156].

The success of cryopreservation may also depend on species-wise optimization of cryoprotectants, additives, freezing method and selection of sample type, either cultured cells or testicular tissue. On comparing fast freezing, slow freezing and vitrification methods in livestock species, slow freezing has been suggested as an ideal freezing protocol for testicular tissue cryopreservation in terms of protecting cellular integrity and resumption of spermatogenesis [153, 157, 158]. However, vitrification was found effective than slow freezing in rodents [159] humans [160] sheep [161] and cats [162]. The post-thaw recovery and transplantation efficiency were better in testicular tissue freezing as compared to cell freezing in bovine and porcine [153, 158].

In bovine, DMSO either alone [163] or in combination with sucrose [148] or trehalose, [153] have been used for cryopreservation of SSCs. DMSO, with sucrose as cryoprotectants in bovine [148], sheep [164], and pig [165], yielded 70% post-thaw viability and cultured with optimum prolificacy. DMSO in combination with FBS and 200 mM trehalose as compared with sucrose and polyethylene glycol are effective cryopreservation media in terms of post-thaw recovery and transplantation efficiency in bovine [153] and porcine [158]. DMSO and glycerol combination was effective for the vitrification of cat testicular tissue [162]. The combination of DMSO (10%), knockout serum replacement (10%) with trehalose (20%) using slow-freezing maintained cell viability, reduced apoptosis, and cell integrity of bovine SSCs [166]. Cryomedia containing DMSO (10%) and knockout serum replacement (KSR) (5–10%) along with FBS (5%) are also used for cryopreservation of bovine SSCs [167]. In horse, DMSO-based cryomedia coupled with the slow-freezing method is equally good for maintaining the structure, function, or colony-forming abilities when compared with ethylene glycol and freezing (fast freezing, vitrification) protocols [168]. Testicular tissue from piglets vitrified in cryomedia containing ethylene glycol, polyvinyl pyrrolidone, and trehalose did not affect the viability. In this tissue, sperm have been produced following xenotransplantation and offspring through assisted reproductive technique [169]. Recombinant human serum albumin (5%) as serum replacement in the cryopreservation media improved proliferation potential and maintained SSC characteristics, and after post-thaw, the cells were cultured and colonized in the testis upon transplantation of SSCs in mice [170].

In rodents, many additives such as quercetin [156], caffeic acid [171], vitamin E [172], catalase [173], and hypotaurine [155] have been incorporated to reduce oxidative stress and improve cyotolerance, post-thaw proliferation, and transplantation capacity. Additives such as vitamin E and C were used for the cryopreservation of sheep SSCs [174]. Incorporation of apoptosis inhibitors as additives during cryopreservation resulted in varied success in terms of the post-thaw recovery rate of SSCs, as one study reported beneficial [175], whereas another study reported no effect [155]. The effect of apoptosis inhibitors in livestock SSCs is yet to be elucidated. To reduce oxidative stress, additives such as melatonin are used in goat SSC cryomedia [176].

In livestock, there is a need for refinement of cryopreservation technique in terms of identifying the best combination of freezing methods and the cell or the tissue type. The ideal method of freezing to be adopted for cryopreservation of testicular cells or tissue is still not clear. Further, spermatogenesis capability of cryopreserved SSC following transplantation, and the genetic and epigenetic stability needs to be established in livestock.

Donor-derived spermatogenesis was possible through SSC transplantation in cattle, sheep, and goats, and live progeny has been produced from sheep and goats [74, 106, 177]. Xenotransplantation of bovine SSCs could colonize and proliferate in the seminiferous tubules of the mouse but did not progress through the complete process of spermatogenesis [153, 178]. The development of appropriate recipients is also essential for colonization of donor SSCs in the recipient testis, donor-derived spermatogenesis, and production of live offspring through the transplantation of SSCs. NANOS2 gene knock models were produced in mice, goats, pigs, and cattle which can be used as an ideal surrogate recipient for the spermatogonial stem cells transplantations [15]. The knockout males lacked the endogenous SSCs and other germ cell population but had intact seminiferous tubule and other somatic cell populations [179, 180]. Donor-derived spermatogenesis through allogenic transplantation was possible in mice, pigs, and goats [15]. Other than germ cell–depleted genome-edited animals, chemotoxic drugs such as busulfan and localized testicular X-ray irradiation can be used for developing recipients for germ cell transplantation [149, 153]; however, these treatments also damage the other cells.

SSC colony characterization and cell morphology

Spermatogonia are round cells with a high nucleus/cytoplasmic ratio. These cells have a spherical nucleus containing one to three dense nucleoli and many cytoplasmic inclusions that are mostly concentrated at one side of the cell in humans [182], rodents [183], and livestock [59]. During the first week of culture, the spermatogonia appeared as single, paired, aligned, and cluster forms [59]. The morphology of 6-week-old mice SSCs cultured with 0.2% BSA plus 10% KSR were grape-shaped colonies. These colonies were positive for specific germ cells markers, αGENA and TRA98. Culturing of SSCs on mouse embryonic fibroblast feeder cells with 10% KSR, substantial cell growth was observed with a doubling time of 5.5±2.7 days, while the addition of 0.2% BSA with 2 or 10% KSR advanced the doubling time to approximately 4 days [184]. Pig SSC colonies were maintained with undifferentiated morphology for more than 2 months and passaged more than 8 times with doubling time between 6 and 7 days. The colony characteristics were similar to mice SSCs [35]. These results revealed the fact that the growth rate of the colony is influenced by the supplements and their concentration in culture.

Maintenance of stemness: problems and possible solutions

The major challenge in establishing the long-term SSC culture system is the maintenance of stemness and preventing differentiation and apoptosis over a period of time. Understanding the pathways influencing stemness is of importance for the in vitro modification of the culture system for successful self-renewal. In addition, the knowledge on SSC differentiation and apoptosis pathways are essential to adopt better strategies for the long-term maintenance of stemness. Few pathways associated with stemness in SSC have been identified in the recent past. For example, the p38 MAPK pathway has a key role in maintaining the self-renewal in mouse mGSCs [185]. Myc-mediated glycolysis increases the SSC self-renewal and proliferation rate of germline stem cells [186]. Blocking of Ras/ERK1/2 pathway by MEK1/2 inhibitor (PD0325901) prevents the proliferation of goat SSCs and downregulated the ETV5 and BCL6B genes [187]. In mice, constitutive WNT signaling from the gonocytes stage onwards stimulates their proliferation, while forced WNT signaling in germ cells causes spermatocytes to undergo apoptosis [188]. SSCs cultured in the presence of BMP4 underwent differentiation (BMP4/Smad signaling pathway), characterized by downregulation of SSC self-renewal markers such as PLZF and upregulation of SSC differentiation marker like c-kit [189].

Several microRNAs, genes and proteins have been upregulated in testicular stem cells and regulating the pathways towards the maintenance of SSCs stemness (Table 1). MicroRNA  (miR-224) overexpression increases the expression of SSCs markers such as PLZF and GFRα1 [190] and enhances the proliferation of cultured SSCs [191]. The pathway regulated by matrix mellatoprotein2 and mediated via Chd1l-miR-486-MMP2 regulatory axis increased β-catenin nuclear translocation and stemness gene expression [192]. So far the complete pathways involved in the maintenance of stemness and inducing differentiation remain elusive particularly in livestock. In-depth analysis on stemness regulators  may help to alter the SSCs fate in terms of self-renewal, differentiation, or apoptosis.

Table 1 Spermatogonial stem cells regulators and their functions in rodents, humans, and livestock
Full size table

Epigenetic modifications in SSCs

Many studies have demonstrated that epigenetic modification plays an important role in maintaining pluripotency of SSCs and its lineage differentiation [193, 194]. A previous study in mice has reported that embryonic stem cells (ESCs) cultured in commercial media gave rise to abnormalities in offspring, which may be due to alteration in imprinting genes [195]. It was predicted that SSCs grown culture may be more prone to epigenetic changes due to exposure to growth factors and media [196]; however, there is no experimental evidence to support the epigenetic changes in SSCs cultured in vitro. Both DNA methylation [197] and histone modification [198] are major changes reported during the isolation and expansion of SSCs. Histone modifications are involved in the differentiation of SSCs to more advanced sperm cells, but only during S-phase the canonical histone synthesis take place, which plays a key role during the cell cycle [199]. Many locus regions involved in pluripotency maintenance and function show a stage-specific differential methylation pattern [200]. Spermatogenesis can proceed with the transcription of certain DNA methylated promoters [201]. However, during spermiogenesis core histone proteins are replaced by transition proteins which are further replaced by protamines resulting in hyper-compaction of chromatin [202].

SSCs platforms: an inexorable technology for future

SSC platforms may play a pivotal role in the male fertility research in humans and for improving productive and reproductive performance in animal husbandry sectors. SSC researches on transgenesis, genomic-level modifications, and in vitro sperm production are the thrust areas in the near future. For the first time, transchromosomic mice were produced by the introduction of megabase-sized large DNA fragments into SSCs using the retro-microcell-mediated chromosome transfer method, with superior chromosomal stability when compared to embryonic stem cells [186]. In vitro sperm production may have significant implications in farm animal industries, human fertility restoration, preservation of endangered species, and many other technologies related to mammalian reproduction. The economics of animal management in the cattle industry can be reduced if renewable SSC pools from elite bulls produce high numbers of sperm from small biotechnological facilities [103].

Conclusion

Though SSC research started decades back, the pace of advancement of the technology is very slow, especially in mammals. In-depth knowledge on species-specific factors affecting stemness and differentiation of SSC is required for the development of the long-term culture system and in vitro spermatogenesis in livestock. The identification of stemness regulators that may alter the SSCs fate in terms of self-renewal, differentiation, or apoptosis in the culture system is very much essential. Epigenetic changes definitely can happen during the culture and differentiation of SSCs in vitro; however, the impact of such alterations on fertility needs to be investigated. The success rate of SSCs transplantation using the cultured and cryopreserved SSCs is low. In vitro differentiation of sperm from SSC culture though developed in rodent model remains a challenge in human and farm animals.