p> cells in sorted fraction was statistically non significant (P > 0.05) in both 36% and 40% percoll gradient (Figure 4). Viability of cells in all the fractions were observed and found to be almost similar (68 ± 5.1).
3.5. Effect of Bead:Cell Ratio on Cell Recovery
Although SSCs represent only 4% of the cells in the testis, these were recovered with appreciable efficiency and purity using a target beads:cell ratio of 4:1 (Figure 5). Total number of cells isolated at 4:1 ratio of bead: cells were 69.06 × 105 with 18% of CD9+ cells. If a lower number of beads, 2:1, were used, the recovery of the total cell isolated were 41.43 × 105 that contain 14% of CD9+ SSCs. When the bead:cell ratio was increased to 6:1, a total of 70.68 × 105 cells were isolated but the percentage of CD9+ spermatogonial stem cells recovery was not increased rather its percentage decreased to 8%, as more non target cells were trapped in cell-bead aggregates.
Figure 3. Number of CD9+ cells in different fractions of percoll discontinuous density gradient. The 36% and 40% fraction of the gradient contained the highest percentage of SSCs. Fraction 28% and 30% of the gradient contained the lowest percentage of SSCs. Values reported are the mean ± SEM. Mean showing different letters are significantly different at (P < 0.05).
Figure 4. Magnetic sorting of specific fraction of percoll gradient (36% and 40%). Sorted fractions contain more number of CD9+ SSCs in comparison to unsorted fraction. Values are Mean ± SEM of three experiments. Mean showing different letters are significantly different at (P < 0.05).
Figure 5. Spermatogonial stem cells were isolated at various bead:cell ratios. The optimal ratio was 4:1 at which the yield of SSCs was highest.
3.6. Immunological Characterization of the Isolated Cell Fractions
The live cells in unsorted, sorted and depleted fractions were characterized using antibody directed against CD9 surface protein by immuno microscopy which revealed that the unsorted fraction contained 5.8% ± 0.66% CD9+ cells. The sorted fraction contained 18.2% ± 1.2% CD9+ cells, indicating three fold enrichment from unsorted fraction (Figure 6). The depleted fraction had no significant (P > 0.05) depletion of CD9+ cells as compared to the unsorted fraction.
The isolation of undifferentiated SSCs from mammalian testicular tissue will expand the knowledge of male fertility and aid in developing technologies to enhance reproductive efficiency along with further exploration of SSC characteristics and mechanisms involved in cell fate decisions between self-renewal and differentiation. The conventional techniques that were used in recent years for spermatogonial cell enrichment was either elutriation  or velocity sedimentation in a BSA gradient at unit gravity [21,22]. Out of the large population of differentiating germ cells within seminiferous tubules, only a small population of spermatogonia resides at the basement membrane of the adult and fully active seminiferous tubule. The isolation of this small spermatogonial population is technically challenging because of their small number, so magnetic beads are specifically useful for their isolation including stem cells from various tissues [23-25] and organ such as bone marrow, muscle and liver [26-28]. Many molecular markers have been used to identify and study undifferentiated spermatogonia and gonocytes such as promyelocytic leukemia zinc finger protein (PLZF) in rodents, non human primates and pigs [29-32], ubiquitin carboxyl esterase L1 (UCHL1) in the bull  and boar [32,34,35], VASA in many species
Figure 6. Bar chart of the number of CD9+ cells in the cell fractions obtained after magnetic separation using anti-CD9 antibody conjugated magnetic beads. Cells were isolated from goat testis. The results are shown as mean ± SEM. The unsorted fraction and depleted fraction consisted of low CD9+ cells. The sorted fraction showed high level of CD9+ cells.
including bulls , boars , primates  & mice , CD9 in mouse and rat . The sorting efficiency of intact cells mainly rely on the availability of specific or particular surface markers on the membrane of stem cells. CD9 acts as a surface marker on the spermatogonial stem cells as several literatures reveals its presence [4,5]. Zou et al. reported enrichment of female germline stem cells using short-type pituitary gland and brain-cadherin (Stpbc), CD9 and interferon inducible transmembrane protein 3 (Iftm3, Fragilis) . However, spermatogonial stem cells can be enriched by selection with an antibody against CD9 of these cell surface molecules. Magnetic cell separation has been described as a successful tool for enrichment of SSCs [40,41].
In the Present study, we sought to isolate enriched spermatogonial stem cell from goat testis. Our results clearly demonstrated that three step purification viz differential plating, percoll discontinuous density gradient followed by magnetic activated cell sorting (MACS) not only decontaminated mature spermatids, spermatozoa and other somatic cell but also substantially enriched the pool of proliferative SSCs from the enzymatic digested heterogeneous testicular cell population. Spermatogonial stem cells were enriched by conventional method of differential plating leading to 7% purification of CD9+ cells by eliminating the somatic cells (myoid and sertoli cells). This is in correlation with PLZF-positive cells of ovine testis study . Thus, suggesting that efficiency of isolation and/or enrichment of cells appears to depend on the maturity of the testis, as at maturity CD9+ cells seem to be abundant . Further an enrichment of SSCs by using discontinuous percoll gradients were performed and showed maximum enrichment at 36% and 40% fraction which was 8% and 9% respectively. Our result is very much consistent with other enrichment studies such as Polychromatin erythroblasts (PCE) from rat bone marrow  and Cardiomyocytes derived from human embryonic stem cells  in which percentage of PCE and cardiomyocytes were highest at (1.040/1.058 = 36%) and (40.5%/58.5% and within 58.5%) percoll fraction respectively. The maximum enriched percoll fraction namely 36% and 40% fraction were further enriched by immunomagnetic beads, showing significantly (P < 0.05) high SSCs enrichment in sorted fraction as compared to unsorted and depleted fraction. Thus, giving an overall enrichment upto 15% - 18% which is in correlation with other study in which spermatogonial cells were enriched upto 25% - 54% when normal testes from Djungarian hamsters, mice and marmoset monkeys were used . The magnetic separation of these two fractions (36% and 40%) of percoll gradient did not show much more enrichment of CD9+ SSCs in comparison to magnetic separation after enzymatic digestion, which indicates that the efficiency of recovery is independent of the number of SSCs in the starting material but was dependent on the ratio of magnetic beads to SSCs. Hence, SSCs were recovered with appreciable efficiency and purity using a target bead cell ratio of 4:1. If a lower number of beads, 2:1, was used, the recovery decreased significantly. When the bead:cell ratio was increased to 6:1, the CD9+ SSCs recovery was not increased, but more non target cells were trapped in cell-bead aggregates. This is consistent with rat mast cell isolation studies where the optimal bead:cell ratio was 3:1 which gave the highest yield and purity of mast cell . We therefore calculated and characterized the unsorted, sorted and depleted fractions indicating that the CD9+ cells were enriched upto three fold as compared to the presorted fraction and the depleted fraction is widely identical to the unsorted fraction because it contains approximately 98% of all the isolated cells and approximately 90% of the CD9+ cells that were present in the unsorted total cell suspension. This is in correlation with enrichment studies of GFRα1 + spermatogonia from adult primate testes where monkey and human testicular cells enriched GFRα1 + cells were threefold and fivefold respectively . Although the procedure to isolate and to enrich SSCs takes about 5 - 6 hr, the viability of the cells is still high ranging between 65% - 70%. This makes these cells suitable for analysis of mRNA expression and protein synthesis and can also be used for culture, preservation and transplantation.
In this study it has been shown that spermatogonial stem cells were enriched upto 15% - 18% when testes from goats were used. The efficiency of separation is probably determined by the binding affinity of the antibody. Magnetic cell sorting is specifically useful for separation of a few cells from a larger number of unwanted cells in the cell preparation [47-49]. Magnetic cell sorting allows the separation of rare target cells with frequencies down to 1 in 1 × 108. These methodological features render this approach appropriate for the isolation of spermatogonia from mature testes. The availability of isolation and enrichment technique would help in studying underlying molecular mechanisms that regulate germ cell development, mitotic proliferation and differentiation of stem cells, meiosis and their regulation in a vertebrate. Additionally, these tools could provide a novel avenue for genetic modification of the male germline and subsequent generation of transgenic livestock with favourable traits such as disease resistance and production of meat or milk containing components beneficial for human consumption. This method will enable the preparation of enriched spermatogonial suspensions for exploration of physiology, reproductive medicine and therapeutic approaches for fertility preservation.
The authors are grateful to Indian Council of Agricultural Research for providing funds for this piece of work.
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