The well documented source for adult multipotent stem cells is spermatogonial stem cells (SSCs) of mammalian testis. It is foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. SSCs isolation from mammalian testis is difficult because of their scarcity and the lack of well characterized cell surface markers. Thus, the isolation of SSCs is of great interest for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. CD9 is a surface marker expressed in mouse and rat male germline stem cells. In this study, CD9 positive SSCs were successfully isolated from the goat testis using enzymatic digestion followed by three step purification: Differential plating, Percoll discontinuous density gradient followed by Magnetic activated cell sorting (MACS). Percoll discontinuous density gradient showed significant differences in the percentage of CD9 + SSCs across individual fraction. The fraction 36% and 40% gave the highest percentage of CD9 + SSCs i.e. 82% ± 1.2 and 9.2% ± 1.3 respectively. Magnetic activated cell sorting of CD9 + cells in the magnetic fraction of goat testes was in the range of 15% - 18% which is upto threefolds. CD9 + SSCs were further recovered with appreciable efficiency after immunomagnetic isolation by using various bead: cells ratio in which 4:1 ratio gave the highest yield of 69.06 × 105 with 18% of CD9 + SSCs. Magnetic activated cell sorting using anti-CD9 antibodies provides an efficient and fast approach as compared to conventional approaches such as differential plating and percoll discontinuous density gradient for enrichment strategy for spermatogonial stem cells from goat testes for undertaking research on basic and applied reproductive biology.
A great excitement and expectation in today’s biomedical world is the study of stem cells, owning to their ability to exist in an undifferentiated state and transforming into differing tissue types, depending on what the cells ambient are. In recent years, interest in spermatogonial stem cells (SSCs) have grown due to development of new research tools, which allow the isolation and culture of these cells. SSCs originate from the primordial germ cells (PGCs) that have ability to self-renew and differentiate into committed progenitors, thus maintaining spermatogenesis by unipotent stem cell system throughout adult life for sustained male fertility. The SSCs reside in stem cell niches located on the basement membrane of seminiferous tubules and among the basal portions of sertoli cells [1-3]. SSCs are very unique among other adult stem cells because they can transmit the parental genetic information to subsequent generation. The ability to study SSCs biology has been difficult because of their rarity in the testes and very limited availability of unique phenotypic markers. Several surface protein markers are commonly found on stem cells such as ITGA6 (also known as α6-integrin), ITGB1 (also known as β1-integrin) [
Testis of normal and healthy goat (5 - 8 month) was collected from slaughter house maintained at karnal, Haryana India. Immediately after slaughter, testis was washed in PBS solution supplemented with 100 IU/ml Penicillin and 100 µg/ml streptomycin. Testis was stored in ice cold PBS and transported to the laboratory. The testis was again washed in sterile PBS containing antibiotic and dissection was done aseptically under laminar airflow hood. Spermatogonial cells (Donor cell) were collected from seminiferous tubule of the testis.
Donor spermatogonial cell from goat testis was collected by following the protocol of Honaramooz et al., 2002 with some minor modifications [
In the cell suspension, SSCs were identified on the basis of their morphology in phase-contrast microscopy. SSCs are large cells with typical spherical shape and a large nucleus/cytoplasm ratio.
The enrichment of spermatogonial cell was done to eliminate contaminating somatic cells (myoid and sertoli cells) from cell suspension. Enrichment was done by incubating the above cell suspension in DMEM containing 10% FBS for overnight at 37˚C, 5% CO2 and 85% relative humidity. During the incubation the sertoli cell and myoid cell were attached to the wall of culture plate due to its anchorage dependency. On the other hand the spermatogonial cells remain in suspension which was removed carefully using pipette. The collected cell suspension was washed in DMEM and viability assessment was done.
The spermatogonial stem cells were enriched from above cell suspension by following the method of van Pelt et al., 1996; Morena et al., 1996 using discontinuous percoll gradient technique [15,16]. In brief, percoll was first sterilized by autoclaving and used for preparation of iso-osmotic percoll suspension. Iso-osmotic percoll was prepared by mixing 0.6% BSA, 45 µg/ml DNase in 82.2% percoll in DMEM. A discontinuous density gradient (28%, 30%, 32%, 36%, 40%, 50% and 65%) of percoll was prepared by diluting iso-osmotic percoll in diluting medium with final densities of 1.0513, 1.054, 1.056, 1.058, 1.061, 1.077 and 1.095 respectively. The gradient was made in 15 ml graduated tube by adding 1 ml each of percoll solution with different density in a sequence that highest density percoll solution comes in bottom and that of lowest in the top of the tube. The cell suspension containing 0.6% BSA, 45 µg DNase in DMEM was layered on the top of the above gradient and centrifuged at 800 ×g for 30 min at 18˚C. The cells were found in the interface between the different density percoll solution were collected and marked as fraction 1 - 8 from top to bottom.
Spermatogonial stem cells from above fractionated cells were done by using antibody coated magnetic beads [
For immunocharacterisation, the sorted fraction was centrifuged at 500 ×g for 15 min. The pellet containing cells were immunostained with anti-CD9 antibody conjugated with phycoerythrin for 1 hr at room temperature [
The results are presented as means ± SEM and statistical analysis was performed. Differences were considered significant when the P-value was < 0.05.
The phase-contrast microscopy was used to identify spermatogonial stem cells in all the fraction on the basis of SSCs morphology. The unsorted fraction consisted of a heterogeneous single-cell fraction. The magnetic fraction (sorted fraction) consisted of relatively homogenous population of cells (
Enzymatic digestion of testicular tissue was done with collagenase in combination with trypsin and hyaluronidase. Sequential enzymatic digestion of the decapsulated testis resulted in a single-cell suspension. After filtration of the cell suspension final cell concentration was found in the range of 5 - 8 × 106 cells/g testicular tissue and a mean of 85.5 × 106 cells were isolated from a single testis. The cell suspension was checked for CD9+ spermatogonial stem cells and found to be 5.8 ± 1.09 percent. Enrichment of cells by differential plating resulted significant (P < 0.05) increase in CD9+ cells (6.9 ± 1.2,
Second phase of enrichment of spermatogonial stem cells was done by using a gradient of percoll ranges from
28% to 65%. For enrichment gradients were made, cells from differential plating was applied on top and centrifuged. At 28% enrichment of SSC was found to be minimum (0.8 ± 0.22) among all gradient. With increase in percentage of percoll in gradient, the number of CD9+ cells increased significantly upto 36% percoll and decreased gradually upto 65% (
Cell suspension of 36% and 40% fraction were further enriched by immunomagnetic beads tagged with anti-CD9 antibody, showing significantly higher percentage of CD9+ cells in sorted fraction (36%: 17.05 ± 0.5; 40%: 18.2 ± 1.2) compared to unsorted (36%: 8.2 ± 1.2; 40%: 9.2 ± 1.1) and depleted fraction (36%: 4.5 ± 1.02; 40%: 4.4 ± 1.08) in both 36% and 40% percoll gradient. Percentage of CD9+ cells in sorted fraction was statistically non significant (P > 0.05) in both 36% and 40% percoll gradient (
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 (
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 (
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 [
including bulls [
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 [
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.