A unique population of stem cells in mammalian testes is spermatogonial stem cells (SSCs) that have the capacity for gamete production and transfer of genetic material to subsequent generations (
SSCs are located close to several supporting somatic cells, including peritubular myoid and Sertoli cells, which may contribute to the formation of an SSC niche. Sertoli cells embrace germ cells of all stages and are joined continuously around the tubule by tight junctions (
This study was initiated to compare Sertoli cells derived from neonatal (NSCs) and adult (ASCs) mice with the intent to examine the effect of Sertoli cell age on the properties of mouse SSCs
All animal care and surgical interventions were undertaken in accordance with the approval of the Royan Institutional Review Board and Institutional Ethical Committee. Male mice (NMRI strain, Pasteur Institute, Tehran, Iran) of various ages (neonatal: 6 days; adult: 6-8 weeks) were used to isolate Sertoli cells. Six-day-old mice were used to isolate germ cells. 6-8week-old NMRI mice were used to make busulfan-induced infertile mice.
Isolation of TCs was performed as previously described (
Dissociated testis cells from testes of neonatal mice were allocated to six-well culture plates (1,000,000 cells/9 cm2). TCs derived from neonatal and adult mice were isolated by two-step enzymatic digestion with 1 mg/ml of collagenase (
Germ cells were isolated from the testes of six-day-old neonatal mice and cultured
Analysis of spermatogonial colonies was undertaken in two steps: the first step determined formed colonies ten days after primary culture, and the second step was accomplished five days after transferring the colonies onto three different feeder layers in order to compare the effects of feeder cells on the colonies’ properties.
The number of colonies in each six-well plate was counted by invert microscopy. Surface area of the colonies was measured with Image J software (National Institutes of Health); the number of cells per colony was measured with 100-150 colonies per group.
To evaluate cloning efficiency [(number of colonies/number of seeded cells) × 100], we analyzed the number of colonies of dissociated single spermatogonial cells. The colonies were mechanically separated from culture plates ten days after primary culture of TCs and singled by an enzyme solution that included trypsin-EDTA (0.05%; Invitrogen) and collagenase IV (1 mg/ml). Subsequently, 200,000 cells per group per replicate were seeded and colony efficiency was evaluated during a ten day period.
Total RNA was isolated using RNXtm (Cinagene, Tehran, Iran) and treated with a DNaseI, RNase-Free Kit (Fermentas) to remove genomic DNA contamination. One μg of total RNA was used for reverse transcription reaction with the Superscript II Reverse Transcriptase (Invitrogen) and random hexamer primer, according to the manufacturer’s instructions.
The primers used in this study were F: 5' CTT ATC CAA GTT CAC CAG TTC 3', R: 5' TGT ATA AGC CGG AGG TAT 3' for Dazl; F: 5' ACT CCA TTA AAC CAG GAA CCA 3', R: 5' CCC ATT TAA TCT CCT CCT TCT C 3' for Stra-8; and F: 5' GAT AAT CAT TTA GCA CAG CCT C 3', R: 5' GTC AAC AGA TGC AAA CAC AG 3' for mvh (vasa). As a positive control, Gapdh was used.
Single cells were obtained by trypsin from picked up SSC colonies on three different feeder layers and fixed in 4% paraformaldehyde in PBS (pH 7.4) for 20 minutes. Single cells were rinsed with washing buffer (2% FBS in PBS plus 0.029 g EDTA) for 5 minutes prior to blocking in 10% normal goat serum in PBS for 15 minutes, followed by incubation with antibody solution overnight at 4°C. Primary antibodies were rat polyclonal anti-α6-integrin (1:100; Sigma), rat polyclonal anti-β1-integrin (1:100; Sigma-Aldrich) and mouse polyclonal anti-c-kit (1:200; Santa Cruz, CA). The following day, cells were washed twice with washing buffer for 5 minutes and incubated with the appropriate secondary antibody [goat anti-rat and goat anti-mouse labeled with fluorescein isothiocyanate (FITC; 1:200; Sigma-Aldrich)] for 45 minutes, and finally washed twice for 5 minutes before analysis. All steps were performed on ice.
Spermatogonial colonies that were cultured on three different feeder layers and confluent Sertoli cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS, pH=7.4) for 20 minutes. Cells were washed twice with 0.1% Tween 20 in PBS prior to blocking in 10% normal goat serum (Vector, Burlingame, CA) in PBS for 15 minutes, followed by incubation with antibody solution against α6 and β1 integrins overnight at 4°C. The following day, cells were washed twice with 0.1% Tween-20 in PBS for 5 minutes, incubated with the appropriate secondary antibody for two hours, and subsequently washed with 0.1% Tween 20 in PBS for 5 minutes. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) in PBS.
To prepare recipient infertile mice testes, busulfan was administered by intraperitoneal injection to mice greater than 4 weeks of age, at a dose of 40 mg/kg, which would almost completely abolish spermatogenesis in most mouse strains (
Cells for transplantation were obtained from cultured SSC colonies ten days after primary culture and from cultured SSC colonies five days after transfer onto NSCs. Cells were incorporated for 24 hours with BrdU (0.1 mM; Sigma-Aldrich) in culture medium to trace the transplanted cells. Colonies were picked up manually, singled by trypsin and counted. About 100,000 cells per 10 μl medium (plus 5 μl trypan blue to visualize the injection) were used to transplant into each testis.
The abdomens of the recipient mice were opened with a 1.5 cm midline incision. Male mice were placed on the platform and the left testis was exposed. After fixation of the testis, the fat pad of the testes was separated and the position of the rete-testis located. The pipette was inserted into the rete-testis and cells were injected into the tubules. Tubular filling was monitored by observing the movement of the cell suspension which was facilitated by the addition of a small amount of trypan blue to the injection medium. Finally, the skin was sutured.
Recipient males were maintained for 4 to 5 weeks before analysis. Testes were removed and dissected from fat for analysis. The transplanted testes of the recipient mice were fixed in Bouin’s solution for 3-4 days, dehydrated and then embedded in paraffin. The sections were immunostained with a primary anti-BrdU (Sigma-Aldrich) such that donor cell-derived spermatogenesis could be visualized.
The results were expressed as mean±SD. All statistical analyses were conducted using SPSS (version 16) software (SPSS, Inc., Chicago, IL; http://www.spss.com). The statistical significance between the mean values was determined by one-way ANOVA and Duncan’s post-test. P ≤ 0.05 was considered significant.
We cultured 1×106 TC in six-well culture plates for ten days in the presence of 1% FBS, 40 ng/ml GDNF and other growth factors. SSC colonies appeared around three to five days after initial plating of the TCs. These colonies were round; however they contained individually detectable cells (Fig 1A, B). After ten days, the numbers of the colonies were 349.5 ± 58.5 and surface area was 23377.3 ± 1364.5 μm2.
Spermatogonial cells were characterized by immunofluorescence and flow cytometry before transferring the colonies to feeder layers (
These spermatogonial cells showed the ability to migrate to the seminiferous basal membrane (
To evaluate the effect of feeder layer on spermatogonial cell culture, ten days after primary culture of TCs, we manually separated spermatogonial colonies from the culture plates without dissociation and then transferred them to Sertoli cells that were either isolated by DSA lectin from neonatal or adult mice and/or STO feeder cells. The morphological appearance of the colonies five days after transfer to different feeder layers showed that the feeder layers did not affect the shape of SSC colonies. Numerous single cells around surrounded the colonies that were cultured on NSCs, which were absent from the two other groups (
After transfer to feeder layers for five days; the numbers of colonies, cell counts per colony and surface areas of the colonies were measured. The numbers of colonies on NSCs were 384.3±42.9, ASCs were 193.2 ± 42.5, and STO were 197.8 ± 38.8 (
Flow cytometry analysis showed a significant increase in the number of α6-integrin-positive cells which were cultured on NSCs (72.9 ± 14.9%) in contrast to those which were cultured on ASCs (29.5 ± 6.5%) and STO (32.5 ± 20.7%); however, there was no significant difference in the number of c-kit-negative colonies (
RT-PCR analysis was used to evaluate the expression of germ cell markers such as Stra8, Dazl, and Mvh in the colonies of the different groups with TCs as a positive control (
Colony efficiency results showed that the number of colonies during the ten days after culture decreased in all groups (
Primary culture of SSC colonies for ten days (A, B). Immunofluorescence staining of spermatogonial colonies for α6-integrin and β1-integrin (C). Flow cytometry analysis of the percentage of a6-integrin-, ß1-integrin-, and c-kit-positive cells (D). Immunohistological tissue section by anti-BrdU shows transplanted BrdU-positive SSCs that are located in the base of seminiferous tubules four weeks after transplantation into the rete-testis of infertile mouse models (E) and the control group (F). H&E staining of testes of infertile mice before transplantation (G).
Morphology of SSC colonies five days after culture on STO, ASCs, and NSCs. (A) Colony analysis five days after colonies were transferred to three different feeders. a: NSCs vs. ASCs and STO, p≤0.05. b: ASCs vs. STO, p≤0.05 (B and C).
Expression of germ cell markers, including Stra-8, Dazl and Vasa in different groups; TC: testicular cells (A). Immunofluorescence staining of colonies on NSCs by anti-α6-integrin and β1-integrin (B). Flow cytometry results of spermatogonial cell markers (α6-integrin, ß1-integrin and c-kit) for spermatogonial colonies five days after co-culture with three different feeders a: NSC vs. ASC, p<0.05, b: NSC vs. ASC and STO, p<0.05 (C). Transplanted BrdU positive SSCs located in the base of seminiferous tubules four weeks after transplantation into the rete-testis of infertile mice models (D).
However, the number of colonies on NSCs was significantly higher in comparison with those on STO (p<0.05) and in comparison with the two other groups (p<0.05). There was no significant difference in the number of colonies at day ten among the different groups. In addition, after transplantation into the testes of busulfan-induced infertile adult mice, the spermatogonial cells cultured on NSCs showed functionality, as verified by their ability to migrate to the seminiferous basal membrane, with no tumor formation (
Colony counts from 2x105 cells per well during ten days culture in different groups. The numbers of cultured colonies were: 114.6±20 (day 4), 113±32 (day 6), 68 ± 25 (day 8), and 40 ± 19.4 (day 10) on NSCs; 85.5 ± 6.3 (day 4), 48.5 ± 6.3 (day 6), 30.3 ± 9.8 (day 8), and 10.5 ± 1.7 (day 10) on ASCs; and 64 ± 15.5 (day 4), 45.3 ± 7.8 (day 6), 25.3 ± 4.9 (day 8), and 12.3 ± 4.2 (day 10), a:NSC vs. ASC, p<0.05, b: NSC vs. ASC and STO, p<0.01.
In this study, we report the positive influence of NSCs in comparison of STO and ASCs in colony number, cell number per colony, and area of colonies, expression of á6- and â1-positive integrins, and cloning efficiency. These phenomena, in co-culture with NSCs, could be a result of the difference in microenvironments that NSCs and ASCs provide for
By considering the biological differences between NSCs and ASCs, such as significantly higher expression of cathepsin L in ASCs when compared to NSCs; the expression of transferrin during maturation and aging of rat Sertoli cells; and increased responsiveness of NSCs to follicle stimulating hormone and decreased responsiveness to androgens (