Journal: International Journal of Molecular Sciences
Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice
doi: 10.3390/ijms22041672
Figure Lengend Snippet: Cyclophosphamide significantly decreased the testicular weight and seminiferous tubule normal histology, VASA cells GFR-α-1, α-6-Integrin, CD9, and C-KIT cells counts in the tubules of immature mice: cyclophosphamide (CP) was intraperitoneally injected (i.p; 100 mg/kg in 100 uL; see methodology section) (CP) or PBS (control, CT; 100 uL). One to 5 weeks after the last injection, mice were sacrificed, and testes were removed, weighed, and fixed in Bouin’s solution for histological evaluation. Changes in the testes weight following CP treatment (CP) compared to control (Control) is presented ( A ). The histology of the seminiferous tubules was examined by hematoxylin-eosin staining ( B ) and a summary of seminiferous tubule damage after 1–5 weeks post CP (CP) treatment compared to the CT is presented ( C ). Ten days post-treatment, the histology of the seminiferous tubules was evaluated by H&E staining ( D ), testes were weighed ( E ), and the total number of cells isolated from the seminiferous tubules were counted ( F ). The presence of VASA-, GFR-α-1-, α-6-Integrin-, CD9-, and C-KIT-positive stained cells in the seminiferous tubules of CT and CP-treated immature mice ( G – K ) was examined by immunofluorescence staining (IF) using specific primary antibodies and Cy3 or Alexa-flour 488 with the relevant secondary antibodies (VASA, α-6-Integrin, CD9, and C-KIT red staining and GFR-α-1 green staining). DAPI (blue color) stained the nucleus of the cells. Arrows show the location of stained cells in the testicular tissues. As a negative control (NC), we stained the tissues only with the secondary antibodies (NC for α-6-Integrin, CD9 and C-KIT were similar and therefore, we present only NC for α-6-Integrin). ( B )—X20 light microscope magnification (100 µm scale). ( D )—X40 light microscope magnification (100 µm scale). ( G – K )—X40 fluorescent microscope magnification (100 µm scale). **— p < 0.01 and ***— p < 0.001.
Article Snippet: Following the removal of the blocking buffer, the first antibodies were added, as follows: Monoclonal mouse anti-mouse Vimentin (Novus, Littleton, CO, USA; 1:500), and polyclonal goat anti-mouse α- sma (Abcam, 1:250), Polyclonal goat anti-mouse Integrin α6 (Santa Cruz, CA, USA; 1:40), polyclonal rabbit anti-mouse VASA (Santa Cruz; 1:100), polyclonal rabbit anti-mouse CD9 (Santa Cruz; 1:100), monoclonal mouse anti-mouse GFR-α-1 (Santa Cruz, sc-271546; 1:50), monoclonal mouse anti-mouse α-6-INTEGRIN (Santa Cruz, 1:50), monoclonal mouse anti-mouse CD9 (Santa Cruz, 1:50), and monoclonal mouse anti-mouse C-KIT (Santa Cruz, 1:50), polyclonal rabbit anti-mouse BOULE (Santa Cruz; 1:50), polyclonal rabbit anti-mouse CREM-1 (Santa Cruz; 1:50), and polyclonal rabbit anti-mouse ACROSIN (Santa Cruz; 1:200).
Techniques: Injection, Control, Staining, Isolation, Immunofluorescence, Negative Control, Light Microscopy, Microscopy
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![Effect of 10-day post busulfan (BU) treatment on spermatogonial cell counts and percentages, as examined by fluorescence-activated cell sorter (FACS) analysis, as follows: BU or DMASO (control, CT) were i.p injected, as described in . Ten days after the injection, the testes were removed, seminiferous tubules were separated, and the cells were enzymatically isolated from the seminiferous tubules. Spermatogonial cells with the membrane markers: alpha-6-INTEGRIN (alpha-6-INT), c-KIT, G-CSF-R, and THY1 were identified by FACS using specific antibodies for each marker ( A ). Spermatogenic cells were identified by immunofluorescence staining for specific markers [premeiotic <t>(CD9,</t> VASA, and SALL4), meiotic (CREM, BOULE, and ACROSIN), and postmeiotic (ACROSIN)] ( B ). The above identified spermatogonial cells were counted and calculated per testicle ( C ), and their percentage was evaluated ( D ). ** p < 0.01 and *** p < 0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1353/pmc06321353/pmc06321353__ijms-19-03804-g004a.jpg)
![Development of spermatogenesis in vitro from BU-treated immature mice: Ten days after BU injection (see ), the testes were removed, seminiferous tubules were separated, and cells were enzymatically isolated and cultured in a methylcellulose culture system (MCS). The MCS was composed of 42% methylcellulose, KSR (10%), StemPro, and growth factors (GDNF (glial cell line derived nerve growth factor), LIF (leukemia inhibitory factor), FGF (fibroblast growth factor), and EGF (epidermal growth factor)) (see Methodology). In some wells, we also added TNF-α (20 pg/mL), FSH (7.5 IU/mL), or testicular homogenates from immature mice (60 µg/mL) or testicular homogenates from GFP-adult mice (60 µg/mL). Every 10–14 days, we added new media containing the same composition of factors that was added in the beginning of the culture. After 4–6 weeks, the developed colonies and cells ( A ) were collected, and the cells were fixed using cold methanol and were stained using immunofluorescence staining, using specific antibodies for markers of the premeiotic (CD9 and VASA), meiotic (CREM, BOULE, and ACROSIN), and postmeiotic (ACROSIN) cells. The effect of FSH and TNF-alpha ( B ) testicular homogenates from immature ( C ) or adult ( D ) mice on the development of spermatogenic cells in vitro compared to before the culture or the control (CT; after culture in the presence of growth factors) were examined. In addition, the development of the sperm-like cells in the cultures was examined according to the DAPI staining of sperm head morphology [ E ; E1—positive control of a sperm head (H); E2—a single sperm head developed in the culture; E3—a group of sperm heads developed in the culture] or periodic acid–Schiff (PAS) staining [ F ; F1—a group of sperm-like cells with heads (H) and tails; F2—a few sperm-like cells with complete morphology of head (H), neck (N), and tails (T)]. ×40 light microscope magnification (100 µm scale). * p < 0.05, ** p < 0.01 and *** p < 0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1353/pmc06321353/pmc06321353__ijms-19-03804-g006a.jpg)