Int J Stem Cells.  2015 Nov;8(2):134-145. 10.15283/ijsc.2015.8.2.134.

Induction of Spermatogenesis by Bone Marrow-derived Mesenchymal Stem Cells in Busulfan-induced Azoospermia in Hamster

• Tamadon A
• Mehrabani
• Rahmanifar F
• Jahromi AR
• Panahi M
• Zare S
• Khodabandeh Z
• Jahromi IR
• Tanideh N
• Dianatpour M
• Ramzi M
• Koohi-Hoseinabadi O

• KMID: 2380808
• DOI: http://doi.org/10.15283/ijsc.2015.8.2.134

Abstract

BACKGROUND
Bone marrow-derived mesenchymal stem cells (BM-MSCs) have potential of differentiation and they secrete anti-inflammatory cytokines and growth factors which make them appropriate for cell therapy. AIM OF THE WORK: Were to evaluate the healing effect of BM-MSCs transplantation on germinal cells of busulfan-induced azoospermic hamsters. MATERIAL AND METHODS: In the present experimental case control study, BM-MSCs were isolated from bone marrow of donor albino hamsters. Five mature male recipient hamsters received two doses of 10 mg/kg of busulfan with 21 days interval to stop endogenous spermatogenesis. After induction of azoospermia, right testis of hamsters was injected with 106 BM-MSCs via efferent duct and the left one remained as azoospermia control testis. Five normal mature hamsters were selected as normal intact control. After 35 days, testes and epididymis of three groups were removed for histological evaluation.
RESULTS
Histomorphological analyses of BM-MSCs treated testes and epididymis showed the epithelial tissue of seminiferous tubules had normal morphology and spermatozoa were present in epididymis tubes. Spermatogenesis was observed in most cell-treated seminiferous tubules. The untreated seminiferous tubules were empty.
CONCLUSION
Transplanted BM-MSCs could successfully induce spermatogenesis in seminiferous tubules of azoospermic hamster. Therefore, BM-MSCs can be an attractive candidate in cell transplantation of azoospermia.

Keyword

Bone marrow; Mesenchymal stem cell; Azoospermia; Cell therapy; Busulfan; Hamster

MeSH Terms

Animals
Azoospermia*
Bone Marrow
Busulfan
Case-Control Studies
Cell Transplantation
Cell- and Tissue-Based Therapy
Cricetinae*
Cytokines
Epididymis
Humans
Intercellular Signaling Peptides and Proteins
Male
Mesenchymal Stromal Cells*
Seminiferous Tubules
Spermatogenesis*
Spermatozoa
Testis
Tissue Donors
Transplants
Busulfan
Cytokines
Intercellular Signaling Peptides and Proteins

Figure

• 

  Fig. 1 (A) Designed micro-injector consistent of a 1 mL syringe (without needle), tube of a 24 gage butterfly needle, and a pre-pulled glass pipette. (B) To make a good contrast between the translucent efferent duct and the fat tissue and the membrane around it, a triangular hard black plastic card was inserted underneath the duct. (C) Bone marrow-derived mesenchymal stem cells (BM-MSCs) transplantation (106 cells were mixed with trypan blue) into efferent duct of hamster testis. (D) Seminiferous tubules were filled with mixture of BM-MSCs and die.

• 

  Fig. 2 Morphological and phenotypic characteristics of hamster bone marrow mesenchymal stem cells. (A) At passage 0, earlier stage. Diverse morphologies including attached spindle-shaped and flattened cells and round other bone marrow cells. (B) Stem cells exhibited large, flattened or fibroblast-like morphology in passage 3.

• 

  Fig. 3 Bone marrow-derived mesenchymal stem cells of hamster cultivated in (A) osteogenic medium and stained with Alizarin Red (×100), (B) in adipogenic medium and were stained with Oil Red O at day 21 after induction (×200), and (C) in culture media without differentiation medium as control (×100). (D) Activation of specific mesenchymal marker (CD29 and CD73) compared with deactivation of specific hematopoietic marker (CD45) of hamster bone marrow-derived stem cells.

• 

  Fig. 4 (A) Sections of seminiferous tubules of normal control hamster. Tubules have condensed spermatogenic epithelium. (B) Sections of seminiferous tubules of treated hamster with bone marrow mesenchymal stem cells. Most of tubules appeared to be filled up with spermatogenic cells. (C) Presence of different kind of germinal epithelial cells in stem cell treated seminiferous tubules. (D) Busulfan treated azoospermic control seminiferous tubules. The seminiferous tubules were partially empty and without vacuolated germinal layer cells indicating the absence of spermatogenesis. Hematoxylin and eosin staining.

• 

  Fig. 5 Sections of epididymis tubules of (A) normal control, (B) busulfan treated azoospermic control, and (C) bone marrow mesenchymal stem cells treated hamsters. Hematoxylin and eosin staining.

• 

  Fig. 6 Mean and standard error of histomorphometric indices of seminiferous tubules in busulfan-induced azoospermic testis treated with bone marrow mesenchymal stem cells (cell therapy) in comparison with busulfan treated testes (azoospermia) and intact normal testis (normal) in hamster. (A) Lumen diameter (μm), (B) Luminal area (μm2), (C) Cellular diameter (μm), (D) Cellular area (μm2), (E) Total diameters (μm), and (F) Cross sectional area of the tubule (μm2). a,b,cdifferent superscript letters show significant differences between groups (p<0.05).

• 

  Fig. 7 Mean and standard error of spermatogenesis index of seminiferous tubules in busulfan-induced azoospermic testis treated with bone marrow mesenchymal stem cells (cell therapy) in comparison with busulfan treated testes (azoospermia) and intact normal testis (normal) in hamster. a,bdifferent superscript letters show significant differences between groups (p<0.05).

Reference

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