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Evaluation of mesenchymal stem cells in treatment of infertility in male rats.

Hassan AI, Alam SS - Stem Cell Res Ther (2014)

Bottom Line: Lead nitrate caused degeneration, necrosis, interstitial edema, and reduction in spermatogenic activity in some seminiferous tubules.It was concluded that lead is a gonadotoxic with a tendency of suppressing semen characteristics and testosterone levels of animals, the presence of MSCs was found to alleviate the toxic effects of lead.We conclude that MSCs derived from the bone marrow of rats can be an effective therapy of LN induced gonado toxicity, thus can contribute to the treatment of infertility.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: The present study aimed to elucidate the therapeutic effects of mesenchymal stem cells (MSCs) derived from the bone marrow of rats (BM) against toxic effects of lead (Pb) on the male gonads of experimental rats.

Methods: The experimental animals were exposed to lead in the form of lead nitrate (LN) one quarter of the LD50. The efficacy of MSCs to reduce gonado-totoxicity induced by lead nitrate at 21, 30 and 60 days, was evaluated experimentally in male rats.

Results: The results showed that testosterone levels and semen quality ameliorated following treatment with MSCs. Also, superoxide dismutase, glutathione peroxidase and catalase levels were increased 21, 30 and 60 days post treatment of MSCs. Moreover, a decrease in genomic DNA alteration and percentage of fragmented DNA was recorded after MSCs treatment. Lead nitrate caused degeneration, necrosis, interstitial edema, and reduction in spermatogenic activity in some seminiferous tubules. The LN-induced changes in histopathologic findings of testis were partially reversed by treatment with MSCs. Histological examination of testis showed deformities in morphology of testis in test animals with gross damage within the seminiferous tubules in Lead nitrate group. The LN-induced changes in histopathologic findings of testis were partially reversed by treatment of MSCs.

Conclusions: It was concluded that lead is a gonadotoxic with a tendency of suppressing semen characteristics and testosterone levels of animals, the presence of MSCs was found to alleviate the toxic effects of lead. We conclude that MSCs derived from the bone marrow of rats can be an effective therapy of LN induced gonado toxicity, thus can contribute to the treatment of infertility.

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Morphological and histological staining of differentiated MSCs/BM. A. Undifferentiated MSCs. B. Differentiated MSC osteoblasts after addition of growth factors. C. MSCs differentiated into osteoblasts stained with Alizarin red. D. Arrows for differentiated MSC chondrocytes after addition of growth factors. E. MSCs differentiated into chondrocytes stained with Alcian blue. MSCs/BM, bone marrow-derived mesenchymal stem cells.
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Fig1: Morphological and histological staining of differentiated MSCs/BM. A. Undifferentiated MSCs. B. Differentiated MSC osteoblasts after addition of growth factors. C. MSCs differentiated into osteoblasts stained with Alizarin red. D. Arrows for differentiated MSC chondrocytes after addition of growth factors. E. MSCs differentiated into chondrocytes stained with Alcian blue. MSCs/BM, bone marrow-derived mesenchymal stem cells.

Mentions: Bone marrow was harvested by flushing the tibiae and femurs of male albino rats with (Dulbecco’s) modified Eagle’s medium ((D)MEM, Gibco BRL, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO/BRL). Nucleated cells were isolated with a density gradient (Ficoll/Paque (Pharmacia, Uppsala, Sweden)) and resuspended in complete culture medium supplemented, then incubated at 37°C in 5% humidified CO2 for 12 to 14 days as the primary culture or until formation of large colonies. When large colonies developed (80% to 90% confluence), the cultures were washed twice with phosphate-buffered saline (PBS) and the cells were trypsinized with 0.25% trypsin in 1 mM ethylenediaminetetraactic acid (EDTA) (GIBCO/BRL) for five minutes at 37°C. After centrifugation, the cells were resuspended with serum-supplemented medium and incubated in 50 cm2 culture flasks (Falcon, Pharmacia, Uppsala, Sweden). The resulting cultures were referred to as first-passage cultures [20]. MSCs in culture were characterized by their adhesiveness and fusiform shape [21]. The resulting cultures were referred to as first-passage cultures [22]. On day 14, the adherent colonies of cells were trypsinized and counted. Cells were identified as being MSCs by their morphology, adherence, and power to differentiate into osteocytes [23] (Figure 1B,C) and chondrocytes [24] (Figure 1D,E). Differentiation into osteocytes was achieved by adding 1 to 1,000 nM dexamethasone, 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate to the medium. Differentiation of MSCs into osteoblasts was confirmed through morphological changes, Alzarin red staining of differentiated osteoblasts and RT-PCR gene expression of osteonectin in differentiated cells. Differentiation into chondrocytes was achieved by adding 500 ng/mL bone morphogenetic protein-2 (BMP-2; R&D Systems, Minneapolis, MN, USA) and 10 ng/ml transforming growth factor β3 (TGFβ3) (Peprotech, London, UK) for three weeks [24]. After passage 3 (P3), stem cells were harvested. Immunophenotyping using 100 ml of the cell suspension was performed by flow cytometry (Accuri, BD Accuri C6; Becton Dickinson San Jose, CA, USA). The MSCs are positive for CD29 (Sigma, San Diego, CA, USA, SAB 4501582) and negative for CD45 (Sigma, OX-1 84112004) (Figure 2A-D).Figure 1


Evaluation of mesenchymal stem cells in treatment of infertility in male rats.

Hassan AI, Alam SS - Stem Cell Res Ther (2014)

Morphological and histological staining of differentiated MSCs/BM. A. Undifferentiated MSCs. B. Differentiated MSC osteoblasts after addition of growth factors. C. MSCs differentiated into osteoblasts stained with Alizarin red. D. Arrows for differentiated MSC chondrocytes after addition of growth factors. E. MSCs differentiated into chondrocytes stained with Alcian blue. MSCs/BM, bone marrow-derived mesenchymal stem cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4528845&req=5

Fig1: Morphological and histological staining of differentiated MSCs/BM. A. Undifferentiated MSCs. B. Differentiated MSC osteoblasts after addition of growth factors. C. MSCs differentiated into osteoblasts stained with Alizarin red. D. Arrows for differentiated MSC chondrocytes after addition of growth factors. E. MSCs differentiated into chondrocytes stained with Alcian blue. MSCs/BM, bone marrow-derived mesenchymal stem cells.
Mentions: Bone marrow was harvested by flushing the tibiae and femurs of male albino rats with (Dulbecco’s) modified Eagle’s medium ((D)MEM, Gibco BRL, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO/BRL). Nucleated cells were isolated with a density gradient (Ficoll/Paque (Pharmacia, Uppsala, Sweden)) and resuspended in complete culture medium supplemented, then incubated at 37°C in 5% humidified CO2 for 12 to 14 days as the primary culture or until formation of large colonies. When large colonies developed (80% to 90% confluence), the cultures were washed twice with phosphate-buffered saline (PBS) and the cells were trypsinized with 0.25% trypsin in 1 mM ethylenediaminetetraactic acid (EDTA) (GIBCO/BRL) for five minutes at 37°C. After centrifugation, the cells were resuspended with serum-supplemented medium and incubated in 50 cm2 culture flasks (Falcon, Pharmacia, Uppsala, Sweden). The resulting cultures were referred to as first-passage cultures [20]. MSCs in culture were characterized by their adhesiveness and fusiform shape [21]. The resulting cultures were referred to as first-passage cultures [22]. On day 14, the adherent colonies of cells were trypsinized and counted. Cells were identified as being MSCs by their morphology, adherence, and power to differentiate into osteocytes [23] (Figure 1B,C) and chondrocytes [24] (Figure 1D,E). Differentiation into osteocytes was achieved by adding 1 to 1,000 nM dexamethasone, 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate to the medium. Differentiation of MSCs into osteoblasts was confirmed through morphological changes, Alzarin red staining of differentiated osteoblasts and RT-PCR gene expression of osteonectin in differentiated cells. Differentiation into chondrocytes was achieved by adding 500 ng/mL bone morphogenetic protein-2 (BMP-2; R&D Systems, Minneapolis, MN, USA) and 10 ng/ml transforming growth factor β3 (TGFβ3) (Peprotech, London, UK) for three weeks [24]. After passage 3 (P3), stem cells were harvested. Immunophenotyping using 100 ml of the cell suspension was performed by flow cytometry (Accuri, BD Accuri C6; Becton Dickinson San Jose, CA, USA). The MSCs are positive for CD29 (Sigma, San Diego, CA, USA, SAB 4501582) and negative for CD45 (Sigma, OX-1 84112004) (Figure 2A-D).Figure 1

Bottom Line: Lead nitrate caused degeneration, necrosis, interstitial edema, and reduction in spermatogenic activity in some seminiferous tubules.It was concluded that lead is a gonadotoxic with a tendency of suppressing semen characteristics and testosterone levels of animals, the presence of MSCs was found to alleviate the toxic effects of lead.We conclude that MSCs derived from the bone marrow of rats can be an effective therapy of LN induced gonado toxicity, thus can contribute to the treatment of infertility.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: The present study aimed to elucidate the therapeutic effects of mesenchymal stem cells (MSCs) derived from the bone marrow of rats (BM) against toxic effects of lead (Pb) on the male gonads of experimental rats.

Methods: The experimental animals were exposed to lead in the form of lead nitrate (LN) one quarter of the LD50. The efficacy of MSCs to reduce gonado-totoxicity induced by lead nitrate at 21, 30 and 60 days, was evaluated experimentally in male rats.

Results: The results showed that testosterone levels and semen quality ameliorated following treatment with MSCs. Also, superoxide dismutase, glutathione peroxidase and catalase levels were increased 21, 30 and 60 days post treatment of MSCs. Moreover, a decrease in genomic DNA alteration and percentage of fragmented DNA was recorded after MSCs treatment. Lead nitrate caused degeneration, necrosis, interstitial edema, and reduction in spermatogenic activity in some seminiferous tubules. The LN-induced changes in histopathologic findings of testis were partially reversed by treatment with MSCs. Histological examination of testis showed deformities in morphology of testis in test animals with gross damage within the seminiferous tubules in Lead nitrate group. The LN-induced changes in histopathologic findings of testis were partially reversed by treatment of MSCs.

Conclusions: It was concluded that lead is a gonadotoxic with a tendency of suppressing semen characteristics and testosterone levels of animals, the presence of MSCs was found to alleviate the toxic effects of lead. We conclude that MSCs derived from the bone marrow of rats can be an effective therapy of LN induced gonado toxicity, thus can contribute to the treatment of infertility.

Show MeSH
Related in: MedlinePlus