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Retinol Improves In Vitro Differentiation of Pre-Pubertal Mouse Spermatogonial Stem Cells into Sperm during the First Wave of Spermatogenesis

View Article: PubMed Central - PubMed

ABSTRACT

Testicular tissue freezing has been proposed for fertility preservation in pre-pubertal boys. Thawed frozen testicular tissue must undergo a maturation process to restore sperm production. The purpose of the current study was to evaluate the ability of retinol to improve the in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm. Testes from pre-pubertal mice, aged 2.5 and 6.5 days post-partum, were cultured on agarose gel at a gas-liquid interphase for 34, 38 and 60 days (D) and for 16, 30 and 36 D respectively. Assessment of basal medium (BM) supplemented with retinol (RE) alone, FSH/LH alone or a combination of both, was performed. Stereological analyses and tissue lesion scoring were performed at the culture time points indicated above. Sperm production was quantified at D30 and D34 after mechanical dissection of the testicular tissues. FSH/LH significantly increased the percentage of round spermatids at D30 and D38, when compared to BM alone. However, RE significantly increased the percentages of round but also elongated spermatids at D30 and D34. Moreover, RE significantly increased the number of spermatozoa per milligram of tissue at D30 and D34 when compared to BM. Therefore, RE improved the in vitro production of spermatids and spermatozoa from pre-pubertal SSCs during the first wave of spermatogenesis. The use of RE could be a useful tool for in vitro spermatogenesis from pre-pubertal human testicular tissue.

No MeSH data available.


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Lesion score assessment in seminiferous tubules during organotypic culture of pre-pubertal mice testicular tissue under the tested conditions.(A) Histological evaluation of alterations in the seminiferous epithelium after staining with PAS and haematoxylin. Photomicrographs were captured at a ×500 magnification, and the scale bar represents 20 μm. Gap formation (vacuole) is represented by red asterisks (*). (B) Mean global lesion score of seminiferous tubules at D0, D38 and D60 of culture (2.5 dpp) and C) D0, D30 and D36 of culture (6.5 dpp) for BM, RE, FSH/LH and in vivo conditions. The results are represented as the mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference with p<0.05. Footnotes: BM: Basal Medium, D: Day, dpp: day post-partum, FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, n: Number of mice testes used in each condition, PAS: Periodic Acid-Schiff, RE: Retinol, s.e.m.: Standard Error of the Mean
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pone.0116660.g003: Lesion score assessment in seminiferous tubules during organotypic culture of pre-pubertal mice testicular tissue under the tested conditions.(A) Histological evaluation of alterations in the seminiferous epithelium after staining with PAS and haematoxylin. Photomicrographs were captured at a ×500 magnification, and the scale bar represents 20 μm. Gap formation (vacuole) is represented by red asterisks (*). (B) Mean global lesion score of seminiferous tubules at D0, D38 and D60 of culture (2.5 dpp) and C) D0, D30 and D36 of culture (6.5 dpp) for BM, RE, FSH/LH and in vivo conditions. The results are represented as the mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference with p<0.05. Footnotes: BM: Basal Medium, D: Day, dpp: day post-partum, FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, n: Number of mice testes used in each condition, PAS: Periodic Acid-Schiff, RE: Retinol, s.e.m.: Standard Error of the Mean

Mentions: Cell detachment and gap formation in seminiferous tubules increased significantly from D0 to D60 (Fig. 3B) regardless of the tested conditions (p = 0.0046) and from D0 to D36 (Fig. 3C) for BM (p = 0.04), FSH/LH (p = 0.0046), RE (p = 0.0046) and in vivo (p = 0.0046). The tissue alteration scores were significantly higher for FSH/LH when compared with BM at D36 (p = 0.02), D38 (p = 0.02) and D60 (p = 0.01). RE significantly reduced the cell detachment and gap formation of seminiferous tubules compared with BM (D30 [p = 0.02] and D60 [p = 0.01]) or FSH/LH regardless of the culture time points tested (p = 0.01) (Fig. 3A, 3C). Morphological changes in the seminiferous tubules were significantly higher in vitro than those observed for the corresponding in vivo controls at all culture time points and under all tested conditions (p = 0.01).


Retinol Improves In Vitro Differentiation of Pre-Pubertal Mouse Spermatogonial Stem Cells into Sperm during the First Wave of Spermatogenesis
Lesion score assessment in seminiferous tubules during organotypic culture of pre-pubertal mice testicular tissue under the tested conditions.(A) Histological evaluation of alterations in the seminiferous epithelium after staining with PAS and haematoxylin. Photomicrographs were captured at a ×500 magnification, and the scale bar represents 20 μm. Gap formation (vacuole) is represented by red asterisks (*). (B) Mean global lesion score of seminiferous tubules at D0, D38 and D60 of culture (2.5 dpp) and C) D0, D30 and D36 of culture (6.5 dpp) for BM, RE, FSH/LH and in vivo conditions. The results are represented as the mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference with p<0.05. Footnotes: BM: Basal Medium, D: Day, dpp: day post-partum, FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, n: Number of mice testes used in each condition, PAS: Periodic Acid-Schiff, RE: Retinol, s.e.m.: Standard Error of the Mean
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4340963&req=5

pone.0116660.g003: Lesion score assessment in seminiferous tubules during organotypic culture of pre-pubertal mice testicular tissue under the tested conditions.(A) Histological evaluation of alterations in the seminiferous epithelium after staining with PAS and haematoxylin. Photomicrographs were captured at a ×500 magnification, and the scale bar represents 20 μm. Gap formation (vacuole) is represented by red asterisks (*). (B) Mean global lesion score of seminiferous tubules at D0, D38 and D60 of culture (2.5 dpp) and C) D0, D30 and D36 of culture (6.5 dpp) for BM, RE, FSH/LH and in vivo conditions. The results are represented as the mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference with p<0.05. Footnotes: BM: Basal Medium, D: Day, dpp: day post-partum, FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, n: Number of mice testes used in each condition, PAS: Periodic Acid-Schiff, RE: Retinol, s.e.m.: Standard Error of the Mean
Mentions: Cell detachment and gap formation in seminiferous tubules increased significantly from D0 to D60 (Fig. 3B) regardless of the tested conditions (p = 0.0046) and from D0 to D36 (Fig. 3C) for BM (p = 0.04), FSH/LH (p = 0.0046), RE (p = 0.0046) and in vivo (p = 0.0046). The tissue alteration scores were significantly higher for FSH/LH when compared with BM at D36 (p = 0.02), D38 (p = 0.02) and D60 (p = 0.01). RE significantly reduced the cell detachment and gap formation of seminiferous tubules compared with BM (D30 [p = 0.02] and D60 [p = 0.01]) or FSH/LH regardless of the culture time points tested (p = 0.01) (Fig. 3A, 3C). Morphological changes in the seminiferous tubules were significantly higher in vitro than those observed for the corresponding in vivo controls at all culture time points and under all tested conditions (p = 0.01).

View Article: PubMed Central - PubMed

ABSTRACT

Testicular tissue freezing has been proposed for fertility preservation in pre-pubertal boys. Thawed frozen testicular tissue must undergo a maturation process to restore sperm production. The purpose of the current study was to evaluate the ability of retinol to improve the in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm. Testes from pre-pubertal mice, aged 2.5 and 6.5 days post-partum, were cultured on agarose gel at a gas-liquid interphase for 34, 38 and 60 days (D) and for 16, 30 and 36 D respectively. Assessment of basal medium (BM) supplemented with retinol (RE) alone, FSH/LH alone or a combination of both, was performed. Stereological analyses and tissue lesion scoring were performed at the culture time points indicated above. Sperm production was quantified at D30 and D34 after mechanical dissection of the testicular tissues. FSH/LH significantly increased the percentage of round spermatids at D30 and D38, when compared to BM alone. However, RE significantly increased the percentages of round but also elongated spermatids at D30 and D34. Moreover, RE significantly increased the number of spermatozoa per milligram of tissue at D30 and D34 when compared to BM. Therefore, RE improved the in vitro production of spermatids and spermatozoa from pre-pubertal SSCs during the first wave of spermatogenesis. The use of RE could be a useful tool for in vitro spermatogenesis from pre-pubertal human testicular tissue.

No MeSH data available.


Related in: MedlinePlus