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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.


Related in: MedlinePlus

Detection and enumeration of flagellated sperm after the dissection of testes explants (at 6.5 dpp) cultured for 30 days.(A) Seminiferous tubules with spermatids as the most advanced stage at D30 of culture. Photomicrographs were captured at a ×200 (A1) and ×500 (A2) magnifications and the scale bars represent 100 μm or 40 μm, respectively. (A1) Multiple elongated spermatids obtained in different seminiferous tubules (black asterisks) in the presence of RE, are shown in thin sections after PAS and haematoxylin staining. Black box in A1 is enlarged in A2. Red box that shows elongated spermatids is enlarged in the inset of the photomicrograph. (A3) Mean proportion of seminiferous tubules containing differentiated germ cells as the most advanced stage under all conditions tested at D30 of culture. (B) Detection and enumeration of flagellated sperm under all conditions tested at D30 of culture. Photomicrographs were captured at a ×500 (Shorr staining) and ×600 (α-tubulin detection) magnifications and the scale bars represent 40 μm. (B1) Flagellated sperm derived in vitro at D30 of culture in the presence of RE with the in vivo-matched age (36.5 dpp), as a control sperm are shown using Shorr staining. (B2) Genuine sperm flagella are shown in green using a specific anti-acetylated α-tubulin antibody, and nuclei are stained in blue by DAPI. A negative control was performed with a pre-immune mouse IgG and is shown in the inset of the photomicrograph. (B3) Mean number of spermatozoa produced in 6.5 dpp testicular tissues cultured for 30 days under the conditions tested in the present study.
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pone.0116660.g006: Detection and enumeration of flagellated sperm after the dissection of testes explants (at 6.5 dpp) cultured for 30 days.(A) Seminiferous tubules with spermatids as the most advanced stage at D30 of culture. Photomicrographs were captured at a ×200 (A1) and ×500 (A2) magnifications and the scale bars represent 100 μm or 40 μm, respectively. (A1) Multiple elongated spermatids obtained in different seminiferous tubules (black asterisks) in the presence of RE, are shown in thin sections after PAS and haematoxylin staining. Black box in A1 is enlarged in A2. Red box that shows elongated spermatids is enlarged in the inset of the photomicrograph. (A3) Mean proportion of seminiferous tubules containing differentiated germ cells as the most advanced stage under all conditions tested at D30 of culture. (B) Detection and enumeration of flagellated sperm under all conditions tested at D30 of culture. Photomicrographs were captured at a ×500 (Shorr staining) and ×600 (α-tubulin detection) magnifications and the scale bars represent 40 μm. (B1) Flagellated sperm derived in vitro at D30 of culture in the presence of RE with the in vivo-matched age (36.5 dpp), as a control sperm are shown using Shorr staining. (B2) Genuine sperm flagella are shown in green using a specific anti-acetylated α-tubulin antibody, and nuclei are stained in blue by DAPI. A negative control was performed with a pre-immune mouse IgG and is shown in the inset of the photomicrograph. (B3) Mean number of spermatozoa produced in 6.5 dpp testicular tissues cultured for 30 days under the conditions tested in the present study.

Mentions: (ii) Cultures of 6.5 dpp mice testes. The highest rates of elongated spermatids were obtained with RE at D30 (Table 2). Therefore, 6.5 day-old testes were cultured for sperm enumeration during 30 days of culture with BM, FSH/LH or RE. Multiple elongated spermatids were obtained in several seminiferous tubules with RE (Fig. 6A1 and 6A2). Compared with BM, RE increased the mean proportion of seminiferous tubules containing elongated spermatids as the most advanced stage (p = 0.02) (Fig. 6A3). Spermatozoa obtained at D30 were confirmed by Shorr staining (Fig. 6B1), and genuine flagella were detected by immunofluorescence with an anti-acetylated tubulin antibody (Fig. 6B2). FSH/LH significantly increased the mean number of flagellated spermatozoa compared with BM (p = 0.02). However, more numerous spermatozoa were obtained with RE compared with BM or FSH/LH (p = 0.02) (Fig. 6B3). Regardless of the culture medium tested, the mean number of flagellated spermatozoa produced in vitro was significantly lower than that observed at the corresponding in vivo age, namely 36.5 dpp (p = 0.01) (Fig. 6B3).


Retinol Improves In Vitro Differentiation of Pre-Pubertal Mouse Spermatogonial Stem Cells into Sperm during the First Wave of Spermatogenesis
Detection and enumeration of flagellated sperm after the dissection of testes explants (at 6.5 dpp) cultured for 30 days.(A) Seminiferous tubules with spermatids as the most advanced stage at D30 of culture. Photomicrographs were captured at a ×200 (A1) and ×500 (A2) magnifications and the scale bars represent 100 μm or 40 μm, respectively. (A1) Multiple elongated spermatids obtained in different seminiferous tubules (black asterisks) in the presence of RE, are shown in thin sections after PAS and haematoxylin staining. Black box in A1 is enlarged in A2. Red box that shows elongated spermatids is enlarged in the inset of the photomicrograph. (A3) Mean proportion of seminiferous tubules containing differentiated germ cells as the most advanced stage under all conditions tested at D30 of culture. (B) Detection and enumeration of flagellated sperm under all conditions tested at D30 of culture. Photomicrographs were captured at a ×500 (Shorr staining) and ×600 (α-tubulin detection) magnifications and the scale bars represent 40 μm. (B1) Flagellated sperm derived in vitro at D30 of culture in the presence of RE with the in vivo-matched age (36.5 dpp), as a control sperm are shown using Shorr staining. (B2) Genuine sperm flagella are shown in green using a specific anti-acetylated α-tubulin antibody, and nuclei are stained in blue by DAPI. A negative control was performed with a pre-immune mouse IgG and is shown in the inset of the photomicrograph. (B3) Mean number of spermatozoa produced in 6.5 dpp testicular tissues cultured for 30 days under the conditions tested in the present study.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116660.g006: Detection and enumeration of flagellated sperm after the dissection of testes explants (at 6.5 dpp) cultured for 30 days.(A) Seminiferous tubules with spermatids as the most advanced stage at D30 of culture. Photomicrographs were captured at a ×200 (A1) and ×500 (A2) magnifications and the scale bars represent 100 μm or 40 μm, respectively. (A1) Multiple elongated spermatids obtained in different seminiferous tubules (black asterisks) in the presence of RE, are shown in thin sections after PAS and haematoxylin staining. Black box in A1 is enlarged in A2. Red box that shows elongated spermatids is enlarged in the inset of the photomicrograph. (A3) Mean proportion of seminiferous tubules containing differentiated germ cells as the most advanced stage under all conditions tested at D30 of culture. (B) Detection and enumeration of flagellated sperm under all conditions tested at D30 of culture. Photomicrographs were captured at a ×500 (Shorr staining) and ×600 (α-tubulin detection) magnifications and the scale bars represent 40 μm. (B1) Flagellated sperm derived in vitro at D30 of culture in the presence of RE with the in vivo-matched age (36.5 dpp), as a control sperm are shown using Shorr staining. (B2) Genuine sperm flagella are shown in green using a specific anti-acetylated α-tubulin antibody, and nuclei are stained in blue by DAPI. A negative control was performed with a pre-immune mouse IgG and is shown in the inset of the photomicrograph. (B3) Mean number of spermatozoa produced in 6.5 dpp testicular tissues cultured for 30 days under the conditions tested in the present study.
Mentions: (ii) Cultures of 6.5 dpp mice testes. The highest rates of elongated spermatids were obtained with RE at D30 (Table 2). Therefore, 6.5 day-old testes were cultured for sperm enumeration during 30 days of culture with BM, FSH/LH or RE. Multiple elongated spermatids were obtained in several seminiferous tubules with RE (Fig. 6A1 and 6A2). Compared with BM, RE increased the mean proportion of seminiferous tubules containing elongated spermatids as the most advanced stage (p = 0.02) (Fig. 6A3). Spermatozoa obtained at D30 were confirmed by Shorr staining (Fig. 6B1), and genuine flagella were detected by immunofluorescence with an anti-acetylated tubulin antibody (Fig. 6B2). FSH/LH significantly increased the mean number of flagellated spermatozoa compared with BM (p = 0.02). However, more numerous spermatozoa were obtained with RE compared with BM or FSH/LH (p = 0.02) (Fig. 6B3). Regardless of the culture medium tested, the mean number of flagellated spermatozoa produced in vitro was significantly lower than that observed at the corresponding in vivo age, namely 36.5 dpp (p = 0.01) (Fig. 6B3).

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