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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

Assessment of in vitro spermatogenesis initiation after immunodetection of Sall-4, c-kit and Stra-8 proteins in CD-1 prepubertal mice testes (6.5 dpp) at D4 and Sall-4 immunodetection at D16 and D30 of culture, in the absence or presence of RE.(A) Immunohistochemical detection of Sall-4, c-kit and Stra-8 on seminiferous tubule sections of prepubertal mice testes at D4 of culture in the absence (A1–A4–A7) or presence (A2–A5–A8) of RE and their corresponding in vivo ages (10.5 dpp) (A3–A6–A9). Brown staining was considered as a positive immunodetection of Sall-4, c-kit and Stra-8 in seminiferous tubule sections counterstained with haematoxylin. Negative control is shown on the right of each positive immunostaining. Photomicrographs were captured at a × 1000 magnification and the scale bars represent 40 μm. (B) Effect of RE on Sall-4, c-kit and Stra-8 expression in seminiferous tubules of prepubertal mice testes (6.5 dpp) cultured for 4 days in vitro. (B1) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column). (B2) Mean proportion of c-kit positive cells per seminiferous tubule section (blue column) and mean proportion of c-kit positive tubules (red column). (B3) Mean proportion of Stra-8 positive cells per seminiferous tubule section (blue column) and mean proportion of Stra-8 positive tubules (red column). Values are represented as mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference between BM and RE or between BM and in vivo condition (p<0.05). (C) Immunohistochemical detection of Sall-4 on seminiferous tubule sections of cultured prepubertal mice testes at D16 and D30 in the absence (C1–C2) or presence (C3–C4) of RE and their corresponding in vivo ages (22.5 dpp and 36.5 dpp, respectively) (C5–C6). Brown staining was considered as a positive immunodetection of Sall-4 in seminiferous tubule sections counterstained with haematoxylin. Negative control is shown on the right of each positive immunostaining. Photomicrographs were captured at a × 1000 magnification and the scale bars represent 40 μm. (D) Assessment of in vitro maintenance of undifferentiated type A spermatogonia after immunodetection of Sall-4 at D16 and D30 of culture, in the presence or absence of RE. (D1) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column) at D16 of culture. (D2) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column) at D30 of culture. Values are represented as mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference between BM and RE or between BM and in vivo condition (p < 0.05). Footnotes: BM: Basal Medium, D: day, dpp: days post partum, n: Number of mice testes used in each condition, NS: No statistical Significance, RE: Retinol, s.e.m.: Standard Error of Mean, IgG: Immunoglobulin G
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pone.0116660.g004: Assessment of in vitro spermatogenesis initiation after immunodetection of Sall-4, c-kit and Stra-8 proteins in CD-1 prepubertal mice testes (6.5 dpp) at D4 and Sall-4 immunodetection at D16 and D30 of culture, in the absence or presence of RE.(A) Immunohistochemical detection of Sall-4, c-kit and Stra-8 on seminiferous tubule sections of prepubertal mice testes at D4 of culture in the absence (A1–A4–A7) or presence (A2–A5–A8) of RE and their corresponding in vivo ages (10.5 dpp) (A3–A6–A9). Brown staining was considered as a positive immunodetection of Sall-4, c-kit and Stra-8 in seminiferous tubule sections counterstained with haematoxylin. Negative control is shown on the right of each positive immunostaining. Photomicrographs were captured at a × 1000 magnification and the scale bars represent 40 μm. (B) Effect of RE on Sall-4, c-kit and Stra-8 expression in seminiferous tubules of prepubertal mice testes (6.5 dpp) cultured for 4 days in vitro. (B1) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column). (B2) Mean proportion of c-kit positive cells per seminiferous tubule section (blue column) and mean proportion of c-kit positive tubules (red column). (B3) Mean proportion of Stra-8 positive cells per seminiferous tubule section (blue column) and mean proportion of Stra-8 positive tubules (red column). Values are represented as mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference between BM and RE or between BM and in vivo condition (p<0.05). (C) Immunohistochemical detection of Sall-4 on seminiferous tubule sections of cultured prepubertal mice testes at D16 and D30 in the absence (C1–C2) or presence (C3–C4) of RE and their corresponding in vivo ages (22.5 dpp and 36.5 dpp, respectively) (C5–C6). Brown staining was considered as a positive immunodetection of Sall-4 in seminiferous tubule sections counterstained with haematoxylin. Negative control is shown on the right of each positive immunostaining. Photomicrographs were captured at a × 1000 magnification and the scale bars represent 40 μm. (D) Assessment of in vitro maintenance of undifferentiated type A spermatogonia after immunodetection of Sall-4 at D16 and D30 of culture, in the presence or absence of RE. (D1) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column) at D16 of culture. (D2) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column) at D30 of culture. Values are represented as mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference between BM and RE or between BM and in vivo condition (p < 0.05). Footnotes: BM: Basal Medium, D: day, dpp: days post partum, n: Number of mice testes used in each condition, NS: No statistical Significance, RE: Retinol, s.e.m.: Standard Error of Mean, IgG: Immunoglobulin G

Mentions: At D4, undifferentiated type A spermatogonia, immunostained with Sall-4, were detected in the whole cross-sectioned tubules with all tested conditions (Fig. 4A1, 4A2 and 4A3). No statistical differences were observed between BM and RE considering the proportion of Sall-4 positive spermatogonia per tubule cross section (Fig. 4B1). However, c-kit and Stra-8 immunostaining appeared stronger with RE (Fig. 4A5, 4A8), when compared with BM (Fig. 4A4, 4A7). C-kit and Stra-8 immunostaining appeared similar between RE (Fig. 4A5, 4A8) and in vivo (Fig. 4A6, 4A9). Moreover, RE significantly increased the proportion of c-kit positive-cells per tubule cross section in comparison to BM (p = 0.02) (Fig. 4B2), but also the proportion of c-kit (p = 0.01) and Stra-8 (p = 0.01) positive tubules when compared to BM alone (Fig. 4B2 and 4B3). RE maintains a similar expression of c-kit and Stra-8 as observed in vivo.


Retinol Improves In Vitro Differentiation of Pre-Pubertal Mouse Spermatogonial Stem Cells into Sperm during the First Wave of Spermatogenesis
Assessment of in vitro spermatogenesis initiation after immunodetection of Sall-4, c-kit and Stra-8 proteins in CD-1 prepubertal mice testes (6.5 dpp) at D4 and Sall-4 immunodetection at D16 and D30 of culture, in the absence or presence of RE.(A) Immunohistochemical detection of Sall-4, c-kit and Stra-8 on seminiferous tubule sections of prepubertal mice testes at D4 of culture in the absence (A1–A4–A7) or presence (A2–A5–A8) of RE and their corresponding in vivo ages (10.5 dpp) (A3–A6–A9). Brown staining was considered as a positive immunodetection of Sall-4, c-kit and Stra-8 in seminiferous tubule sections counterstained with haematoxylin. Negative control is shown on the right of each positive immunostaining. Photomicrographs were captured at a × 1000 magnification and the scale bars represent 40 μm. (B) Effect of RE on Sall-4, c-kit and Stra-8 expression in seminiferous tubules of prepubertal mice testes (6.5 dpp) cultured for 4 days in vitro. (B1) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column). (B2) Mean proportion of c-kit positive cells per seminiferous tubule section (blue column) and mean proportion of c-kit positive tubules (red column). (B3) Mean proportion of Stra-8 positive cells per seminiferous tubule section (blue column) and mean proportion of Stra-8 positive tubules (red column). Values are represented as mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference between BM and RE or between BM and in vivo condition (p<0.05). (C) Immunohistochemical detection of Sall-4 on seminiferous tubule sections of cultured prepubertal mice testes at D16 and D30 in the absence (C1–C2) or presence (C3–C4) of RE and their corresponding in vivo ages (22.5 dpp and 36.5 dpp, respectively) (C5–C6). Brown staining was considered as a positive immunodetection of Sall-4 in seminiferous tubule sections counterstained with haematoxylin. Negative control is shown on the right of each positive immunostaining. Photomicrographs were captured at a × 1000 magnification and the scale bars represent 40 μm. (D) Assessment of in vitro maintenance of undifferentiated type A spermatogonia after immunodetection of Sall-4 at D16 and D30 of culture, in the presence or absence of RE. (D1) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column) at D16 of culture. (D2) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column) at D30 of culture. Values are represented as mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference between BM and RE or between BM and in vivo condition (p < 0.05). Footnotes: BM: Basal Medium, D: day, dpp: days post partum, n: Number of mice testes used in each condition, NS: No statistical Significance, RE: Retinol, s.e.m.: Standard Error of Mean, IgG: Immunoglobulin G
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pone.0116660.g004: Assessment of in vitro spermatogenesis initiation after immunodetection of Sall-4, c-kit and Stra-8 proteins in CD-1 prepubertal mice testes (6.5 dpp) at D4 and Sall-4 immunodetection at D16 and D30 of culture, in the absence or presence of RE.(A) Immunohistochemical detection of Sall-4, c-kit and Stra-8 on seminiferous tubule sections of prepubertal mice testes at D4 of culture in the absence (A1–A4–A7) or presence (A2–A5–A8) of RE and their corresponding in vivo ages (10.5 dpp) (A3–A6–A9). Brown staining was considered as a positive immunodetection of Sall-4, c-kit and Stra-8 in seminiferous tubule sections counterstained with haematoxylin. Negative control is shown on the right of each positive immunostaining. Photomicrographs were captured at a × 1000 magnification and the scale bars represent 40 μm. (B) Effect of RE on Sall-4, c-kit and Stra-8 expression in seminiferous tubules of prepubertal mice testes (6.5 dpp) cultured for 4 days in vitro. (B1) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column). (B2) Mean proportion of c-kit positive cells per seminiferous tubule section (blue column) and mean proportion of c-kit positive tubules (red column). (B3) Mean proportion of Stra-8 positive cells per seminiferous tubule section (blue column) and mean proportion of Stra-8 positive tubules (red column). Values are represented as mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference between BM and RE or between BM and in vivo condition (p<0.05). (C) Immunohistochemical detection of Sall-4 on seminiferous tubule sections of cultured prepubertal mice testes at D16 and D30 in the absence (C1–C2) or presence (C3–C4) of RE and their corresponding in vivo ages (22.5 dpp and 36.5 dpp, respectively) (C5–C6). Brown staining was considered as a positive immunodetection of Sall-4 in seminiferous tubule sections counterstained with haematoxylin. Negative control is shown on the right of each positive immunostaining. Photomicrographs were captured at a × 1000 magnification and the scale bars represent 40 μm. (D) Assessment of in vitro maintenance of undifferentiated type A spermatogonia after immunodetection of Sall-4 at D16 and D30 of culture, in the presence or absence of RE. (D1) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column) at D16 of culture. (D2) Mean proportion of Sall-4-positive cells per seminiferous tubule section (blue column) and mean proportion of Sall-4 positive tubules (red column) at D30 of culture. Values are represented as mean ± s.e.m., n = 4. Asterisk indicates a statistically significant difference between BM and RE or between BM and in vivo condition (p < 0.05). Footnotes: BM: Basal Medium, D: day, dpp: days post partum, n: Number of mice testes used in each condition, NS: No statistical Significance, RE: Retinol, s.e.m.: Standard Error of Mean, IgG: Immunoglobulin G
Mentions: At D4, undifferentiated type A spermatogonia, immunostained with Sall-4, were detected in the whole cross-sectioned tubules with all tested conditions (Fig. 4A1, 4A2 and 4A3). No statistical differences were observed between BM and RE considering the proportion of Sall-4 positive spermatogonia per tubule cross section (Fig. 4B1). However, c-kit and Stra-8 immunostaining appeared stronger with RE (Fig. 4A5, 4A8), when compared with BM (Fig. 4A4, 4A7). C-kit and Stra-8 immunostaining appeared similar between RE (Fig. 4A5, 4A8) and in vivo (Fig. 4A6, 4A9). Moreover, RE significantly increased the proportion of c-kit positive-cells per tubule cross section in comparison to BM (p = 0.02) (Fig. 4B2), but also the proportion of c-kit (p = 0.01) and Stra-8 (p = 0.01) positive tubules when compared to BM alone (Fig. 4B2 and 4B3). RE maintains a similar expression of c-kit and Stra-8 as observed in vivo.

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