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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

Histological evaluation of seminiferous tubules after in vitro maturation of prepubertal (2.5 dpp and 6.5 dpp) CD-1 mice testes.(A) Spermatid formation during culture from gonocytes or spermatogonia of CD-1 mice testes. Photomicrographs were captured at a ×500 (A1 to A22) and ×1000 (Insets) magnification. The scale bar represents 40 μm or 5 μm in the photomicrograph or inset, respectively, which apply to all other photomicrographs and insets. Corresponding in vivo ages for the culture time points tested are also shown (A5–A9–A14–A18–A22). At D0 of culture, gonocytes (A1) (green box) or spermatogonia (A10) (blue box) were the only germ cells present in the seminiferous tubules. Red boxes represent round spermatids produced in each culture condition tested (BM, FSH/LH and RE) at D16, D30, D36, D38 and D60 of culture, and are enlarged in the lower inset of each photomicrograph. Black boxes represent elongated spermatids generated at each culture condition tested (BM, FSH/LH and RE) at D30, D36, D38, and D60 of culture, and are enlarged at the upper inset of each photomicrograph. (B) Histological evaluation of spermatogenic cell stages after Tra98, CREM-1 and Acrosin immunostaining in all conditions tested (BM, FSH/LH and RE) at D30 of culture with their corresponding in vivo ages (36.5 dpp). Photomicrographs were captured at a ×1000 magnification and the scale bars represent 20 μm. Negative controls are shown on the right of each positive immunostaining tested. (B1–B2–B3–B4) Brown staining indicates spermatogonia as well as leptotene/zygotene and early pachytene spermatocytes I with positive nuclear expression of Tra98. Sertoli cells, mid-pachytene and late pachytene spermatocytes I and round-to-elongated spermatids counterstain with hematoxylin only (no Tra98). (B5–B6–B7–B8) Immunodetection of CREM-1 as a specific marker of round spermatids that are enlarged at the inset of each photomicrograph for all culture conditions tested (BM, FSH/LH and RE). (B9–B10–B11–B12) Round spermatids developed a genuine acrosomal cap in all culture conditions tested (BM, FSH/LH and RE) at D30 of culture, which stains in brown using an anti-acrosin antibody, and is enlarged at the inset of each photomicrograph. Footnotes: CREM: cAMP-Responsive Element Modulator, D: Day, dpp: day post-partum, ES: Elongated Spermatid, E-P: Early Pachytene Spermatocyte I, HES: Hemalun Eosin Saffron, L/Z: Leptotene/Zygotene Spermatocyte I, L-P: Late Pachytene Spermatocyte I, PAS: Periodic Acid-Schiff, RS: Round Spermatid, S: Sertoli cell, Sg: Spermatogonia, IgG: Immunoglobulin G
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pone.0116660.g005: Histological evaluation of seminiferous tubules after in vitro maturation of prepubertal (2.5 dpp and 6.5 dpp) CD-1 mice testes.(A) Spermatid formation during culture from gonocytes or spermatogonia of CD-1 mice testes. Photomicrographs were captured at a ×500 (A1 to A22) and ×1000 (Insets) magnification. The scale bar represents 40 μm or 5 μm in the photomicrograph or inset, respectively, which apply to all other photomicrographs and insets. Corresponding in vivo ages for the culture time points tested are also shown (A5–A9–A14–A18–A22). At D0 of culture, gonocytes (A1) (green box) or spermatogonia (A10) (blue box) were the only germ cells present in the seminiferous tubules. Red boxes represent round spermatids produced in each culture condition tested (BM, FSH/LH and RE) at D16, D30, D36, D38 and D60 of culture, and are enlarged in the lower inset of each photomicrograph. Black boxes represent elongated spermatids generated at each culture condition tested (BM, FSH/LH and RE) at D30, D36, D38, and D60 of culture, and are enlarged at the upper inset of each photomicrograph. (B) Histological evaluation of spermatogenic cell stages after Tra98, CREM-1 and Acrosin immunostaining in all conditions tested (BM, FSH/LH and RE) at D30 of culture with their corresponding in vivo ages (36.5 dpp). Photomicrographs were captured at a ×1000 magnification and the scale bars represent 20 μm. Negative controls are shown on the right of each positive immunostaining tested. (B1–B2–B3–B4) Brown staining indicates spermatogonia as well as leptotene/zygotene and early pachytene spermatocytes I with positive nuclear expression of Tra98. Sertoli cells, mid-pachytene and late pachytene spermatocytes I and round-to-elongated spermatids counterstain with hematoxylin only (no Tra98). (B5–B6–B7–B8) Immunodetection of CREM-1 as a specific marker of round spermatids that are enlarged at the inset of each photomicrograph for all culture conditions tested (BM, FSH/LH and RE). (B9–B10–B11–B12) Round spermatids developed a genuine acrosomal cap in all culture conditions tested (BM, FSH/LH and RE) at D30 of culture, which stains in brown using an anti-acrosin antibody, and is enlarged at the inset of each photomicrograph. Footnotes: CREM: cAMP-Responsive Element Modulator, D: Day, dpp: day post-partum, ES: Elongated Spermatid, E-P: Early Pachytene Spermatocyte I, HES: Hemalun Eosin Saffron, L/Z: Leptotene/Zygotene Spermatocyte I, L-P: Late Pachytene Spermatocyte I, PAS: Periodic Acid-Schiff, RS: Round Spermatid, S: Sertoli cell, Sg: Spermatogonia, IgG: Immunoglobulin G

Mentions: (i) Cultures of 2.5 dpp mice testes. At D0, seminiferous tubules contained only gonocytes and Sertoli cells (Fig. 5A1, S1A1 Fig.). Round and elongated spermatids were detected in all conditions tested at D34 (S1A2–S1A4 Fig.), D38 and D60 (Fig. 5A2–5A9). At D34, the mean percentage of elongated spermatids was significantly increased in presence of FSH/LH when compared to BM (p = 0.02). However, RE significantly increased the mean percentage of round (p = 0.01) but also elongated spermatids (p = 0.01) when compared with BM (S1A5 and S1A6 Fig.). At D38, FSH/LH significantly increased the mean proportion of round spermatids compared with BM (p = 0.02) (Table 1). However, the mean proportion of elongated spermatids was not significantly different between these two conditions at D38 or at D60 (Table 1). RE had no effect on the percentage of round spermatids observed at D38 but significantly increased the mean proportion of elongated spermatids compared with BM (p = 0.02). The mean proportion of round spermatids did not increase at D60 compared with D38 of culture whatever the condition tested (p = 0.34). The mean proportions of round and elongated spermatids are not statistically different between FSH/LH and RE at D38 and D60. Regardless of the culture medium, the proportions of round or elongated spermatids obtained were significantly lower in vitro at D34, D38 and D60 compared with the corresponding in vivo ages, i.e., 36.5 dpp (p = 0.01), 40.5 dpp (p = 0.01) and 62.5 dpp (p = 0.01).


Retinol Improves In Vitro Differentiation of Pre-Pubertal Mouse Spermatogonial Stem Cells into Sperm during the First Wave of Spermatogenesis
Histological evaluation of seminiferous tubules after in vitro maturation of prepubertal (2.5 dpp and 6.5 dpp) CD-1 mice testes.(A) Spermatid formation during culture from gonocytes or spermatogonia of CD-1 mice testes. Photomicrographs were captured at a ×500 (A1 to A22) and ×1000 (Insets) magnification. The scale bar represents 40 μm or 5 μm in the photomicrograph or inset, respectively, which apply to all other photomicrographs and insets. Corresponding in vivo ages for the culture time points tested are also shown (A5–A9–A14–A18–A22). At D0 of culture, gonocytes (A1) (green box) or spermatogonia (A10) (blue box) were the only germ cells present in the seminiferous tubules. Red boxes represent round spermatids produced in each culture condition tested (BM, FSH/LH and RE) at D16, D30, D36, D38 and D60 of culture, and are enlarged in the lower inset of each photomicrograph. Black boxes represent elongated spermatids generated at each culture condition tested (BM, FSH/LH and RE) at D30, D36, D38, and D60 of culture, and are enlarged at the upper inset of each photomicrograph. (B) Histological evaluation of spermatogenic cell stages after Tra98, CREM-1 and Acrosin immunostaining in all conditions tested (BM, FSH/LH and RE) at D30 of culture with their corresponding in vivo ages (36.5 dpp). Photomicrographs were captured at a ×1000 magnification and the scale bars represent 20 μm. Negative controls are shown on the right of each positive immunostaining tested. (B1–B2–B3–B4) Brown staining indicates spermatogonia as well as leptotene/zygotene and early pachytene spermatocytes I with positive nuclear expression of Tra98. Sertoli cells, mid-pachytene and late pachytene spermatocytes I and round-to-elongated spermatids counterstain with hematoxylin only (no Tra98). (B5–B6–B7–B8) Immunodetection of CREM-1 as a specific marker of round spermatids that are enlarged at the inset of each photomicrograph for all culture conditions tested (BM, FSH/LH and RE). (B9–B10–B11–B12) Round spermatids developed a genuine acrosomal cap in all culture conditions tested (BM, FSH/LH and RE) at D30 of culture, which stains in brown using an anti-acrosin antibody, and is enlarged at the inset of each photomicrograph. Footnotes: CREM: cAMP-Responsive Element Modulator, D: Day, dpp: day post-partum, ES: Elongated Spermatid, E-P: Early Pachytene Spermatocyte I, HES: Hemalun Eosin Saffron, L/Z: Leptotene/Zygotene Spermatocyte I, L-P: Late Pachytene Spermatocyte I, PAS: Periodic Acid-Schiff, RS: Round Spermatid, S: Sertoli cell, Sg: Spermatogonia, IgG: Immunoglobulin G
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pone.0116660.g005: Histological evaluation of seminiferous tubules after in vitro maturation of prepubertal (2.5 dpp and 6.5 dpp) CD-1 mice testes.(A) Spermatid formation during culture from gonocytes or spermatogonia of CD-1 mice testes. Photomicrographs were captured at a ×500 (A1 to A22) and ×1000 (Insets) magnification. The scale bar represents 40 μm or 5 μm in the photomicrograph or inset, respectively, which apply to all other photomicrographs and insets. Corresponding in vivo ages for the culture time points tested are also shown (A5–A9–A14–A18–A22). At D0 of culture, gonocytes (A1) (green box) or spermatogonia (A10) (blue box) were the only germ cells present in the seminiferous tubules. Red boxes represent round spermatids produced in each culture condition tested (BM, FSH/LH and RE) at D16, D30, D36, D38 and D60 of culture, and are enlarged in the lower inset of each photomicrograph. Black boxes represent elongated spermatids generated at each culture condition tested (BM, FSH/LH and RE) at D30, D36, D38, and D60 of culture, and are enlarged at the upper inset of each photomicrograph. (B) Histological evaluation of spermatogenic cell stages after Tra98, CREM-1 and Acrosin immunostaining in all conditions tested (BM, FSH/LH and RE) at D30 of culture with their corresponding in vivo ages (36.5 dpp). Photomicrographs were captured at a ×1000 magnification and the scale bars represent 20 μm. Negative controls are shown on the right of each positive immunostaining tested. (B1–B2–B3–B4) Brown staining indicates spermatogonia as well as leptotene/zygotene and early pachytene spermatocytes I with positive nuclear expression of Tra98. Sertoli cells, mid-pachytene and late pachytene spermatocytes I and round-to-elongated spermatids counterstain with hematoxylin only (no Tra98). (B5–B6–B7–B8) Immunodetection of CREM-1 as a specific marker of round spermatids that are enlarged at the inset of each photomicrograph for all culture conditions tested (BM, FSH/LH and RE). (B9–B10–B11–B12) Round spermatids developed a genuine acrosomal cap in all culture conditions tested (BM, FSH/LH and RE) at D30 of culture, which stains in brown using an anti-acrosin antibody, and is enlarged at the inset of each photomicrograph. Footnotes: CREM: cAMP-Responsive Element Modulator, D: Day, dpp: day post-partum, ES: Elongated Spermatid, E-P: Early Pachytene Spermatocyte I, HES: Hemalun Eosin Saffron, L/Z: Leptotene/Zygotene Spermatocyte I, L-P: Late Pachytene Spermatocyte I, PAS: Periodic Acid-Schiff, RS: Round Spermatid, S: Sertoli cell, Sg: Spermatogonia, IgG: Immunoglobulin G
Mentions: (i) Cultures of 2.5 dpp mice testes. At D0, seminiferous tubules contained only gonocytes and Sertoli cells (Fig. 5A1, S1A1 Fig.). Round and elongated spermatids were detected in all conditions tested at D34 (S1A2–S1A4 Fig.), D38 and D60 (Fig. 5A2–5A9). At D34, the mean percentage of elongated spermatids was significantly increased in presence of FSH/LH when compared to BM (p = 0.02). However, RE significantly increased the mean percentage of round (p = 0.01) but also elongated spermatids (p = 0.01) when compared with BM (S1A5 and S1A6 Fig.). At D38, FSH/LH significantly increased the mean proportion of round spermatids compared with BM (p = 0.02) (Table 1). However, the mean proportion of elongated spermatids was not significantly different between these two conditions at D38 or at D60 (Table 1). RE had no effect on the percentage of round spermatids observed at D38 but significantly increased the mean proportion of elongated spermatids compared with BM (p = 0.02). The mean proportion of round spermatids did not increase at D60 compared with D38 of culture whatever the condition tested (p = 0.34). The mean proportions of round and elongated spermatids are not statistically different between FSH/LH and RE at D38 and D60. Regardless of the culture medium, the proportions of round or elongated spermatids obtained were significantly lower in vitro at D34, D38 and D60 compared with the corresponding in vivo ages, i.e., 36.5 dpp (p = 0.01), 40.5 dpp (p = 0.01) and 62.5 dpp (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