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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

Time course of spermatogenesis in mice and in vitro culture procedure.(A) Schematic overview of the timeline of spermatogenesis during post-natal mouse development (A1) and procedure for the in vitro culture of prepubertal mice testes used in the present study (A2). The culture media tested for the culture of 2.5 and 6.5 dpp mice testis tissues are shown with (*) and (#) symbols, respectively. (B) Set of experiments performed after culture of 2.5 (B1) and 6.5 dpp (B2) mice testis tissues. The days of culture are mentioned according to the experiment performed for each age group (B1 and B2). Footnotes: BM: Basal Medium, D: Day, dpp: day post-partum, KSR: Knock-out Serum Replacement, FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, α-MEM: alpha-Minimum Essential Medium, PAS: Periodic Acid-Schiff, RE: Retinol, RIA: Radioimmunoassay, SSCs: Spermatogonial Stem Cells
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pone.0116660.g001: Time course of spermatogenesis in mice and in vitro culture procedure.(A) Schematic overview of the timeline of spermatogenesis during post-natal mouse development (A1) and procedure for the in vitro culture of prepubertal mice testes used in the present study (A2). The culture media tested for the culture of 2.5 and 6.5 dpp mice testis tissues are shown with (*) and (#) symbols, respectively. (B) Set of experiments performed after culture of 2.5 (B1) and 6.5 dpp (B2) mice testis tissues. The days of culture are mentioned according to the experiment performed for each age group (B1 and B2). Footnotes: BM: Basal Medium, D: Day, dpp: day post-partum, KSR: Knock-out Serum Replacement, FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, α-MEM: alpha-Minimum Essential Medium, PAS: Periodic Acid-Schiff, RE: Retinol, RIA: Radioimmunoassay, SSCs: Spermatogonial Stem Cells

Mentions: A schematic overview of the time course of spermatogenesis in mice and the experimental procedure can be found in Fig. 1A1. Testicular tissues were partially inserted into small holes in 1.5% (w/v) agarose gels (A6013–10G, Sigma-Aldrich) that had been pre-soaked overnight in BM to replace water. Each agarose gel strand was 7 mm in height and 1.3 ml of the culture medium was used for each Petri dish (Center-Well Organ Culture Dish-BDFalcon; BD Biosciences, Franklin Lakes, New Jersey, USA). Two pieces of agarose gels, each containing two testicular tissues, were then placed into a small Petri dish containing the basal culture medium alone or supplemented with FSH/LH and RE as described above (see “Culture media and reagents”). The incubator used for cultures contained 5% CO2 and 95% air and was maintained at 34°C (Fig. 1A2).


Retinol Improves In Vitro Differentiation of Pre-Pubertal Mouse Spermatogonial Stem Cells into Sperm during the First Wave of Spermatogenesis
Time course of spermatogenesis in mice and in vitro culture procedure.(A) Schematic overview of the timeline of spermatogenesis during post-natal mouse development (A1) and procedure for the in vitro culture of prepubertal mice testes used in the present study (A2). The culture media tested for the culture of 2.5 and 6.5 dpp mice testis tissues are shown with (*) and (#) symbols, respectively. (B) Set of experiments performed after culture of 2.5 (B1) and 6.5 dpp (B2) mice testis tissues. The days of culture are mentioned according to the experiment performed for each age group (B1 and B2). Footnotes: BM: Basal Medium, D: Day, dpp: day post-partum, KSR: Knock-out Serum Replacement, FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, α-MEM: alpha-Minimum Essential Medium, PAS: Periodic Acid-Schiff, RE: Retinol, RIA: Radioimmunoassay, SSCs: Spermatogonial Stem Cells
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116660.g001: Time course of spermatogenesis in mice and in vitro culture procedure.(A) Schematic overview of the timeline of spermatogenesis during post-natal mouse development (A1) and procedure for the in vitro culture of prepubertal mice testes used in the present study (A2). The culture media tested for the culture of 2.5 and 6.5 dpp mice testis tissues are shown with (*) and (#) symbols, respectively. (B) Set of experiments performed after culture of 2.5 (B1) and 6.5 dpp (B2) mice testis tissues. The days of culture are mentioned according to the experiment performed for each age group (B1 and B2). Footnotes: BM: Basal Medium, D: Day, dpp: day post-partum, KSR: Knock-out Serum Replacement, FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, α-MEM: alpha-Minimum Essential Medium, PAS: Periodic Acid-Schiff, RE: Retinol, RIA: Radioimmunoassay, SSCs: Spermatogonial Stem Cells
Mentions: A schematic overview of the time course of spermatogenesis in mice and the experimental procedure can be found in Fig. 1A1. Testicular tissues were partially inserted into small holes in 1.5% (w/v) agarose gels (A6013–10G, Sigma-Aldrich) that had been pre-soaked overnight in BM to replace water. Each agarose gel strand was 7 mm in height and 1.3 ml of the culture medium was used for each Petri dish (Center-Well Organ Culture Dish-BDFalcon; BD Biosciences, Franklin Lakes, New Jersey, USA). Two pieces of agarose gels, each containing two testicular tissues, were then placed into a small Petri dish containing the basal culture medium alone or supplemented with FSH/LH and RE as described above (see “Culture media and reagents”). The incubator used for cultures contained 5% CO2 and 95% air and was maintained at 34°C (Fig. 1A2).

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