Fig 1.
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
Fig 2.
Seminiferous tubule growth (A1, A2), intra-tubular cell density (A3, A4), germ/Sertoli cells ratio (A5 and A6) and intra-tubular cell proliferation (B1, B2) during organotypic culture of prepubertal mice testes under different culture conditions.
The results are presented as the mean ± s.e.m., with n = 4. Asterisk indicates a statistically significant difference (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, PCNA: Proliferating Cell Nuclear Antigen, RE: Retinol, s.e.m.: Standard Error of the Mean
Fig 3.
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
Fig 4.
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
Fig 5.
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
Table 1.
Assessment of germ cell differentiation using Tra-98 immunostaining of seminiferous tubules obtained after in vitro culture of 2.5 dpp testes for 38 and 60 days compared with age-matched in vivo tissues.
Table 2.
Assessment of germ cell differentiation using Tra98 immunostaining of seminiferous tubules obtained after in vitro culture of 6.5 dpp testes for 30 and 36 days compared with age-matched in vivo tissues.
Fig 6.
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.