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