Achievement of complete in vitro spermatogenesis in testicular tissues from prepubertal mice exposed to mono- or polychemotherapy

Abstract

The assessment of the impact of chemotherapies on in vitro spermatogenesis in experimental models is required before considering the application of this fertility restoration strategy to prepubertal boys who received these treatments before testicular tissue cryopreservation. The present work investigated the effects of exposure of prepubertal mice to mono- (vincristine or cyclophosphamide) and polychemotherapy (a combination of vincristine and cyclophosphamide) on the first wave of in vitro spermatogenesis. When testicular tissue exposed to monochemotherapy was preserved, polychemotherapy led to severe alterations of the seminiferous epithelium and increased apoptosis in prepubertal testes prior in vitro maturation, suggesting a potential additive gonadotoxic effect. These alterations were also found in the testicular tissues of polychemotherapy-treated mice after 30 days of organotypic culture and were associated with a reduction in the germ cell/Sertoli cell ratio. The different treatments neither altered the ability of spermatogonia to differentiate in vitro into spermatozoa nor the yield of in vitro spermatogenesis. However, more spermatozoa with morphological abnormalities and fragmented DNA were produced after administration of polychemotherapy. This work therefore shows for the first time the possibility to achieve a complete in vitro spermatogenesis after an in vivo exposure of mice to a mono- or polychemotherapy before meiotic entry.

Figure 1

Impact of chemotherapy prepubertal exposure on mouse testis before organotypic culture. 5923915316674500Histograms representing (a) mean ratios of testicular weight to body weight and (b) mean ratios of spermatogonia to Sertoli cells per seminiferous tubule in 6 dpp mice treated with VCR, CYP or VCR+CYP or untreated (control) at 3 dpp. The ratio of the mean number of spermatogonia counted per seminiferous tubule to the mean number of Sertoli cells observed per tubule was specified above each bar. TRA98 immunostaining was performed to detect spermatogonia (c). Intratubular cells were identified as: S for Sertoli cells (irregular TRA98− blue nuclei) and Sg for spermatogonia (smooth spherical TRA98+ brown nuclei). Morphological alterations of the seminiferous tubules were assessed semi-quantitatively by calculating a global lesional score including nuclear and epithelial alterations (d). Representative microscopy images of Hemalun Eosin Saffron-stained 6 dpp mouse testicular tissues are shown at a ×400 magnification (e). Panels on the upper right corner represent enlarged views of pyknotic nuclei () and vacuoles (*). Data are expressed as means ± SEM with n = 4 mice for all groups. A value of *P < 0.05 was considered statistically significant. ^aP < 0.05 compared to control; ^bP < 0.05 compared to VCR and ^cP < 0.05 compared to CYP.

Figure 2

Impact of chemotherapy prepubertal exposure on intratubular cell before organotypic culture. (a) Representative images of Ki67 (A1-D1) and cleaved caspase-3 (A2-D2) immunofluorescence and combined detection of γH₂A.X and DDX4 (A3-D3) in testicular tissues from 6 dpp control mice or VCR, CYP or VCR+CYP-treated mice. Testicular tissue sections were counterstained with DAPI (blue). (b) The percentage of intratubular cell proliferation (Ki67+ tubules in solid colour and Ki67+ cells in hatch colour) and (c) apoptosis (Caspase 3+ tubules in solid colour and cleaved caspase 3+ cells in hatch colour) were quantified for control, VCR, CYP and VCR+CYP groups. Double γH₂A.X and DDX4 immunofluorescence was performed to determine the percentage of spermatogonia with DNA double-strand breaks for each condition (d). Data are expressed as means ± SEM with n = 4 mice for all groups. A value of *P < 0.05 was considered statistically significant. Magnification ×400.

Figure 3

Progression of spermatogenesis following in vitro maturation of mouse testicular tissues treated by chemotherapy. (a) Microscopy images showing the general appearance of testicular tissue exposed to monochemotherapy (VCR or CYP), polychemotherapy (VCR+CYP) or unexposed (control) following 30 days of in vitro maturation. The necrotic area in cultured tissues has been delimited by dotted lines. (b) TRA98 immunostaining associated with Periodic Acid Schiff reaction was performed to visualize the progression of spermatogenesis after 30 days of organotypic culture, by detecting TRA98+ germ cells (spermatogonia, leptotene/zygotene and pachytene spermatocytes) and the pink-labelled acrosome (PAS+) of spermatids in control, CYP, VCR and VCR+CYP-treated mice. Intratubular cells were identified as: S for Sertoli cells (irregular TRA98- blue nuclei close to the basement membrane), Sg for spermatogonia (smooth spherical TRA98+ brown nuclei close to the basement membrane), L/Z for leptotene/zygotene spermatocytes (irregular spherical TRA98+ brown nuclei with condensed chromatin), P for pachytene spermatocytes (irregular spherical TRA98+ brown nuclei with highly condensed chromatin), RS for round spermatids (regular small round TRA98- blue nuclei and PAS+ acrosomal cap) or ES for elongated spermatids (elongated TRA98- blue nuclei with highly condensed chromatin and PAS+ acrosomal cap). Panels on the lower right corner represent a higher magnification of elongated spermatids (). (c) Percentage of seminiferous tubules at the most advanced differentiation stage of spermatogenesis after in vitro maturation of testicular tissues from mice exposed to VCR, CYP, VCR+CYP or unexposed (control) during the prepubertal period. Data are presented as means ± SEM with n = 4 testicular explants for each group. A value of *P < 0.05 was considered statistically significant.

Figure 4

Impact of chemotherapy prepubertal exposure on intratubular cell following 30 days of in vitro maturation. (a) Representative images of Ki67 (A1–D1), cleaved caspase-3 (A2–D2) and γH₂A.X (A3–D3) immunofluorescence in testicular tissues from control, VCR, CYP or VCR+CYP-treated mice after 30 days of culture. Testicular tissue sections were counterstained with DAPI (blue). (b) Histogram representing the mean ratio of germ cells to Sertoli cells per seminiferous tubule in cultured testicular fragments for each group. Ki67, cleaved caspase-3 and γH₂A.X immunodetection was performed to determine (c) the percentage of intratubular cell proliferation (Ki67+ tubules in solid colour and Ki67+ cells in hatch colour), (d) apoptosis (Caspase 3+ tubules in solid colour and cleaved caspase 3+ cells in hatch colour) and (e) intratubular cells with DNA double-strand breaks for each condition. The results are presented as means ± SEM with n = 4 testicular explants for all groups. A value of *P < 0.05 was considered statistically significant. Magnification ×400.

Figure 5

Impact of chemotherapy prepubertal exposure on in vitro produced spermatozoa. (a) Histogram representing the mean number of spermatozoa per milligram of tissue produced after in vitro maturation of testicular tissues from mice exposed to monochemotherapy (VCR or CYP), polychemotherapy (VCR+CYP) or unexposed (control) during the prepubertal period. (b) Percentage of spermatozoa with one or multiple (2, 3 or 4) flagella among the flagellated spermatozoa recovered from testicular tissues of control, VCR, CYP or VCR+CYP-treated mice after 30 days of culture. (c) Histogram representing the proportion of in vitro produced spermatozoa with normal or abnormal heads in testicular tissues exposed to VCR, CYP, VCR+CYP or unexposed (control). Sperm heads were assigned to two categories based on morphological observations: normal (elongated and hook-shaped) and abnormal. Abnormal heads included calyculate, microcephalic (small rounded head) and macrocephalic (large rounded head). The mean number of spermatozoa counted per milligram of tissue is indicated in brackets for each category of sperm head morphology. (d) Percentage of in vitro produced spermatozoa with fragmented DNA (TUNEL+) in testicular tissues from control, VCR, CYP or VCR+CYP-treated mice. For each condition, hundred spermatozoa were examined. The results are presented as means ± SEM with n = 6 pooled cultured testes per group. ^aP < 0.05 compared to control; ^bP < 0.05 compared to VCR and ^cP < 0.05 compared to CYP.