Establishment, maintenance and functional integrity of the blood-testis barrier in organotypic cultures of fresh and frozen/thawed prepubertal mouse testes.

STUDY QUESTION: Can the spatio-temporal formation of an intact blood-testis barrier (BTB), which is essential for the progression of spermatogenesis, be reproduced in cultures of fresh or frozen/thawed prepubertal mouse testes? SUMMARY ANSWER: Organotypic cultures allow the establishment and maintenance of major BTB components and the formation of a functional BTB in mouse testicular tissues. WHAT IS KNOWN ALREADY: In vitro maturation of prepubertal testicular tissues is a promising approach to restore fertility in adult survivors of childhood cancer. Although gametes can be successfully obtained from prepubertal mouse testes in organotypic cultures, the spermatogenic yield remains low compared to in vivo controls. STUDY DESIGN, SIZE, DURATION: Mouse testicular tissues were frozen using controlled slow freezing (CSF) or solid surface vitrification (SSV) procedures. A total of 158 testes (fresh n = 58, CSF n = 58 or SSV n = 42) from 6 to 7 days postpartum (dpp) mice were cultured at 34°C in basal medium (α-MEM, 10% KnockOut Serum Replacement, 5 µg/ml gentamicin) at a gas-liquid interphase (under 20% O2), with or without 10-6 M retinol, for 9, 16 and 30 days. In addition, 32 testes from 6-7, 15-16, 22-23 and 36-37 dpp mice were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: The mRNA levels of BTB genes (Claudin 3, Claudin 11, Zonula occludens 1 and Connexin-43), germ cell-specific genes (Sal-like protein 4, Kit oncogene, Stimulated by retinoic acid gene 8, Synaptonemal complex protein 3, Transition protein 1 and Protamine 2), markers of Sertoli cell immaturity/maturity (anti-Mullerian hormone, androgen receptor, cyclin-dependent kinase inhibitor 1b) and the androgen-regulated gene Reproductive homeobox 5 (Rhox5) were measured by quantitative RT-PCR (RT-qPCR). The localization of BTB proteins in seminiferous tubules was studied by immunohistochemistry and spermatogenic progression was evaluated histologically. The integrity of the BTB was assessed using a biotin tracer. MAIN RESULTS AND THE ROLE OF CHANCE: Modest differences in Claudin 11 (Cldn11), Zonula occludens 1 (Zo-1), Connexin-43 (Cx43) transcript levels and in the localization of the corresponding proteins were found between in vitro cultures of fresh or frozen/thawed testes and in vivo controls (P < 0.05). However, a 32-77-fold decrease in Claudin 3 (Cldn3) mRNA levels and a lack of CLDN3 immunolabelling in 36-44% of seminiferous tubules were observed in 30-day organotypic cultures (P < 0.05). Although Sertoli cell maturation and the completion of a full spermatogenic cycle were achieved after 30 days of culture, meiotic and postmeiotic progression was altered in cultured testicular tissues (P < 0.05). Moreover, an increased BTB permeability and a decreased expression of Rhox5 were observed at the end of the culture period in comparison with in vivo controls (P < 0.05). Completion of spermatogenesis occurred in vitro in seminiferous tubules with an intact BTB, and in those expressing or lacking CLDN3. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Further studies will be needed to determine whether the expression of other BTB components is altered and to decipher the reason for lower Cldn3 and Rhox5 mRNA levels in organotypic cultures. WIDER IMPLICATIONS OF THE FINDINGS: This work contributes to a better understanding of the molecular mechanisms occurring in in vitro matured prepubertal testes. The organotypic culture system will have to be developed further and optimized for human tissue, before potential clinical applications can be envisaged. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Rouen University Hospital, Ligue contre le Cancer (to L.D.), and co-supported by European Union and Région Normandie (to A.O.). Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest.

Assessment of sperm nuclear quality after in vitro maturation of fresh or frozen/thawed mouse pre-pubertal testes.

STUDY QUESTION: Is nuclear quality of in vitro generated spermatozoa from fresh or frozen/thawed pre-pubertal mouse testes similar to that of their in vivo counterparts? SUMMARY ANSWER: The production of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was not significantly increased in organotypic cultures compared to in vivo controls. WHAT IS KNOWN ALREADY: Although murine spermatozoa have been produced in vitro from pre-pubertal testes, their nuclear DNA integrity has never been investigated. STUDY DESIGN, SIZE, DURATION: Fresh and frozen/thawed testicular fragments from 6 to 7 days postpartum (dpp) mice were cultured for 30 days. Testicular tissues were frozen by controlled slow freezing (CSF) or solid surface vitrification (SSV). In total, 30 fresh, 30 CSF, 30 SSV testes were used for in vitro maturation and 6 testes from 36 to 37 dpp mice were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine spermatozoa were extracted from pooled in vitro cultured testicular fragments and from in vivo controls. Sperm aneuploidy was analyzed by fluorescence in situ hybridization (FISH), DNA fragmentation by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, chromatin condensation by aniline blue staining, telomere length and number by quantitative FISH, DNA oxidation by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Because of the low spermatogenic yield in cultures, a hundred spermatozoa extracted from pooled tissues were examined and compared to their in vivo counterparts. MAIN RESULTS AND THE ROLE OF CHANCE: Most of spermatozoa generated in vitro and in vivo were haploid, contained unfragmented DNA and normally condensed chromatin. A similar proportion of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was found in cultures and in vivo. No significant difference in telomere length was found within the nuclei of in vitro and in vivo generated spermatozoa. However, the number of telomere spots was lower in gametes obtained from cultures of fresh, CSF and SSV testes than in their natural counterparts (P < 0.01). Moreover, the proportion of spermatozoa containing 8-OHdG was significantly increased in frozen/thawed tissues in comparison to fresh tissues and in vivo controls (P < 0.05). LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: Further studies will be needed to enhance the production of spermatozoa in organotypic cultures while preserving their quality, to investigate epigenetic modifications and embryonic development. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study comparing the nuclear quality of in vitro and in vivo generated murine spermatozoa. The organotypic culture system will have to be adapted for human tissue and extensive analyses of human gamete quality will have to be performed before potential clinical applications can be envisaged. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Rouen University Hospital, Ligue contre le Cancer, Agence de la Biomédecine, Association Laurette Fugain, France Lymphome Espoir, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.

Vitamin A prevents round spermatid nuclear damage and promotes the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Does vitamin A (retinol, Rol) prevent round spermatid nuclear damage and increase the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue? SUMMARY ANSWER: The supplementation of an in vitro culture of ~0.75 mm3 testicular explants from pre-pubertal mice with Rol enhances spermatogenesis progression during the first spermatogenic wave. WHAT IS KNOWN ALREADY: The production of functional spermatozoa in vitro has only been achieved in the mouse model and remains a rare event. Establishing an efficient culture medium for vitrified pre-pubertal testicular tissue is now a crucial step to improve the spermatic yield obtained in vitro. The role of Rol in promoting the differentiation of spermatogonia and their entry into meiosis is well established; however, it has been postulated that Rol is also required to support their full development into elongated spermatids. STUDY DESIGN, SIZE, DURATION: A total of 60 testes from 6.5 days post-partum (dpp) mice were vitrified/warmed, cut into fragments and cultured for 30 days: 20 testes were used for light microscopy and histological analyses, 20 testes for DNA fragmentation assessment in round spermatids and 20 testes for induced sperm motility assessment. Overall, 16 testes of 6.5 dpp were used as in vitro fresh tissue controls and 12 testes of 36.5 dpp mice as in vivo controls. Testes were vitrified with the optimal solid surface vitrification procedure and cultured with an in vitro organ culture system until Day 30 (D30). Histological analysis, cell death, degenerating round spermatids, DNA fragmentation in round spermatids and induced sperm motility were assessed. Testosterone levels were measured in media throughout the culture by radioimmunoassay. MAIN RESULTS AND THE ROLE OF CHANCE: At D30, better tissue development together with higher differentiation of spermatogonial stem cells, and higher global cell division ability were observed for vitrified/warmed testicular fragments of ~0.75 mm3 with a culture medium supplemented with Rol compared to controls. During in vitro culture of vitrified pre-pubertal testicular tissue, Rol enhanced and maintained the entry of spermatogonia into meiosis and promoted a higher spermatic yield. Furthermore, decreased round spermatid nuclear alterations and DNA damage combined with induced sperm motility comparable to in vivo highlight the crucial role of Rol in the progression of spermatogenesis during the first wave. LIMITATIONS, REASONS FOR CAUTION: Despite our promising results, the culture media will have to be further improved and adapted within the context of a human application. WIDER IMPLICATIONS OF THE FINDINGS: The results have potential implications for the handling of human pre-pubertal testicular tissues cryopreserved for fertility preservation. However, because some alterations in round spermatids persist after in vitro culture with Rol, the procedure needs to be optimized before human application, bearing in mind that the murine and human spermatogenic processes differ in many respects. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by a Ph.D. grant from the Normandy University and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Rouen University Hospital, Institute for Research and Innovation in Biomedicine (IRIB) and Agence de la Biomédecine. The authors declare that there is no conflict of interest.