In vitro formation of the blood-testis barrier during long-term organotypic culture of human prepubertal tissue: comparison with a large cohort of pre/peripubertal boys.

de Michele F ，Poels J ，Giudice MG ，De Smedt F ，Ambroise J ，Vermeulen M ，Gruson D ，Wyns C ... -  《-》

Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue.

Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)? Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation. Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans. The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm3 tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared. ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3β-hydroxysteroid dehydrogenase (3β-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis. Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either medium. GDNF was expressed until Day 139, demonstrating SC functionality. Moreover, a significant reduction in AMH expression and release indicated SC maturation. Testosterone concentrations in supernatants increased in both culture media, demonstrating LC functionality with paracrine interactions. SG were present up to Day 139, although the ratio between MAGE-A4-positive cells and well-preserved tubules was significantly reduced over the course of culture (P ≤ 0.001). SCs exhibited a decreased proliferation rate over time (P ≤ 0.05). The proliferation rate of SG remained stable until Day 64, but over the total culture period (139 days), it was found to have decreased (P ≤ 0.05). The number of apoptotic cells did not vary during culture, nor was any statistical difference observed between the two culture media for any of the studied parameters. N/A LIMITATIONS, REASONS FOR CAUTION: Loss of SG constitutes a limitation for evaluating full functionality of spermatogonial stem cells and warrants further investigation. The scarcity of human immature material is the reason for the limited amount of tissue available for experiments, precluding more comprehensive analysis. Our culture system, mimicking the peripubertal testicular microenvironment with SC maturation, LC functionality and preserved paracrine interactions, and the first to use human ITT, opens the door to a deeper understanding of niche and culture conditions to obtain sperm from cryostored ITT, with the ultimate goal of restoring fertility after gonadotoxic treatments. This project was supported by a grant from the Fond National de la Recherche Scientifique de Belgique (grant Télevie N° 7.4554.14F and N° 7.4512.15F) and the Fondation Salus Sanguinis. No conflict of interest is declared.

de Michele F ，Poels J ，Weerens L ，Petit C ，Evrard Z ，Ambroise J ，Gruson D ，Wyns C ... -  《-》

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

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? Organotypic cultures allow the establishment and maintenance of major BTB components and the formation of a functional BTB in mouse testicular tissues. 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. 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. 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. 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. None. 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. 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. 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.

Rondanino C ，Maouche A ，Dumont L ，Oblette A ，Rives N ... -  《-》

Assessment of fresh and cryopreserved testicular tissues from (pre)pubertal boys during organ culture as a strategy for in vitro spermatogenesis.

Can the organ culture method be applied to both fresh and cryopreserved human (pre)pubertal testicular tissue as a strategy for in vitro spermatogenesis? Although induction of spermatogenesis was not achieved in vitro, testicular architecture, endocrine function and spermatogonial proliferation were maintained in both fresh and cryopreserved testicular tissues. Cryopreservation of a testicular biopsy is increasingly offered as a fertility preservation strategy for prepubertal cancer patients. One of the proposed experimental approaches to restore fertility is the organ culture method, which, in the mouse model, successfully allows for in vitro development of spermatozoa from testicular biopsies. However, complete spermatogenesis from human prepubertal testicular tissue in such an organ culture system has not been demonstrated. Testicular tissue was collected from nine (pre)pubertal boys diagnosed with cancer (ranging from 6 to 14 years of age) admitted for fertility preservation before treatment. Testicular biopsies were either immediately processed for culture or first cryopreserved, using a controlled slow freezing protocol, and thawed before culture. Organ culture of testicular fragments was performed in two different media for a maximum period of 5 weeks, targeting early cellular events (viability, meiosis and somatic differentiation) in vitro. Fresh and cryopreserved-thawed testis fragments (1-2 mm3) were cultured at a gas-liquid interphase (34°C, 5% CO2) in Minimum Essential Medium alpha + 10% knock-out serum replacement medium containing 10-7 M melatonin and 10-6 M retinoic acid, with or without 3 IU/L FSH/LH supplementation. The effect of culture conditions on testicular fragments was weekly assessed by histological evaluation of germ cell development and immunohistochemical identification of spermatogonia (using MAGEA4), proliferative status of spermatogonia and Sertoli cells (using proliferating cell nuclear antigen [PCNA]), intratubular cell apoptosis (by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) and Sertoli cells maturation (using Anti-Müllerian Hormone [AMH] versus Androgen Receptor [AR]). Additionally, Leydig cells' functionality was determined by measuring testosterone concentration in the culture media supernatants. Neither fresh nor cryopreserved human (pre)pubertal testicular fragments were able to initiate spermatogenesis in our organ culture system. Nonetheless, our data suggest that fresh and cryopreserved testicular fragments have comparable functionality in the described organ culture conditions, as reflected by the absence of significant differences in any of the weekly evaluated functional parameters. Additionally, no significant differences were found between the two tested media when culturing fresh and cryopreserved human testicular fragments. Although spermatogonia survived and remained proliferative in all culture conditions, a significant reduction of the spermatogonial population (P ≤ 0.001) was observed over the culture period, justified by a combined reduction of proliferation activity (P ≤ 0.001) and increased intratubular cell apoptosis (P ≤ 0.001). We observed a transient increase in Sertoli cell proliferative activity, loss of AMH expression (P ≤ 0.001) but no induction of AR expression. Leydig cell endocrine function was successfully stimulated in vitro as indicated by increased testosterone production in all conditions throughout the entire culture period (P ≤ 0.02). N/A. Although not noticeable in this study, we cannot exclude that if an optimized culture method ensuring complete spermatogenesis in human testicular fragments is established, differences in functional or spermatogenic efficiency between fresh and cryopreserved tissue might be found. The current inability to initiate spermatogenesis in vitro from cryopreserved human testicular fragments should be included in the counselling of patients who are offered testicular tissue cryopreservation to preserve fertility. This project was funded by EU-FP7-PEOPLE-2013-ITN 603568 `Growsperm'. None of the authors have competing interests. Not applicable.

Portela JMD ，de Winter-Korver CM ，van Daalen SKM ，Meißner A ，de Melker AA ，Repping S ，van Pelt AMM ... -  《-》

Gonadotropin suppression in men leads to a reduction in claudin-11 at the Sertoli cell tight junction.

Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5α-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), β-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and β-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. Claudin-11 formed a continuous staining pattern at the BTB in control men. Regardless of gonadotropin suppression treatment, claudin-11 localization was markedly disrupted and was broadly associated with the extent of meiotic/post-meiotic germ cell suppression; claudin-11 staining was (i) punctate (i.e. 'spotty' appearance) at the basal aspect of tubules when the average numbers of adluminal germ cells were <15% of control, (ii) presented as short fragments with cytoplasmic extensions when numbers were 15-25% of control or (iii) remained continuous when numbers were >40% of control. Changes in localization of connexin-43 and vinculin were also observed (smaller effects than for claudin-11) but ZO-1, β-catenin and β-actin did not differ, compared with control. Claudin-11 was the only Sertoli cell TJ protein investigated, but it is considered to be the most pivotal of constituent proteins given its known implication in infertility and BTB function. We were limited to testis samples which had been gonadotropin-suppressed for 8 weeks, shorter than the 74-day spermatogenic wave, which may account for the heterogeneity in claudin-11 and germ cell response observed among the men. Longer suppression (12-24 weeks) is known to suppress germ cells further and claudin-11 disruption may be more uniform, although we could not access such samples. These findings are important for our understanding of the sites of action of male hormonal contraception, because they suggest that BTB function could be ablated following long-term hormone suppression treatment. National Health and Medical Research Council (Australia) Program Grants 241000 and 494802; Research Fellowship 1022327 (to R.I.M.) and the Victorian Government's Operational Infrastructure Support Program. None of the authors have any conflicts to disclose. Not applicable.

McCabe MJ ，Tarulli GA ，Laven-Law G ，Matthiesson KL ，Meachem SJ ，McLachlan RI ，Dinger ME ，Stanton PG ... -  《-》