BackgroundTesticular tissue cryopreservation before gonadotoxic treatments allows fertility preservation in children suffering from cancer. Fertility restoration strategies, in particular in vitro maturation of prepubertal testicular tissue, are being developed mainly in animal models. The rat, widely used in biomedical research, including in reproductive biology, is a relevant model. ObjectivesTo determine whether sequential two-step culture protocols can improve the efficiency of rat in vitro spermatogenesis. Materials and methodsRat prepubertal testicular tissues were cultured on agarose gels with either a one-step or two-step protocol with or without polydimethylsiloxane (PDMS) ceiling chips. The progression of spermatogenesis, germ/Sertoli cell ratio, cell proliferation, seminiferous tubule area, and intratubular cell density were assessed by histological and immunohistochemical analyses. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays and Peanut Agglutinin (PNA) lectin labeling were performed to analyze the Deoxyribonucleic Acid (DNA) integrity and differentiation step of in vitro-produced spermatids. ResultsSequential two-step protocols allowed the production of spermatids with a higher efficiency compared with the one-step culture protocol. However, the efficiency was low, as less than 1.5% of tubules contained spermatids. Most of the in vitro-produced spermatids contained unfragmented DNA and were at an early step of differentiation. Rare elongating spermatids could be detected in the cultured explants. Although complete in vitro spermatogenesis could not be obtained with PDMS ceiling chips, entry into meiosis was promoted in one-step organotypic cultures. Discussion and conclusionComplete in vitro meiosis and the beginning of the elongation phase of spermiogenesis were obtained in a rat model using sequential culture methods. Because of their low efficiency, further work will be necessary to identify the culture conditions allowing the completion of spermiogenesis. These optimizations could pave the way for future applications, including the development of an in vitro fertility restoration procedure for childhood cancer survivors, which is still far from being clinically available.

In vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest.

Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones being essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cell and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated in clinics for childhood cancer survivors.

La préservation de la fertilité des enfants présentant un cancer est depuis plusieurs années devenue uneétape importante dans la prise en charge de la qualité de vie après le cancer. La chimiothérapie et la radiothérapieutilisées dans le traitement du cancer peuvent entraîner une infertilité à l’âge adulte. Un protocole de congélationlente contrôlée mis au point au sein au laboratoire permet actuellement de préserver la structure et la fonctionnalitédu tissu testiculaire de ces enfants. Afin de restaurer la fertilité de ces patients, différentes méthodes de maturationin vitro du tissu testiculaire prépubère ont vu le jour. La culture organotypique sur gel d’agarose permet d’obtenirune spermatogenèse complète à partir de tissus testiculaires prépubères de souris. Cependant, un blocage de laspermatogenèse est observé lorsque ce même protocole est utilisé chez le rat ou chez l’homme. Le rat, dont ladurée de la spermatogenèse est intermédiaire entre la souris et l’homme, représente un modèle adéquat pour étudierce blocage méiotique.Après validation d’une procédure de congélation chez le rat prépubère, nous avons comparé l’effet demilieux de culture de composition différente sur la progression de la spermatogenèse in vitro. Nos résultatsmontrent une incapacité des milieux testés à soutenir une première vague complète de la spermatogenèse. Desanalyses histologiques ont montré un maintien de la fonctionnalité des tissus testiculaires de rats prépubères enculture. Nos résultats suggèrent plutôt une incapacité des milieux de culture à soutenir la différenciation descellules ayant initié leur méiose. Les analyses RNA-seq réalisées sur ces tissus mettent en évidence unedérégulation importante de l’expression des gènes en lien avec la spermatogenèse, la stéroïdogenèse ainsi que leprocessus inflammatoire.Afin de pouvoir envisager une application chez l’homme, il est indispensable de continuer ces travauxpar la réalisation de cultures séquentielles, permettant de mimer au mieux les conditions physiologiques, et parl’ajout de molécules anti-inflammatoires dans les milieux de culture. Par ailleurs, l’aplatissement des tissus entreune matrice de PDMS et un gel d’agarose pourrait être envisagé afin d’assurer une meilleure oxygénation etdiffusion des nutriments au sein des explants tissulaires.

Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones being essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cell and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated in clinics for childhood cancer survivors.