Impact of low- or moderate-risk gonadotoxic chemotherapy prior to testicular tissue freezing on spermatogonia quantity in human (pre)pubertal testicular tissue.

STUDY QUESTION: What is the impact of low- or moderate-risk gonadotoxic chemotherapy received prior to testicular tissue freezing (TTF), and of the cancer itself, on spermatogonia quantity in testicular tissue from (pre)pubertal boys? SUMMARY ANSWER: Vincristine, when associated with alkylating agents, has an additional adverse effect on spermatogonia quantity, while carboplatin has no individual contribution to spermatogonia quantity, in testicular tissue of (pre)pubertal boys, when compared to patients who have received non-alkylating chemotherapy. WHAT IS KNOWN ALREADY: The improved survival rates after cancer treatment necessitate the inclusion of fertility preservation procedures as part of the comprehensive care for patients, taking into consideration their age. Sperm cryopreservation is an established procedure in post-pubertal males while the TTF proposed for (pre)pubertal boys remains experimental. Several studies exploring testicular tissue of (pre)pubertal boys after TTF have examined the tubular fertility index (TFI, percentage of seminiferous tubule cross-sections containing spermatogonia) and the number of spermatogonia per seminiferous tubule cross-section (S/T). All studies have demonstrated that TFI and S/T always decrease after the introduction of chemotherapeutic agents, especially those which carry high gonadotoxic risks such as alkylating agents. STUDY DESIGN, SIZE, DURATION: Testicular tissue samples from 79 (pre)pubertal boys diagnosed with cancer (from 6 months to 16 years of age) were cryopreserved between May 2009 and June 2014. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of (pre)pubertal testicular tissue to elucidate whether the chemotherapy or the primary diagnosis affects mainly TFI and S/T. PARTICIPANTS/MATERIALS, SETTING, METHODS: (Pre)pubertal boys with cancer diagnosis who had been offered TTF prior to conditioning treatment for hematopoietic stem cell transplantation were included in the study. All the patients had previously received chemotherapy with low- or moderate-risk for future fertility. We have selected patients for whom the information on the chemotherapy received was complete. The quantity of spermatogonia and quality of testicular tissue were assessed by both morphological and immunohistochemical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant reduction in the number of spermatogonia was observed in boys treated with alkylating agents. The mean S/T values in boys exposed to alkylating agents were significantly lower compared to boys exposed to non-alkylating agents (P = 0.018). In contrast, no difference was observed for patients treated with carboplatin as the sole administered alkylating agent compared to the group of patients exposed to non-alkylating agents. We observed an increase of S/T with age in the group of patients who did not receive any alkylating agent and a decrease of S/T with age when patients received alkylating agents included in the cyclophosphamide equivalent dose (CED) formula (r = 0.6166, P = 0.0434; r = -0.3759, P = 0.0036, respectively). The TFI and S/T decreased further in the group of patients who received vincristine in combination with alkylating agents (decrease of 22.4%, P = 0.0049 and P < 0.0001, respectively), but in this group the CED was also increased significantly (P < 0.0001). Multivariate analysis, after CED adjustment, showed the persistence of a decrease in TFI correlated with vincristine administration (P = 0.02). LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study of testicular tissues obtained from (pre)pubertal boys who were at risk of infertility. The study population is quite heterogeneous, with a small number of patients in each sub-group. Our results are based on comparisons between patients receiving alkylating agents compared to patients receiving non-alkylating agents rather than chemotherapy-naive patients. The French national guidelines for fertility preservation in cancer patients recommend TTF before highly gonadotoxic treatment. Therefore, all the patients had received low- or moderate-risk gonadotoxic chemotherapy before TTF. Access to testicular tissue samples from chemotherapy-naive patients with comparable histological types of cancer was not possible. The functionality of spermatogonia and somatic cells could not be tested by transplantation or in vitro maturation due to limited sample sizes. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes the spermatogonial quantity of (pre)pubertal boys prior to TTF. We confirmed a negative correlation between the cumulative exposure to alkylating agents and spermatogonial quantity. In addition, the synergistic use of vincristine in combination with alkylating agents showed a cumulative deleterious effect on the TFI. For patients for whom fertility preservation is indicated, TTF should be proposed for chemotherapy with a predicted CED above 4000 mg/m2. However, the data obtained from vincristine and carboplatin use should be confirmed in a subsequent study including more patients. STUDY FUNDING/COMPETING INTEREST(S): This study had financial support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. The sponsors played no role in the study. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Experience, and gynaecological and reproductive health follow-up of young adult women who have undergone ovarian tissue cryopreservation.

RESEARCH QUESTION: What are the experience, and gynaecological and reproductive health outcomes in young adult women who have undergone ovarian tissue cryopreservation (OTC)? DESIGN: A retrospective observational study was conducted at a single institution between May 2019 and February 2021 including 87 women aged over 18 years undergoing OTC. Medical characteristics and questionnaire data collected more than 18 months after OTC were analysed. RESULTS: Close to 74% (n = 64/87) of women had a follow-up consultation and completed the questionnaire. Most women found the information provided on the OTC technique and the strategies proposed to restore fertility with ovarian tissue understandable and useful. The majority of patients thought that OTC had a positive impact on their well-being during disease treatment. Anti-Müllerian hormone serum concentration decreased significantly after treatment (P < 0.0001) and was significantly lower when patients received chemotherapy before OTC (P = 0.0039). The total cyclophosphamide equivalent dose was significantly higher in women with FSH concentrations above 25 IU/l after treatment (P = 0.0004). More than 70% of women who planned a pregnancy after the end of treatment succeeded, with a natural pregnancy rate close to 53%. Only nine patients (8.0%) underwent ovarian tissue transplantation for fertility restoration and six of them became pregnant and delivered at least once. CONCLUSION: Young adult women expressed a good satisfaction rate with OTC and that their experience had been beneficial. The usage rate of cryopreserved ovarian tissue remains low. The gynaecological and reproductive health follow-up consultation should be included in the supportive care provided following OTC.

Understanding the Underlying Molecular Mechanisms of Meiotic Arrest during In Vitro Spermatogenesis in Rat Prepubertal Testicular Tissue.

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.

Cannabis consumption might exert deleterious effects on sperm nuclear quality in infertile men.

RESEARCH QUESTION: Can cannabis consumption alter sperm nuclear integrity in infertile men? DESIGN: A retrospective cross-sectional study conducted between July 2003 and December 2013, which included 54 men who consulted for male-factor infertility. Twenty-seven infertile men who were regular cannabis users were matched to 27 infertile men who were cannabis non-users. To complement the conventional semen parameter and plasma hormone level assessments, sperm nuclear alterations were explored using fluorescence in-situ hybridization to assess numerical chromosomal abnormalities, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling to investigate DNA fragmentation, aniline blue staining to examine chromatin condensation and a motile sperm organelle morphology examination to detect vacuoles in sperm heads. RESULTS: The rates of sperm aneuploidy (P = 0.0044), diploidy (P = 0.037), total chromosome abnormalities (P = 0.0027) and DNA fragmentation (P = 0.027) were significantly higher in cannabis users than in non-cannabis users. CONCLUSIONS: Cannabis consumption might have deleterious effects on sperm nuclear quality in infertile men by increasing numerical chromosome abnormalities and DNA fragmentation. Cannabis consumption induces these detrimental effects on the progression of spermatogenesis from meiotic stages to spermiogenesis and potentially on post-testicular sperm maturation in infertile men. Any potential findings, however, need to be validated with larger sample size, and our data are only exploratory findings.

Exposure to Chemotherapy During Childhood or Adulthood and Consequences on Spermatogenesis and Male Fertility.

Over the last decade, the number of cancer survivors has increased thanks to progress in diagnosis and treatment. Cancer treatments are often accompanied by adverse side effects depending on the age of the patient, the type of cancer, the treatment regimen, and the doses. The testicular tissue is very sensitive to chemotherapy and radiotherapy. This review will summarize the epidemiological and experimental data concerning the consequences of exposure to chemotherapy during the prepubertal period or adulthood on spermatogenic progression, sperm production, sperm nuclear quality, and the health of the offspring. Studies concerning the gonadotoxicity of anticancer drugs in adult survivors of childhood cancer are still limited compared with those concerning the effects of chemotherapy exposure during adulthood. In humans, it is difficult to evaluate exactly the toxicity of chemotherapeutic agents because cancer treatments often combine chemotherapy and radiotherapy. Thus, it is important to undertake experimental studies in animal models in order to define the mechanism involved in the drug gonadotoxicity and to assess the effects of their administration alone or in combination on immature and mature testis. These data will help to better inform cancer patients after recovery about the risks of chemotherapy for their future fertility and to propose fertility preservation options.

DNA methylation and histone post-translational modifications in the mouse germline following in-vitro maturation of fresh or cryopreserved prepubertal testicular tissue.

RESEARCH QUESTION: Do cryopreservation and in-vitro culture procedures affect the expression of DNA methyltransferases (DNMT) and histone-modifying enzymes, as well as the establishment of DNA methylation and histone post-translational modifications (PTM) in germ cells in prepubertal mouse testicular tissue? DESIGN: This study investigated the expression of epigenetic modification enzymes, DNA methylation and histone PTM, and the spermatogenic progression after in-vitro maturation of fresh or cryopreserved mouse prepubertal testicular tissue. Fresh or cryopreserved testicular fragments from 6-7 days post-partum mice were cultured for 30 days in the presence of retinol with or without FSH. RESULTS: The in-vitro maturation of fresh or cryopreserved tissue allowed the differentiation of spermatogonia into spermatozoa. Differences in the levels of transcripts encoding epigenetic modification enzymes (Dnmt1, Dnmt3a, Jarid1b, Src1, Sirt1, Hdac1) were found between 30-day tissue cultures and age-matched in-vivo controls. DNMT1/DNMT3a expression and the presence of 5-methylcytosine (5mC) were detected in spermatogonia and leptotene/zygotene spermatocytes in cultures. The relative 5mC fluorescence intensity was similar in spermatozoa produced in cultures of cryopreserved tissues or in vivo. H3K4me3, H3K9ac and H4K8ac were present in all germ cell types but differences in the proportion of germ cells containing these epigenetic marks were found after cultures. CONCLUSIONS: Despite differences with the in-vivo situation, DNA methylation and histone methylation and acetylation occur in the mouse germline in in-vitro matured fresh or cryopreserved mouse prepubertal testicular tissue, and the expression of the enzymes catalysing these epigenetic modifications are maintained in vitro.

Vitamin E but Not GSH Decreases Reactive Oxygen Species Accumulation and Enhances Sperm Production during In Vitro Maturation of Frozen-Thawed Prepubertal Mouse Testicular Tissue.

Freezing-thawing procedures and in vitro culture conditions are considered as a source of stress associated with increased reactive oxygen species (ROS) generation, leading to a damaged cell aerobic metabolism and consequently to oxidative stress. In the present study, we sought to investigate whether vitamin E (Vit E) or reduced glutathione (GSH) enhances sperm production by decreasing ROS accumulation during in vitro maturation of prepubertal mice testes. Testes of prepubertal mice were cryopreserved using a freezing medium supplemented or not supplemented with Vit E and were cultured after thawing. In presence of Rol alone in culture medium, frozen-thawed (F-T) testicular tissues exhibited a higher ROS accumulation than fresh tissue during in vitro culture. However, Vit E supplementation in freezing, thawing, and culture media significantly decreased cytoplasmic ROS accumulation in F-T testicular tissue during in vitro maturation when compared with F-T testicular tissue cultured in the presence of Rol alone, whereas GSH supplementation in culture medium significantly increased ROS accumulation associated with cytolysis and tissue disintegration. Vit E but not GSH promoted a better in vitro sperm production and was a suitable ROS scavenger and effective molecule to improve the yield of in vitro spermatogenesis from F-T prepubertal mice testes. The prevention of oxidative stress in the cytoplasmic compartment should be regarded as a potential strategy for improving testicular tissue viability and functionality during the freeze-thaw procedure and in vitro maturation.

Does soaking temperature during controlled slow freezing of pre-pubertal mouse testes influence course of in vitro spermatogenesis?

The banking of testicular tissue before highly gonadotoxic treatment is a prerequisite for the preservation of fertility in pre-pubertal boys not yet producing sperm. The aim of the current study is to evaluate the impact of a soaking temperature performed at -7 °C, -8 °C or -9 °C on the ability of frozen-thawed mouse spermatogonial stem cells (SSCs) to generate haploid germ cells after in vitro maturation. Testes of 6.5-day-old post-partum CD-1 mice were cryopreserved by using a controlled slow freezing protocol with soaking at -7 °C, -8 °C or -9 °C. Frozen-thawed pre-pubertal testicular tissues were cultured in vitro on agarose gel for 30 days. Histological evaluations were performed and flagellated late spermatids were counted after mechanical dissection of the cultured tissues. The differentiation of frozen SSCs into elongated spermatids was more efficient after treatment at -9 °C than at -7 °C and -8 °C. After dissection, flagellated late spermatids were observed by using Shorr staining. The number of flagellated late spermatids was significantly decreased after slow freezing when compared with a fresh tissue control. Therefore, the soaking temperature during slow freezing of pre-pubertal mouse testicular tissue might positively influence the course of in vitro spermatogenesis. Our slow freezing protocol with a soaking temperature at -9 °C was the optimal condition in terms of the achievement of in vitro spermatogenesis with a higher production of elongated spermatids, although the effectiveness of the maturation process was reduced compared with the fresh tissue control.

Evaluation of sperm nuclear integrity in patients with different percentages of decapitated sperm in ejaculates.

The decapitated sperm defect is a rare type of teratozoospermia responsible for male infertility. Spermatozoa from patients affected by this syndrome are used for intracytoplasmic sperm injection (ICSI) although little is known about their DNA integrity. This study evaluated sperm nuclear alterations in four patients and ten fertile men (control group). Sperm samples were examined by light, transmission electron and high-magnification contrast microscopy and analysed after terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling, aniline blue staining and fluorescence in-situ hybridization. Spermatozoa from patients presented varying degrees of decapitation, along with morphological and ultrastructural head abnormalities. Whereas the proportion of spermatozoa with fragmented DNA and numerical chromosome abnormalities was similar in patients 1-3 and controls, the percentage of spermatozoa with hypocondensed chromatin was higher in patients 1-3 than in fertile men. Patient 4 presented a distinct phenotype, with an increased proportion of flagellated spermatozoa with DNA strand breaks as well as increased aneuploidy and diploidy rates compared with controls and with patients 1-3. No successful pregnancy resulted from ICSI although embryos were obtained for three patients. The morphological defects and the nuclear alterations observed in spermatozoa of patients with the decapitated sperm syndrome may have contributed to ICSI failures.

Genetic identification of intracellular trafficking regulators involved in Notch-dependent binary cell fate acquisition following asymmetric cell division.

Notch signalling is involved in numerous cellular processes during development and throughout adult life. Although ligands and receptors are largely expressed in the whole organism, activation of Notch receptors only takes place in a subset of cells and/or tissues and is accurately regulated in time and space. Previous studies have demonstrated that endocytosis and recycling of both ligands and/or receptors are essential for this regulation. However, the precise endocytic routes, compartments and regulators involved in the spatiotemporal regulation are largely unknown. In order to identify intracellular trafficking regulators of Notch signalling, we have undertaken a tissue-specific dsRNA genetic screen of candidates potentially involved in endocytosis and recycling within the endolysosomal pathway. dsRNA against 418 genes was induced in the Drosophila melanogaster sensory organ lineage in which Notch signalling regulates binary cell fate acquisition. Gain or loss of Notch signalling phenotypes were observed in adult sensory organs for 113 of them. Furthermore, 26 genes were found to regulate the steady state localisation of Notch, Sanpodo, a Notch co-factor, and/or Delta in the pupal lineage. In particular, we identified 20 genes with previously unknown function in D. melanogaster intracellular trafficking. Among them, we identified CG2747 and we show that it regulates the localisation of clathrin adaptor AP-1 complex, a negative regulator of Notch signalling. Together, our results further demonstrate the essential function of intracellular trafficking in regulating Notch-signalling-dependent binary cell fate acquisition and constitute an additional step toward the elucidation of the routes followed by Notch receptor and ligands during signalling.

Complete meiosis in rat prepubertal testicular tissue under in vitro sequential culture conditions.

BACKGROUND: Testicular 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. OBJECTIVES: To determine whether sequential two-step culture protocols can improve the efficiency of rat in vitro spermatogenesis. MATERIALS AND METHODS: Rat 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. RESULTS: Sequential 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 CONCLUSION: Complete 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.

Throughout in vitro first spermatogenic wave: Next-generation sequencing gene expression patterns of fresh and cryopreserved prepubertal mice testicular tissue explants.

Introduction: Suitable cryopreservation procedures of pre-pubertal testicular tissue associated with efficient culture conditions are crucial in the fields of fertility preservation and restoration. In vitro spermatogenesis remains a challenging technical procedure to undergo a complete spermatogenesis.The number of haploid cells and more specifically the spermatic yield produced in vitro in mice is still extremely low compared to age-matched in vivo controls and this procedure has never yet been successfully transferred to humans. Methods: To evaluate the impact of in vitro culture and freezing procedure, pre-pubertal testicular mice testes were directly cultured until day 4 (D4), D16 and D30 or cryopreserved by controlled slow freezing then cultured until D30. Testes composed of a panel of 6.5 dpp (days postpartum), 10.5 dpp, 22.5 dpp, and 36.5 dpp mice were used as in vivo controls. Testicular tissues were assessed by histological (HES) and immunofluorescence (stimulated by retinoic acid gene 8, STRA8) analyses. Moreover, a detailed transcriptome evaluation study has been carried out to study the gene expression patterns throughout the first in vitro spermatogenic wave. Results: Transcriptomic analyses reveal that cultured tissues expression profiles are almost comparable between D16 and D30; highlighting an abnormal kinetic throughout the second half of the first spermatogenesis during in vitro cultures. In addition, testicular explants have shown dysregulation of their transcriptomic profile compared to controls with genes related to inflammation response, insulin-like growth factor and genes involved in steroidogenesis. Discussion: The present work first shows that cryopreservation had very little impact on gene expression in testicular tissue, either directly after thawing or after 30 days in culture. Transcriptomic analysis of testis tissue samples is highly informative due to the large number of expressed genes and identified isoforms. This study provides a very valuable basis for future studies concerning in vitro spermatogenesis in mice.

Steroidogenesis and androgen/estrogen signaling pathways are altered in in vitro matured testicular tissues of prepubertal mice.

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 are 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 cells 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 into clinics for childhood cancer survivors.

Identification and characterization of endogenous galectins expressed in Madin Darby canine kidney cells.

Madin Darby canine kidney (MDCK) cells are a well characterized epithelial cell line used to study mechanisms of polarized delivery. As glycans on apically expressed proteins have been identified as targeting signals, and crosslinking by the abundant galectin-3 has been implicated in the mechanism of glycan-dependent sorting, we wanted to identify other members of the galectin (Gal) family expressed in MDCK cells. By analyzing intron-exon boundaries, we identified canine genes that were highly homologous to mammalian Gal-1, 2, 3, 4, 7, 8, 9, and 12, and galectin-related HSPC159 and GRIFIN. Transcripts for Gal-2 and -12 were not detected in MDCK cells, but we found transcript levels for Gal-3 > Gal-9 > Gal-8 > Gal-1 ⋙ Gal-4 > Gal-7. Canine Gal-1, -2, -3, -4, -7, -8, -9, and -12 were cloned and expressed in Escherichia coli as GST fusion proteins to characterize binding specificities on arrays of synthetic glycans on glass slides from Core H of the NIH Consortium for Functional Glycomics. Individual expression of the N-terminal (GST-Gal-9N) and C-terminal (GST-Gal-9C) carbohydrate recognition domains greatly improved protein yield and the ability to characterize Gal-9 binding on the array. Canine galectins differentially bound sulfated disaccharides as well as human blood groups A, B, and H on both N-glycans and linear glycan structures on the array. Analysis of GST-Gal-1, -3, -4, -7, -8, -9N, and -9C binding to immunopurified human MUC1 expressed in MDCK cells revealed a preference for binding GST-Gal-3 and -9, which interestingly reflects the two most abundant galectins expressed in MDCK cells.

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

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.

Galectin-7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells.

Galectins (Gal) are ß-galactoside-binding proteins that function in epithelial development and homeostasis. An overlapping role for Gal-3 and Gal-7 in wound repair was reported in stratified epithelia. Although Gal-7 was thought absent in simple epithelia, it was reported in a proteomic analysis of cilia isolated from cultured human airway, and we recently identified Gal-7 transcripts in Madin-Darby canine kidney (MDCK) cells (Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. J Biol Chem 286: 6780-6790, 2011). We now report that Gal-7 is localized exclusively on the primary cilium of MDCK, LLC-PK(1) (pig kidney), and mpkCCD(c14) (mouse kidney) cells as well as on cilia in the rat renal proximal tubule. Gal-7 is also present on most cilia of multiciliated cells in human airway epithelia primary cultures. Interestingly, exogenous glutathione S-transferase (GST)-Gal-7 bound the MDCK apical plasma membrane as well as the cilium, while the lectin Ulex europeaus agglutinin, with glycan preferences similar to Gal-7, bound the basolateral plasma membrane as well as the cilium. In pull-down assays, ß1-integrin isolated from either the basolateral or apical/cilia membranes of MDCK cells was similarly bound by GST-Gal-7. Selective localization of Gal-7 to cilia despite the presence of binding sites on all cell surfaces suggests that intracellular Gal-7 is specifically delivered to cilia rather than simply binding to surface glycoconjugates after generalized secretion. Moreover, depletion of Gal-7 using tetracycline-induced short-hairpin RNA in mpkCCD(c14) cells significantly reduced cilia length and slowed wound healing in a scratch assay. We conclude that Gal-7 is selectively targeted to cilia and plays a key role in surface stabilization of glycoconjugates responsible for integrating cilia function with epithelial repair.

Improving Freezing Protocols and Organotypic Culture: A Histological Study on Rat Prepubertal Testicular Tissue.

Testicular tissue freezing before gonadotoxic treatments allows the preservation of fertility for children suffering from cancer. Recently, the testis organ culture method was presented as a relevant method to restore the fertility of these patients. However, the yield of spermatozoa production is low in the mouse model and no gamete has been obtained in vitro in the rat model. Here, we assess different cryopreservation protocols and culture conditions to improve the efficiency of in vitro maturation of rat prepubertal testes. Testes from male rats aged 5 or 8 days post-partum were cultured onto agarose gels of different percentages. After determining the best culture conditions, different cryopreservation protocols were assessed. Finally, testicular tissues were cultured with media of various compositions and analyzed at different time points. Our results show that the cryopreservation protocols allow the preservation of tissue architecture, cell proliferation, with no or moderate increase of cell death. In vitro spermatogenesis did not proceed beyond the pachytene spermatocyte stage. Only 2 of the 6 tested media allowed the survival of differentiated germ cells over the 45-day culture period. In conclusion, this study highlights the necessity to further improve the organ culture method before applying it into the clinics.

Activation of the cannabinoid receptor type 2 by the agonist JWH133 promotes the first wave of in vitro spermatogenesis.

BACKGROUND: Oncological procedures have irreversible side effects on germ cells for childhood cancer survival boys. In vitro culture of prepubertal testicular tissue has been proposed to restore fertility; however, recent data on animal models showed that meiotic and post-meiotic progression was impaired. OBJECTIVES: As potential key inducers of the mitosis-meiosis switch, type 2 cannabinoid receptor (CB2 ) has been proposed to play a central role in the meiotic entry of male germ cells. Herein, the in vitro first spermatogenesis wave in mice was used to understand the impact of CB2 activation on the differentiation of spermatogonia until elongated spermatids. MATERIALS AND METHODS: A first set of cultured testicular explants of 6.5 days post-partum (dpp) mice was performed to assess the impact of a range of JWH133 supplementation (10 nm, 100 nm, 1 µm, 10 µm). Then, the progressive development of germ cells at key timepoints of spermatogenesis was evaluated throughout (i) in vitro culture (day 2 [D2], D3, D6, D10, D18, and D30) coupled with (ii) in vivo counterparts (8.5, 9.5, 12.5, 16.5, 24.5, and 36.5 dpp). RESULTS: CB2 was detected at the plasma membrane of cells, and a successful completion of spermatogenesis was obtained in vitro. One day after the activation of CB2 by 1 µm of the agonist JWH133, percentage of zygotene spermatocyte I increased. CONCLUSION: After 30 days of culture, (i) an enrichment of haploid germ cells detected by flow cytometry, (ii) a reduced necrotic area, and (iii) an increase in the density of post-meiotic germ cells were observed. We showed that the activation of CB2 improves in vitro entry into meiosis and differentiation of spermatogonia, mimicking physiological meiotic transition.

Dynamics of epigenetic modifications in ICSI embryos from in vitro-produced spermatozoa.

BACKGROUND: In prepubertal boys with cancer, fertility preservation relies on testicular tissue freezing before treatment. In vitro maturation of frozen/thawed tissues could be one of the procedures envisaged to restore the fertility of cured patients. It is necessary to ascertain in the mouse model that in vitro-generated spermatozoa are able to ensure embryo development, without altering the epigenetic processes occurring during the pre-implantation period. OBJECTIVES: The aims of the present study were to investigate the fertilizing ability of in vitro-produced spermatozoa and explore several epigenetic marks at different stages of embryo development. MATERIALS AND METHODS: Fresh or controlled slow-frozen (CSF)/thawed testicular tissues from 6 to 7 days post-partum (dpp) mice were cultured for 30 days. Intracytoplasmic sperm injection (ICSI) experiments were performed using in vitro-produced spermatozoa. Testicular spermatozoa from 36 to 37 dpp mice were used as in vivo controls. DNA methylation/hydroxymethylation and histone post-translational modifications (H3K4me3, H3K27me3 and H3K9ac) were analysed by immunofluorescence from the zygote to the blastocyst stages. RESULTS: The spermatozoa generated in cultures of fresh or CSF testicular tissues were able to initiate embryonic development. The freezing of prepubertal testicular tissues limits the production of spermatozoa in vitro and the fertilization rate after ICSI. Similar levels of H3K4me3, H3K27me3 and H3K9ac were found in ICSI embryos derived from in vitro- and in vivo-produced spermatozoa. DNA methylation levels were increased in 4-cell embryos and morula obtained by ICSI with in vitro-produced spermatozoa. DISCUSSION AND CONCLUSION: Our study shows for the first time that the use of in vitro-produced spermatozoa alters DNA methylation/demethylation dynamics but has little impact on H3K4me3, H3K27me3 and H3K9ac levels in mouse early embryos. Further work will have to be performed to determine whether the use of these gametes is not deleterious for embryo development before considering a human application.

IHC_Tool: An open-source Fiji procedure for quantitative evaluation of cross sections of testicular explants.

Immunohistochemical analysis is a routine procedure for clinical and research studies in male fertility. However, most of the interpretations remain subjective and time-consuming, with inherent intra- and inter-observer variability. Given the prognostic and research implications of testicular assessment, a more objective and less time-consuming method is required. In the current study, we used in vitro matured pre-pubertal murine testes as a model. The main objective was to develop an affordable automated digital immunohistochemistry image analysis tool for an unbiased and quantitative assessment of testicular tissue sections. Testicular explants were fixed, cut, and stained for specific germ cell markers. The classical manual counting procedure was evaluated. Background and noise were reduced on brightfield images. Photomicrographs were stitched (Background_Elimination_Stitching) to create high-quality images. Two procedures were evaluated (IHC_Tool and Stained_Nuclear_Area); then a procedure (Necrotic_Area_Elimination) allowing withdrawal of the necrotic area observed after culture was assessed. Finally, the number of stained nuclei in the unaltered tissue area was extracted. The automated IHC_Tool procedure with images saved as TIFF at a ×200 magnification allowed the most rigorous cell quantification. IHC_Tool developed for testicular sample analysis can be used for various types of tissues. We foresee that this method will minimize inter-observer variations across laboratories and will be helpful for clinical trials and translational initiatives.