Microfluidic and Static Organotypic Culture Systems to Support Ex Vivo Spermatogenesis From Prepubertal Porcine Testicular Tissue: A Comparative Study.

Background: In vitro maturation of immature testicular tissue (ITT) cryopreserved for fertility preservation is a promising fertility restoration strategy. Organotypic tissue culture proved successful in mice, leading to live births. In larger mammals, including humans, efficiently reproducing spermatogenesis ex vivo remains challenging. With advances in biomaterials technology, culture systems are becoming more complex to better mimic in vivo conditions. Along with improving culture media components, optimizing physical culture conditions (e.g., tissue perfusion, oxygen diffusion) also needs to be considered. Recent studies in mice showed that by using silicone-based hybrid culture systems, the efficiency of spermatogenesis can be improved. Such systems have not been reported for ITT of large mammals. Methods: Four different organotypic tissue culture systems were compared: static i.e., polytetrafluoroethylene membrane inserts (OT), agarose gel (AG) and agarose gel with polydimethylsiloxane chamber (AGPC), and dynamic i.e., microfluidic (MF). OT served as control. Porcine ITT fragments were cultured over a 30-day period using a single culture medium. Analyses were performed at days (d) 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity, diameters, and tissue core integrity were evaluated on histology. Immunohistochemistry was used to identify germ cells (PGP9.5, VASA, SYCP3, CREM), somatic cells (SOX9, INSL3) and proliferating cells (Ki67), and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured in supernatants using ELISA. Results: ITT fragments survived and grew in all systems. ST diameters, and Sertoli cell (SOX9) numbers increased, meiotic (SYCP3) and post-meiotic (CREM) germ cells were generated, and testosterone was secreted. When compared to control (OT), significantly larger STs (d10 through d30), better tissue core integrity (d5 through d20), higher numbers of undifferentiated spermatogonia (d30), meiotic and post-meiotic germ cells (SYCP3: d20 and 30, CREM: d20) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cell (Ki67/VASA) numbers, Leydig cell (INSL3) numbers and testosterone levels were not significantly different between systems. Conclusions: Using a modified culture system (AGPC), germ cell survival and the efficiency of porcine germ cell differentiation were moderately improved ex vivo. We assume that further optimization can be obtained with concomitant modifications in culture media components.

Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles.

Avascular transplantation of frozen-thawed testicular tissue fragments represents a potential future technique for fertility restoration in boys with cancer. A significant loss of spermatogonia was observed in xeno-transplants of human tissue most likely due to the hypoxic period before revascularization. To reduce the effect of hypoxia-reoxygenation injuries, several options have already been explored, like encapsulation in alginate hydrogel and supplementation with nanoparticles delivering a necrosis inhibitor (NECINH) or VEGF. While these approaches improved short-term (5 days) vascular surfaces in grafts, neovessels were not maintained up to 21 days; i.e., the time needed for achieving vessel stabilization. To better support tissue grafts, nanoparticles loaded with VEGF, PDGF and NECINH were developed. Testicular tissue fragments from 4-5-week-old mice were encapsulated in calcium-alginate hydrogels, either non-supplemented (control) or supplemented with drug-loaded nanoparticles (VEGF-nanoparticles; VEGF-nanoparticles + PDGF-nanoparticles; NECINH-nanoparticles; VEGF-nanoparticles + NECINH-nanoparticles; and VEGF-nanoparticles + PDGF-nanoparticles + NECINH-nanoparticles) before auto-transplantation. Grafts were recovered after 5 or 21 days for analyses of tissue integrity (hematoxylin-eosin staining), spermatogonial survival (immuno-histo-chemistry for promyelocytic leukemia zinc finger) and vascularization (immuno-histo-chemistry for α-smooth muscle actin and CD-31). Our results showed that a combination of VEGF and PDGF nanoparticles increased vascular maturity and induced a faster maturation of vascular structures in grafts.

Fertility preservation for prepubertal boys: lessons learned from the past and update on remaining challenges towards clinical translation.

BACKGROUND: Childhood cancer incidence and survivorship are both on the rise. However, many lifesaving treatments threaten the prepubertal testis. Cryopreservation of immature testicular tissue (ITT), containing spermatogonial stem cells (SSCs), as a fertility preservation (FP) option for this population is increasingly proposed worldwide. Recent achievements notably the birth of non-human primate (NHP) progeny using sperm developed in frozen-thawed ITT autografts has given proof of principle of the reproductive potential of banked ITT. Outlining the current state of the art on FP for prepubertal boys is crucial as some of the boys who have cryopreserved ITT since the early 2000s are now in their reproductive age and are already seeking answers with regards to their fertility. OBJECTIVE AND RATIONALE: In the light of past decade achievements and observations, this review aims to provide insight into relevant questions for clinicians involved in FP programmes. Have the indications for FP for prepubertal boys changed over time? What is key for patient counselling and ITT sampling based on the latest achievements in animals and research performed with human ITT? How far are we from clinical application of methods to restore reproductive capacity with cryostored ITT? SEARCH METHODS: An extensive search for articles published in English or French since January 2010 to June 2020 using keywords relevant to the topic of FP for prepubertal boys was made in the MEDLINE database through PubMed. Original articles on fertility preservation with emphasis on those involving prepubertal testicular tissue, as well as comprehensive and systematic reviews were included. Papers with redundancy of information or with an absence of a relevant link for future clinical application were excluded. Papers on alternative sources of stem cells besides SSCs were excluded. OUTCOMES: Preliminary follow-up data indicate that around 27% of boys who have undergone testicular sampling as an FP measure have proved azoospermic and must therefore solely rely on their cryostored ITT to ensure biologic parenthood. Auto-transplantation of ITT appears to be the first technique that could enter pilot clinical trials but should be restricted to tissue free of malignant cells. While in vitro spermatogenesis circumvents the risk linked to cancer cell contamination and has led to offspring in mice, complete spermatogenesis has not been achieved with human ITT. However, generation of haploid germ cells paves the way to further studies aimed at completing the final maturation of germ cells and increasing the efficiency of the processes. WIDER IMPLICATIONS: Despite all the research done to date, FP for prepubertal boys remains a relatively young field and is often challenging to healthcare providers, patients and parents. As cryopreservation of ITT is now likely to expand further, it is important not only to acknowledge some of the research questions raised on the topic, e.g. the epigenetic and genetic integrity of gametes derived from strategies to restore fertility with banked ITT but also to provide healthcare professionals worldwide with updated knowledge to launch proper multicollaborative care pathways in the field and address clinical issues that will come-up when aiming for the child's best interest.

The air-liquid interface culture of the mechanically isolated seminiferous tubules embedded in agarose or alginate improves in vitro spermatogenesis at the expense of attenuating their integrity.

Optimization of tissue culture systems able to complete male germ cell maturation to post-meiotic stages is considered as an important matter in reproductive biology. Considering that hypoxia is one of the factors limiting the efficiency of organ culture, the aim of this study was to use isolated seminiferous tubules (STs), having more surface and less thickness, in an organotypic culture system in order to improve oxygen diffusion and reduce hypoxia. The mechanically separated STs embedded in agarose or alginate and 1-3-mm3 testicular tissue fragments of 3 adult mice were separately placed on the flat surface of agarose gel that was half-soaked in the medium. Survival and differentiation of germ cells using PLZF and SCP3 markers, identity of Sertoli cell using GATA4, cell proliferation with the Ki67 marker, and ST integrity using a ST scoring were evaluated up to 36 d at different culture times, each corresponding to the duration of one spermatogenic cycle. We observed a significantly reduced ST integrity in STs embedded in agarose or alginate on day 9 (versus tissue fragments p ≤ 0.05). There was no difference in the number of PLZF-positive cells between groups, but the number of SCP3 (in all-time points) and GATA4-positive cells was significantly higher in the culture of embedded STs. Although embedding STs can be useful for the progress of in vitro spermatogenesis, it makes them sensitive to degeneration. Further improvements are required to modify the air-liquid interface method to maintain ST integrity.

Blood Testis Barrier and Somatic Cells Impairment in a Series of 35 Adult Klinefelter Syndrome Patients.

Klinefelter Syndrome (KS) is the most common genetic cause of infertility in men. Degeneration of the testicular tissue starts in utero and accelerates at puberty with hyalinisation of seminiferous tubules, spermatogonia apoptosis and germ cell maturation arrest. Therefore, fertility preservation in young KS boys has been proposed, although this measure is still debated due to insufficient knowledge of the pathophysiology of the disease. To better understand the underlying mechanisms of testicular failure and germ cell loss, we analysed functional and morphological alterations in the somatic compartment of KS testis, i.e., Sertoli cells, including the blood-testis barrier (BTB) and Leydig cells (LC). We compared three populations: 35 KS 47,XXY non-mosaic patients, 28 Sertoli-cell-only (SCO) syndrome patients and 9 patients with normal spermatogenesis. In KS patients the expression of BTB proteins connexin-43 and claudin-11 assessed with a semi-quantitative scoring system appeared significantly reduced with a disorganised pattern. A significant reduction in seminiferous tubules expressing androgen receptors (AR) was observed in KS compared to normal spermatogenesis controls. INSL3 expression, a marker of LC maturation, was also significantly reduced in KS compared to patients with normal spermatogenesis or SCO. Hence, the somatic compartment impairment in KS could be involved in degeneration of seminiferous tubules.

Role of stem cells in fertility preservation: current insights.

While improvements made in the field of cancer therapy allow high survival rates, gonadotoxicity of chemo- and radiotherapy can lead to infertility in male and female pre- and postpubertal patients. Clinical options to preserve fertility before starting gonadotoxic therapies by cryopreserving sperm or oocytes for future use with assisted reproductive technology (ART) are now applied worldwide. Cryopreservation of pre- and postpubertal ovarian tissue containing primordial follicles, though still considered experimental, has already led to the birth of healthy babies after autotransplantation and is performed in an increasing number of centers. For prepubertal boys who do not produce gametes ready for fertilization, cryopreservation of immature testicular tissue (ITT) containing spermatogonial stem cells may be proposed as an experimental strategy with the aim of restoring fertility. Based on achievements in nonhuman primates, autotransplantation of ITT or testicular cell suspensions appears promising to restore fertility of young cancer survivors. So far, whether in two- or three-dimensional culture systems, in vitro maturation of immature male and female gonadal cells or tissue has not demonstrated a capacity to produce safe gametes for ART. Recently, primordial germ cells have been generated from embryonic and induced pluripotent stem cells, but further investigations regarding efficiency and safety are needed. Transplantation of mesenchymal stem cells to improve the vascularization of gonadal tissue grafts, increase the colonization of transplanted cells, and restore the damaged somatic compartment could overcome the current limitations encountered with transplantation.

Male fertility preservation in DSD, XXY, pre-gonadotoxic treatments - Update, methods, ethical issues, current outcomes, future directions.

This paper aims at reviewing the fertility preservation strategies that could be considered in several conditions at risk of spermatogonial depletion such as 46,XY disorders of sexual development, Klinefelter syndrome and after gonadotoxic treatment in males highlighting current knowledge on diseases and processes involved in infertility as well as future directions along with their specific ethical issues. While sperm cryopreservation after puberty is the only validated technique for fertility preservation, for prepubertal boys facing gonadotoxic therapies or at risk of testicular tissue degeneration where testicular sperm is not present, cryopreservation of spermatogonial cells may be an option to ensure future parenthood. Promising results with transplantation and in vitro maturation of spermatogonial cells were achieved in animals but so far none of the techniques was applied in humans.

Tissue Engineering to Improve Immature Testicular Tissue and Cell Transplantation Outcomes: One Step Closer to Fertility Restoration for Prepubertal Boys Exposed to Gonadotoxic Treatments.

Despite their important contribution to the cure of both oncological and benign diseases, gonadotoxic therapies present the risk of a severe impairment of fertility. Sperm cryopreservation is not an option to preserve prepubertal boys' reproductive potential, as their seminiferous tubules only contain spermatogonial stem cells (as diploid precursors of spermatozoa). Cryobanking of human immature testicular tissue (ITT) prior to gonadotoxic therapies is an accepted practice. Evaluation of cryopreserved ITT using xenotransplantation in nude mice showed the survival of a limited proportion of spermatogonia and their ability to proliferate and initiate differentiation. However, complete spermatogenesis could not be achieved in the mouse model. Loss of germ cells after ITT grafting points to the need to optimize the transplantation technique. Tissue engineering, a new branch of science that aims at improving cellular environment using scaffolds and molecules administration, might be an approach for further progress. In this review, after summarizing the lessons learned from human prepubertal testicular germ cells or tissue xenotransplantation experiments, we will focus on the benefits that might be gathered using bioengineering techniques to enhance transplantation outcomes by optimizing early tissue graft revascularization, protecting cells from toxic insults linked to ischemic injury and exploring strategies to promote cellular differentiation.

Role of LRRK2 in the regulation of dopamine receptor trafficking.

Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1) and D2 (DRD2) trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD.

Update on fertility restoration from prepubertal spermatogonial stem cells: How far are we from clinical practice?

Fertility preservation in prepubertal boys facing gonadotoxic treatment is still at the experimental stage. Nevertheless cryopreservation of immature testicular tissue (ITT) obtained by small testicular biopsy is being increasingly proposed in reproductive care clinics for this purpose. Different approaches to in vivo or in vitro mature spermatogonial stem cells (SSCs) contained in ITT have been studied: autografting of testicular tissue pieces, transplantation of one's own purified germ cell suspensions, and in vitro maturation (IVM) for subsequent use of sperm for intra cytoplasmic sperm injection (ICSI). While complete spermatogenesis yielding fertile offspring has been achieved in a number of animal species after cell and tissue transplantation and IVM, no mature sperm has yet been obtained from human prepubertal SSCs. This review describes research conducted by our team and a number of others working on fertility restoration from SSCs, with special emphasis on debated concerns and progress made towards clinical application of different strategies.

Differential protein-protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways.

Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson's disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1- and LRRK2-specific cellular processes by identifying their distinct interacting proteins. A protein microarray-based interaction screen was performed with recombinant 3xFlag-LRRK1 and 3xFlag-LRRK2 and, in parallel, co-immunoprecipitation followed by mass spectrometry was performed from SH-SY5Y neuroblastoma cell lines stably expressing 3xFlag-LRRK1 or 3xFlag-LRRK2. We identified a set of LRRK1- and LRRK2-specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14-3-3 consensus binding motifs. To assess the functional relevance of these interactions, SH-SY5Y-LRRK1 and -LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14-3-3 binding, or with EGF, an EGF-R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins. LRRK1 and LRRK2 (leucine-rich repeat kinase) interaction partners were identified by two different protein-protein interaction screens. These confirmed epidermal growth factor receptor (EGR-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. Functional analysis of these interactions and the pathways they mediate shows that LRRK1 and LRRK2 signaling do not intersect, reflective of the differential role of both LRRKs in Parkinson's disease.

LRRK2 affects vesicle trafficking, neurotransmitter extracellular level and membrane receptor localization.

The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S) pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S) shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

LRRK2 and vesicle trafficking.

Mutations in LRRK2 (leucine-rich repeat kinase 2) (also known as PARK8 or dardarin) are responsible for the autosomal-dominant form of PD (Parkinson's disease). LRRK2 mutations were found in approximately 3-5% of familial and 1-3% of sporadic PD cases with the highest prevalence (up to 40%) in North Africans and Ashkenazi Jews. To date, mutations in LRRK2 are a major genetic risk factor for familial and sporadic PD. Despite the fact that 8 years have passed from the establishment of the first link between PD and dardarin in 2004, the pathophysiological role of LRRK2 in PD onset and progression is far from clearly defined. Also the generation of different LRRK2 transgenic or knockout animals has not provided new hints on the function of LRRK2 in the brain. The present paper reviews recent evidence regarding a potential role of LRRK2 in the regulation of membrane trafficking from vesicle generation to the movement along cytoskeleton and finally to vesicle fusion with cell membrane.