Scaffold-Based and Scaffold-Free Testicular Organoids from Primary Human Testicular Cells.

Organoid systems take advantage of the self-organizing capabilities of cells to create diverse multi-cellular tissue surrogates that constitute a powerful novel class of biological models. Clearly, the formation of a testicular organoid (TO) in which human spermatogenesis can proceed from a single-cell suspension would exert a tremendous impact on research and development, clinical treatment of infertility, and screening of potential drugs and toxic agents. Recently, we showed that primary adult and pubertal human testicular cells auto-assembled in TOs either with or without the support of a natural testis scaffold. These mini-tissues harboured both the spermatogonial stem cells and their important niche cells, which retained certain specific functions during long-term culture. As such, human TOs might advance the development of a system allowing human in vitro spermatogenesis. Here we describe the methodology to make scaffold-based and scaffold-free TOs.

The Effect of a Unilateral Orchiectomy before Gonadotoxic Treatment on the Contralateral Testis in Adult and Prepubertal Rats.

PURPOSE: Previous studies have shown that the removal of one testis leads to a compensatory mechanism in the contralateral one, but this was species and age dependent. The aim of this study was to check whether this compensation would still occur after the combination of a unilateral orchiectomy and gonadotoxic treatment, since this resembles the clinical situation of patients who have to undergo highly toxic cancer treatment and therefore choose to cryopreserve a testicular biopsy for fertility restoration purposes. MATERIALS & METHODS: Sprague Dawley rats underwent either unilateral orchiectomy, gonadotoxic busulfan treatment, the combination of both or served as fertile control. A comparison of the compensatory effects was made between adult and prepubertal treated rats. Mating experiments were performed, testosterone levels were followed-up, testicular weight was recorded and histology was analysed. RESULTS: Adult treated rats were able to restore fertility spontaneously in all treatment groups. On the other hand, 30% of the rats that underwent a unilateral orchiectomy and gonadotoxic treatment at prepubertal age showed hampered spermatogenesis, low testosterone levels, decreased testicular weights and were not able to reproduce. CONCLUSION: This study emphasizes the need of fertility preservation strategies in prepubertal patients before gonadotoxic interventions.

Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions.

OBJECTIVE: To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cell (mSSC) culture conditions. DESIGN: Experimental basic science study. SETTING: Reproductive biology laboratory. PATIENT(S): Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. INTERVENTION(S): Testicular cells used to create in vitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA-)/epithelial cell surface antigen (EPCAM+) in coculture with inactivated testicular feeders from the same patient. MAIN OUTCOME MEASURE(S): Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. RESULT(S): Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells in vitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA-/EPCAM+ resulted in an enrichment of 27% VASA+/UTF1+ hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm2 after 2 weeks in vitro compared with unsorted cells (61 hSSCs/cm2) and differentially plated cells (49 hSSCS/cm2). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. CONCLUSION(S): We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA-/EPCAM+ sorted cells with testicular feeders improved the germ cell/somatic cell ratio.

Exome sequencing reveals a nonsense mutation in TEX15 causing spermatogenic failure in a Turkish family.

Infertility is a global healthcare problem, and despite long years of assisted reproductive activities, a significant number of cases remain idiopathic. Our currently restricted understanding of basic mechanisms driving human gametogenesis severely limits the improvement of clinical care for infertile patients. Using exome sequencing, we identified a nonsense mutation leading to a premature stop in the TEX15 locus (c.2130T>G, p.Y710*) in a consanguineous Turkish family comprising eight siblings in which three brothers were identified as infertile. TEX15 displays testis-specific expression, maps to chromosome 8, contains four exons and encodes a 2789-amino acid protein with uncertain function. The mutation, which should lead to early translational termination at the first exon of TEX15, co-segregated with the infertility phenotype, and our data strongly suggest that it is the cause of spermatogenic defects in the family. All three affected brothers presented a phenotype reminiscent of the one observed in KO mice. Indeed, previously reported results demonstrated that disruption of the orthologous gene in mice caused a drastic reduction in testis size and meiotic arrest in the first wave of spermatogenesis in males while female KO mice were fertile. The data from our study of one Turkish family suggested that the identified mutation correlates with a decrease in sperm count over time. A diagnostic test identifying the mutation in man could provide an indication of spermatogenic failure and prompt patients to undertake sperm cryopreservation at an early age.