Cancer treatment in childhood and testicular function : the importance of the somatic environment

Testicular function and future fertility may be affected by cancer treatment during childhood. Whilst survival of the germ (stem) cells is critical for ensuring the potential for fertility in these patients, the somatic cell populations also play a crucial role in providing a suitable environment to support germ cell maintenance and subsequent development. Regulation of the spermatogonial germ-stem cell niche involves many signalling pathways with hormonal influence from the hypothalamo-pituitary-gonadal axis. In this review, we describe the somatic cell populations that comprise the testicular germ-stem cell niche in humans and how they may be affected by cancer treatment during childhood. We also discuss the experimental models that may be utilized to manipulate the somatic environment and report the results of studies that investigate the potential role of somatic cells in the protection of the germ cells in the testis from cancer treatment.Peer reviewe

Testicular organoids: a new model to study the testicular microenvironment in vitro?

BACKGROUND: In recent decades, a broad range of strategies have been applied to model the testicular microenvironment in vitro. These models have been utilized to study testicular physiology and development. However, a system that allows investigations into testicular organogenesis and its impact in the spermatogonial stem-cell (SSC) niche in vitro has not been developed yet. Recently, the creation of tissue-specific organ-like structures called organoids has resurged, helping researchers to answer scientific questions that previous in vitro models could not help to elucidate. So far, a small number of publications have concerned the generation of testicular organoids and their application in the field of reproductive medicine and biology. OBJECTIVE AND RATIONALE: Here, we aim to elucidate whether testicular organoids might be useful in answering current scientific questions about the regulation and function of the SSC niche as well as germ cell proliferation and differentiation, and whether or not the existing in vitro models are already sufficient to address them. Moreover, we would like to discuss how an organoid system can be a better solution to address these prominent scientific problems in our field, by the creation of a rationale parallel to those in other areas where organoid systems have been successfully utilized. SEARCH METHODS: We comprehensively reviewed publications regarding testicular organoids and the methods that most closely led to the formation of these organ-like structures in vitro by searching for the following terms in both PubMed and the Web of Science database: testicular organoid, seminiferous tubule 3D culture, Sertoli cell 3D culture, testicular cord formation in vitro, testicular morphogenesis in vitro, germ cell 3D culture, in vitro spermatogenesis, testicular de novo morphogenesis, seminiferous tubule de novo morphogenesis, seminiferous tubule-like structures, testicular in vitro model and male germ cell niche in vitro, with no restrictions to any publishing year. The inclusion criteria were based on the relation with the main topic (i.e. testicular organoids, testicular- and seminiferous-like structures as in vitro models), methodology applied (i.e. in vitro culture, culture dimensions (2D, 3D), testicular cell suspension or fragments) and outcome of interest (i.e. organization in vitro). Publications about grafting of testicular tissue, germ-cell transplantation and female germ-cell culture were excluded. OUTCOMES: The application of organoid systems is making its first steps in the field of reproductive medicine and biology. A restricted number of publications have reported and characterized testicular organoids and even fewer have denominated such structures by this method. However, we detected that a clear improvement in testicular cell reorganization is recognized when 3D culture conditions are utilized instead of 2D conditions. Depending on the scientific question, testicular organoids might offer a more appropriate in vitro model to investigate testicular development and physiology because of the easy manipulation of cell suspensions (inclusion or exclusion of a specific cell population), the fast reorganization of these structures and the controlled in vitro conditions, to the same extent as with other organoid strategies reported in other fields. WIDER IMPLICATIONS: By way of appropriate research questions, we might use testicular organoids to deepen our basic understanding of testicular development and the SSC niche, leading to new methodologies for male infertility treatment

Placental Apoptosis in Small for Gestational Age Babies: A Comparison between Swedish and Pakistani Populations

Background: Foeto-placental growth is regulated by a complex balance of growth promoting and growth inhibiting factors and hormones, namely the insulin-like growth factors (IGF) and the intracellular caspase proteins. Changes in the IGF-axis appear to affect this balance, with deficiencies possibly triggering apoptosis.Aim: To ascertain levels of apoptosis in the placenta of infants born small for gestational age (SGA) and appropriate for gestational age (AGA), comparing samples from two population groups, Pakistani and Swedish, in an attempt to better understand the mechanism behind foetal-placental growth restriction.Methods: Placental samples were taken immediately following delivery in both Karachi and Stockholm. In total 36 samples were included for further analysis (Pakistani: SGA n = 12, AGA n = 12; Swedish: SGA n = 7, AGA n = 5). Protein extraction was conducted for cell-death ELISA, and the remaining tissue samples were then paraffin embedded for further immunohistochemical and immunoflourescent analysis, looking at the apoptotic proteins, p53, caspase 8, and caspase 3. Furthermore, we compared maternal and newborn anthropometry between populations.Results: A higher apoptotic index, for caspase 8 and caspase 3, was seen in the Pakistani samples, as compared to the Swedish samples (p<0.01). TUNEL assays showed higher levels of apoptosis in the Pakistani population as compared to the Swedish population (p<0.01). Cell death ELISA analysis showed greater apoptotic activity in placenta from the Pakistani population as compared to the Swedish groups (p<0.05) as well as increased apoptotic activity in the SGA groups as compared to the AGA groups within each population (ELISA, p<0.05). No differences were seen in p53 levels as assessed by immunohistochemistry. Pakistani mothers were, on average, shorter than their Swedish counterparts (p<0.01).Conclusion: Increased apoptotic activity in placenta of the Pakistani population, as compared to their Swedish counterparts, may be associated with decreased foetal-placental growth seen in this population, particularly in babies born SGA. These findings, along with previously published results of the IGF-axis, and birth weight outcomes, suggests that lower IGF levels may be involved in the extracellular triggering of apoptosis, through caspase 8. This may further suggest a possible mechanism of foetal-placental growth restriction

Testicular organoid generation by a novel in vitro three-layer gradient system.

A system that models the testicular microenvironment and spermatogonial stem-cell (SSC) niche in vitro has not been produced yet. Here, we developed and characterized a novel three-dimensional multilayer model, the Three-Layer Gradient System (3-LGS), which permits the generation of rat testicular organoids with a functional blood-testis barrier (BTB) and germ cell establishment and proliferation. The model is unique as regards the formation of cellular organizations that more closely represent the in vivo germ-to-somatic cell associations in vitro. Moreover, we also verified the roles of retinoic acid (RA), IL-1α, TNFα and RA inhibitors in germ cell maintenance and BTB organization in vitro. Treatment with RA was beneficial for germ cell maintenance, while IL-1α and TNFα were observed to impair the formation of testicular organoids and germ cell maintenance. Taking in account our characterization and validation results, we propose the 3-LGS as a new platform to investigate the SSC niche in vitro and to search for novel unknown factors involved in germ cell proliferation and differentiation. Moreover, we suggest that this model can be used in other scientific fields to study organogenesis and development by the generation of organoids

Self-organising human gonads generated by a Matrigel-based gradient system.

BACKGROUND: Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro. Here we extend the model to human first-trimester embryonic gonadal tissue. RESULTS: Testicular cell suspensions reorganised into testis-like organoids with distinct seminiferous-like cords situated within an interstitial environment after 7 days. In contrast, tissue reorganisation failed to occur when mesonephros, which promotes testicular development in vivo, was included in the tissue digest. Organoids generated from dissociated female gonad cell suspensions formed loosely organised cords after 7 days. In addition to displaying testis-specific architecture, testis-like organoids demonstrated evidence of somatic cell differentiation. Within the 3-LGS, we observed the onset of AMH expression in the cytoplasm of SOX9-positive Sertoli cells within reorganised testicular cords. Leydig cell differentiation and onset of steroidogenic capacity was also revealed in the 3-LGS through the expression of key steroidogenic enzymes StAR and CYP17A1 within the interstitial compartment. While the 3-LGS generates a somatic cell environment capable of supporting germ cell survival in ovarian organoids germ cell loss was observed in testicular organoids. CONCLUSION: The 3-LGS can be used to generate organised whole gonadal organoids within 7 days. The 3-LGS brings a new opportunity to explore gonadal organogenesis and contributes to the development of more complex in vitro models in the field of developmental and regenerative medicine

The gut microbiota and developmental programming of the testis in mice

Nutrients and environmental chemicals, including endocrine disruptors, have been incriminated in the current increase in male reproductive dysfunction, but the underlying mechanisms remain unknown. The gastrointestinal tract represents the largest surface area exposed to our environment and thereby plays a key role in connection with exposure of internal organs to exogenous factors. In this context the gut microbiome (all bacteria and their metabolites) have been shown to be important contributors to body physiology including metabolism, cognitive functions and immunity. Pivotal to male reproduction is a proper development of the testis, including the formation of the blood-testis barrier (BTB) that encapsulates and protects germ cells from stress induced environmental cues, e.g. pathogenic organisms and xenobiotics. Here we used specific pathogen free (SPF) mice and germ-free (GF) mice to explore whether gut microbiota and/or their metabolites can influence testis development and regulation of BTB. Lumen formation in the seminiferous tubules, which coincides with the development of the BTB was delayed in the testes of GF mice at 16 days postpartum. In addition, perfusion experiments (Evans blue) demonstrated increased BTB permeability in these same mice. Reduced expressions of occludin, ZO-2 and E-cadherin in GF testis suggested that the microbiota modulated BTB permeability by regulation of cell-cell adhesion. Interestingly, exposure of GF mice to Clostridium Tyrobutyricum (CBUT), which secrete high levels of butyrate, restored the integrity of the BTB and normalized the levels of cell adhesion proteins. Moreover, the GF mice exhibited lower serum levels of gonadotropins (LH and FSH) than the SPF group. In addition, the intratesticular content of testosterone was lower in GF compared to SPF or CBUT animals. Thus, the gut microbiome can modulate the permeability of the BTB and might play a role in the regulation of endocrine functions of the testis.Scopu

Maintenance of Sertoli Cell Number and Function in Immature Human Testicular Tissues Exposed to Platinum-Based Chemotherapy-Implications for Fertility Restoration

Background: Retrospective studies in adult survivors of childhood cancer show long-term impacts of exposure to alkylating chemotherapy on future fertility. We recently demonstrated germ cell loss in immature human testicular tissues following exposure to platinum-based chemotherapeutic drugs. This study investigated the effects of platinum-based chemotherapy exposure on the somatic Sertoli cell population in human fetal and pre-pubertal testicular tissues. Methods: Human fetal (n = 23; 14–22 gestational weeks) testicular tissue pieces were exposed to cisplatin (0.5 or 1.0 μg/ml) or vehicle for 24 h in vitro and analysed 24–240 h post-exposure or 12 weeks after xenografting. Human pre-pubertal (n = 10; 1–12 years) testicular tissue pieces were exposed to cisplatin (0.5 μg/ml), carboplatin (5 μg/ml) or vehicle for 24 h in vitro and analysed 24–240 h post-exposure; exposure to carboplatin at 10-times the concentration of cisplatin reflects the relative clinical doses given to patients. Immunohistochemistry was performed for SOX9 and anti-Müllerian hormone (AMH) expression and quantification was carried out to assess effects on Sertoli cell number and function respectively. AMH and inhibin B was measured in culture medium collected post-exposure to assess effects on Sertoli cell function. Results: Sertoli cell (SOX9(+ve)) number was maintained in cisplatin-exposed human fetal testicular tissues (7,647 ± 459 vs. 7,767 ± 498 cells/mm(2); p > 0.05) at 240 h post-exposure. No effect on inhibin B (indicator of Sertoli cell function) production was observed at 96 h after cisplatin (0.5 and 1.0 μg/ml) exposure compared to control (21 ± 5 (0.5 μg/ml cisplatin) vs. 23 ± 7 (1.0 μg/ml cisplatin) vs. 25 ± 7 (control) ng/ml, p > 0.05). Xenografting of cisplatin-exposed (0.5 μg/ml) human fetal testicular tissues had no long-term effect on Sertoli cell number or function (percentage seminiferous area stained for SOX9 and AMH, respectively), compared with non-exposed tissues. Sertoli cell number was maintained in human pre-pubertal testicular tissues following exposure to either 0.5 μg/ml cisplatin (6,723 ± 1,647 cells/mm(2)) or 5 μg/ml carboplatin (7,502 ± 627 cells/mm(2)) compared to control (6,592 ± 1,545 cells/mm(2)). Conclusions: This study demonstrates maintenance of Sertoli cell number and function in immature human testicular tissues exposed to platinum-based chemotherapeutic agents. The maintenance of a functional Sertoli cell environment following chemotherapy exposure suggests that fertility restoration by spermatogonial stem cell (SSC) transplant may be possible in boys facing platinum-based cancer treatment

Primary Human Testicular Cells Self-Organize into Organoids with Testicular Properties.

So far, successful de novo formation of testicular tissue followed by complete spermatogenesis in vitro has been achieved only in rodents. Our findings reveal that primary human testicular cells are able to self-organize into human testicular organoids (TOs), i.e., multi-cellular tissue surrogates, either with or without support of a biological scaffold. Despite lacking testis-specific topography, these mini-tissues harbored spermatogonia and their important niche cells, which retained specific functionalities during long-term culture. These observations indicate the posibility of in vitro re-engineering of a human testicular microenvironment from primary cells. Human TOs might help in the development of a biomimetic testicular model that would exert a tremendous impact on research and development, clinical treatment of infertility, and screening in connection with drug discovery and toxicology