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

Fertility preservation in childhood cancer: Endocrine activity in prepubertal human testis xenografts exposed to a pubertal hormone environment

Survivors of childhood cancer are at risk for long-term treatment-induced health sequelae, including gonadotoxicity and iatrogenic infertility. At present, for prepubertal boys there are no viable clinical options to preserve future reproductive potential. We investigated the effect of a pubertal induction regimen with gonadotrophins on prepubertal human testis xenograft development. Human testis tissue was obtained from patients with cancer and non-malignant haematological disorders (n = 6; aged 1–14 years) who underwent testis tissue cryopreservation for fertility preservation. Fresh and frozen-thawed testis fragments were transplanted subcutaneously or intratesticularly into immunocompromised mice. Graft-bearing mice received injections of vehicle or exogenous gonadotrophins, human chorionic gonadotrophin (hCG, 20 IU), and follicle-stimulating hormone (FSH, 12.5 IU) three times a week for 12 weeks. The gross morphology of vehicle and gonadotrophin-exposed grafts was similar for both transplantation sites. Exposure of prepubertal human testis tissue xenografts to exogenous gonadotrophins resulted in limited endocrine function of grafts, as demonstrated by the occasional expression of the steroidogenic cholesterol side-chain cleavage enzyme (CYP11A1). Plasma testosterone concentrations (0.13 vs. 0.25 ng/mL; p = 0.594) and seminal vesicle weights (10.02 vs. 13.93 mg; p = 0.431) in gonadotrophin-exposed recipient mice were comparable to vehicle-exposed controls. Regardless of the transplantation site and treatment, initiation and maintenance of androgen receptor (AR) expression were observed in Sertoli cells, indicating commitment towards a more differentiated status. However, neither exogenous gonadotrophins (in castrated host mice) nor endogenous testosterone (in intact host mice) were sufficient to repress the expression of markers associated with immature Sertoli cells, such as anti-Müllerian hormone (AMH) and Ki67, or to induce the redistribution of junctional proteins (connexin 43, CX43; claudin 11, CLDN11) to areas adjacent to the basement membrane. Spermatogonia did not progress developmentally but remained the most advanced germ cell type in testis xenografts. Overall, these findings demonstrate that exogenous gonadotrophins promote partial activation and maturation of the somatic environment in prepubertal testis xenografts. However, alternative hormone regimens or additional factors for pubertal induction are required to complete the functional maturation of the spermatogonial stem cell (SSC) niche

Experimental approaches to studying human testicular development

Survivors of childhood cancer encounter a broad spectrum of long-term side effects, including infertility after completion of cancer therapy. Prepubertal boys do not yet produce sperm; therefore they cannot benefit from the standard technologies of sperm freezing and currently, there are no established clinical options to preserve their future fertility. Prepubertal testicular tissue and/or cells can be collected and cryopreserved prior to gonadotoxic treatment for future clinical use such as re-transplantation or ex-vivo development of immature testis tissue. However, the factors required to functionally induce human testicular development have not yet been determined. This thesis aimed to study human testicular development by employing in vitro and in vivo approaches. Three studies were conducted using second-trimester human fetal testis (HFT) as a model of immature testis and prepubertal testis tissue. In the first set of studies, HFT fragments were transplanted subcutaneously and were exposed to human chorionic gonadotrophin (hCG). Host mice received for each experiment, testis tissue from one fetus and were randomly allocated to one of three groups: ‘Untreated’, ‘Continuous hCG’ and ‘Withdrawal hCG’. Untreated mice did not receive any treatment for 9-12 months; whilst mice belonging to ‘Continuous hCG’ group received exogenous hCG for 9-12 months. In order to mimic prepuberty, which is characterised by low/undetectable gonadotrophin levels; in ‘Withdrawal hCG’ group mice were exposed to hCG for 7 months followed by 5 months without hCG. This study demonstrated the potential for hCG to induce the acquisition of mature Sertoli cell features associated with androgen responsiveness including androgen receptor expression in Sertoli cells, blood-testis-barrier formation (connexin 43 expression) and lumen development. However, the expression of markers found in undifferentiated Sertoli cells (e.g. anti-Müllerian hormone, AMH) was retained in LTXs suggesting that these cells were partially differentiated. A second set of studies was carried out to determine the effects of gonadotrophins (hCG and follicle stimulating hormone, FSH) and transplantation sites (subcutaneous and intratesticular) on prepubertal human testis graft development. Pre(peri)pubertal human testis fragments were grafted subcutaneously and intratesticularly for 13 weeks. Host mice received subcutaneous injections of either vehicle or gonadotrophins (hCG+FSH) for 12 weeks. This study demonstrated: (i) initiation and maintenance of androgen receptor expression in Sertoli cells in prepubertal xenografts; (ii) induction of steroidogenesis required exogenous gonadotrophins stimulation; (iii) mouse testicular parenchyma provided a better transplantation site than the back skin of host mice in terms of germ cell survival; (iv) germ cell differentiation did not occur in prepubertal human testis xenografts; and (v) poor graft survival was observed in case of transplantation of peripubertal testis tissue containing meiotic cells. The third study was designed to assess in vitro the impact of different factors (such as TNF-alpha, forskolin and adult human testis-derived conditioned media) on human fetal testis development. In this study, TNF-alpha was identified as a candidate factor that exerts an inhibitory effect on AMH expression in human fetal testis tissue with no impact on the expression of the other key somatic cell genes analysed in this study, indicating therefore a specific role of TNF-alpha on AMH downregulation. In conclusion, this thesis provides evidence of the importance of gonadotrophins and TNF-alpha in the stepwise process of Sertoli cell maturation and Leydig cell function. Furthermore, these findings suggest that further work is warranted to determine the factors required to generate functional gametes from prepubertal testis tissue.2020-07-0

Exogenous Gonadotrophin Stimulation Induces Partial Maturation of Human Sertoli Cells in a Testicular Xenotransplantation Model for Fertility Preservation

The future fertility of prepubertal boys with cancer may be irreversibly compromised by chemotherapy and/or radiotherapy. Successful spermatogenesis has not been achieved following the xenotransplantation of prepubertal human testis tissue, which is likely due to the failure of somatic cell maturation and function. We used a validated xenograft model to identify the factors required for Leydig and Sertoli cell development and function in immature human testis. Importantly, we compared the maturation status of Sertoli cells in xenografts with that of human testis tissues (n = 9, 1 year-adult). Human fetal testis (n = 6; 14–21 gestational weeks) tissue, which models many aspects of prepubertal testicular development, was transplanted subcutaneously into castrated immunocompromised mice for ~12 months. The mice received exogenous human chorionic gonadotropin (hCG; 20IU, 3×/week). In xenografts exposed continuously to hCG, we demonstrate the maintenance of Leydig cell steroidogenesis, the acquisition of features of Sertoli cell maturation (androgen receptor, lumen development), and the formation of the blood–testis barrier (connexin 43), none of which were present prior to the transplantation or in xenografts in which hCG was withdrawn after 7 months. These studies provide evidence that hCG plays a role in Sertoli cell maturation, which is relevant for future investigations, helping them generate functional gametes from immature testis tissue for clinical application