Experimental approaches to studying human testicular development

Abstract

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.2020-07-06