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

Abstract

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barrier (connexin 43), none of which were present prior to the transplantation or in xenografts in which hCG was withdrawn after 7 months.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.

Fertility Preservation in Childhood Cancer: Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment

“…We sought to determine the impact of gonadotrophins on AR expression in prepubertal testis grafts. Sertoli cells begin to express AR as they progress from an undifferentiated to a more differentiated status [ 32 ]. Regardless of the transplantation site and treatment received, AR expression in Sertoli cells was induced in testis tissues obtained from two donors (5 and 8 years; Figure 4 a–c,i–k), whilst its expression was maintained in transplanted testis tissues from the remaining patients across all experimental groups ( Figure 4 d–f,l–n).…”

“…The same authors suggested that this result could be attributed to the very few steroid-producing interstitial cells present in 12-36-month-old boys in comparison with testosterone-producing Leydig cells in the testes of young infants. Our previous study using human foetal testis xenografts supports this hypothesis; continuous gonadotrophin (hCG) stimulation for 9-12 months resulted in a significant increase in the seminal vesicle weight of host castrate mice compared with those in which hCG was withdrawn during the grafting period [ 32 ].…”

“…These results concur with those seen in a previous study showing AMH expression in AR-positive prepubertal testis grafts [ 19 ]. Maintenance of AMH expression in AR-expressing seminiferous cords has also been documented in studies involving long-term human foetal testis xenotransplants, in which host mice were exposed to exogenous hCG [ 32 ]. Moreover, it seems that no androgen response elements are present on the human AMH promoter [ 58 ], and studies on Sertoli-cell specific AR knockout and gain-of-function transgenic mouse models demonstrated that androgen action is not required for AMH downregulation [ 37 , 59 ].…”

“…Exposure of prepubertal testis grafts to gonadotrophins did not repress the expression of markers (AMH, Ki67) found in immature Sertoli cells or induce the formation of the BTB. In our previous work, Sertoli cells displayed a mature CX43 expression pattern in human foetal testis grafts exposed to hCG for 9-12 months [ 32 ]. Although the exact mechanism underpinning CX43 expression is not entirely clear, data reported in our previous study indicate that testosterone produced from human foetal testis xenografts may have stimulated the initial expression and localisation of CX43 restricted to the basement membrane, similar to that observed in adult testes.…”

“…For histological analyses, testis sections were stained with Haematoxylin and Eosin (H&E) following standard protocols [ 32 ]. Specific proteins were detected using immunohistochemical methods, as previously described [ 32 , 65 ].…”