Study of the somatic compartment maturation from immature testicular tissue fragments during organotypic in vitro culture

Details

Supervisors

Wyns Christine

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

Chemotherapy and radiotherapy are common and effective cancer treatments in young boys, but can damage the gonads, leading to permanent infertility in adulthood. Fertility preservation is therefore recommended, and the cryopreservation of immature testicular tissue (ITT) is the only option available to date. In vitro maturation (IVM) of cryopreserved ITT to produce spermatozoa ex vivo is one of the most promising experimental fertility restoration strategies. It has been applied successfully in animals but remains challenging in humans. The lesser success using human ITT has been hypothesized to be the result of an inadequate maturation of the somatic compartment which is secondary to the lack of crucial components in the culture medium. The heterogeneity of the initial maturation status of the human tissue has also been hypothesized to impact the results of the IVM. The aim was to study the maturation of the somatic compartment in frozen-thawed ITT (from two maturation statuses), after previous in vitro culture using 2 complex culture media. Briefly, ITT fragments from pre- (n=3) and peripubertal (n=3) boys were cultured for 64 days under two media: M0 (complex medium) and M1 (M0 + supraphysiological testosterone). Analyses were performed on FFPE tissue (immunohistochemistry) and frozen supernatant (immunoassay) at different timepoints to evaluate the maturation of the 3 main somatic cell populations: Sertoli cells (SCs), Leydig cells (LCs), and peritubular myoid cells (PMCs). SCs are cells within seminiferous tubules (STs) expressing SRY-Box transcription factor 9 (SOX9). SCs were considered immature when expressing anti-mullerian hormone (AMH), and mature when expressing the androgen receptor (AR). Maturity of the SCs was further assessed by the establishment of the blood-testis barrier (BTB), an inter-SCs junctional complex, evidenced by zona occludens 1 (ZO1). LCs are interstitial cells expressing cytochrome P450 family 11 subfamily A member 1 (CYP11A1). LCs were considered immature when expressing Delta like homolog 1 (DLK1) and mature when expressing Insulin like 3 (INSL3). Testosterone production by LCs was also assessed. PMCs are interstitial cells expressing actin alpha 2 (ACTA2). ACTA2 expression pattern around STs was assessed by a histologic score (complete, partial, or absent). Immature PMCs have a weak and unorganized ACTA2 staining, while mature PMCs have a strong and organized staining. In prepubertal patients, the number of SCs (SOX9) per ST remained stable (higher number in M1>M0 at day 16, p<0.05), AMH staining decreased and AR expression showed some increase over culture time in M0. The staining for BTB protein ZO1 appeared with culture although it remained unorganized (M0 and M1). The ratio of LCs (CYP11A1) in the interstitium did not change over culture, and no mature LCs (INSL3) developed (M0 and M1). Testosterone secretion (M0) initially increased (until day 14) but was not sustained afterwards. The ratio of PMCs (ACTA2) increased over culture and showed a progressively mature staining pattern around ST (more complete staining pattern in M1>M0, p<0.01). Abnormal CYP11A1 staining appeared in SCs during culture. In peripubertal patients SC numbers per ST decreased, AMH decreased while AR remained stable. The staining for BTB protein (ZO1), initially present, was lost with culture and became unorganized (M0 and M1). The ratio of mature LCs (INSL3+) decreased during culture in both media. PMCs showed a loss of organization in both media during the culture period. An abnormal CYP11A1 staining also appeared in SCs. These results highlight that there is a partial somatic maturation in prepubertal ITT while there is a loss of the initial maturation in peripubertal ITT with a global dysfunction of the somatic compartment in all patients independently of the medium. This confirms that the initial ITT maturation status impacts the IVM outcome. As somatic cells seem to dysfunction in culture, further optimization is needed to improve the culture medium components.