The human Leydig cell: maturation status and function in organotypic culture of immature testicular tissue fragments

Details

Supervisors

Christine Wyns

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

Abstract Chemotherapeutic treatments or radiotherapy can induce germ cell depletion in the gonads of prepubertal boys and subsequent infertility in adulthood. Currently, the only experimental fertility preservation strategy is the cryopreservation of immature testicular tissue (ITT) fragments containing spermatogonial stem cells. In vitro maturation (IVM) of ITT is one of the promising techniques to produce spermatozoa for in vitro fertilization. In recent experiments, we demonstrated that spermatogenesis can be initiated in vitro in ITT cultured in an organotypic tissue culture system. However, spermatogenesis did not progress beyond the round spermatid stage. The loss in adequate testosterone levels noted after two weeks of culture is the most probable cause of this blockade. Indeed, testosterone (produced by Leydig cells (LCs)) is essential for haploid cell differentiation. As the LC number remained stable over culture time, we hypothesized that a LC dysfunction in vitro leads to testosterone loss in culture. These cells might be either undergoing incomplete maturation or dedifferentiating due to the lack of important factors in the medium. Several markers of LC function and maturation have been reported. Delta Like Non-Canonical Notch Ligand 1 (DLK1) is expressed in immature LCs. Insulin-like factor 3(INSL3) and Calretinin (CALB2) are produced increasingly as the LC matures. In this study, the different markers of LCs were analyzed on both formalin-fixed paraffin-embedded tissue and on frozen-thawed culture supernatants (at days 16, 32, and 64) recovered from the previous experiments on the ex-vivo culture of human ITT (using two different culture media) from 3 prepubertal patients (2, 8, and 11 years old). The expression of DLK1, INSL3, and CALB2 was investigated by immunohistochemistry (IHC). The number of positive LCs (for one or both markers) was counted on the total surface for each tissue section. Interstitial cell death was also assessed by caspase 3 activity on IHC sections. Immunoenzymatic assays (ELISA) were performed to quantify the presence of DLK1, INSL3, and IGF1 in the supernatant. The number of mature (INSL3+, CALB2+) was very low all along the culture period while that of immature LCs (DLK1+INSL3-) decreased. INSL3 was evidenced in the supernatants (peaking at day 32) but with levels much lower than the expected intratesticular levels. DLK1 was not present in its soluble form and IGF1, as an important growth factor for LCs maturation, could not be detected. In this study, we showed that most LCs remain in an immature phenotype or are lost during culture. This is in accordance with the loss of testosterone noted. The absence of essential factors such as IGF1 in culture can explain this loss. Indeed, IGF1 deficiency was shown to induce a reduction of mature LCs number in the testis of mice, associated with reduced circulating testosterone. Supplementing the culture medium with factors that promote LC proliferation and differentiation should be considered in future experiments to promote adequate LC function ex-vivo.