Irradiation affects germ and somatic cells in prepubertal monkey testis xenografts.

Study question: Does irradiation evoke adverse effects in germ and somatic cells in testis xenografts from prepubertal monkeys?

Summary answer: In addition to the expected depletion of germ cells, a dose-dependent effect of irradiation was observed at the mRNA and protein level in Sertoli and peritubular myoid cells.

What is known already: Testicular irradiation studies in monkeys have focused on the dose-dependent effects on germ cells. Previous studies using intact animals or xenografts reported that germ cells are highly sensitive to irradiation. Their depletion was demonstrated by morphometric and histological analyses. The effect of irradiation on expression of Sertoli and peritubular myoid cell markers, however, has not yet been described.

Study design, size, duration: The testes of two prepubertal macaques (Macaca fascicularis) were dissected into testicular fragments. Fragments were randomly exposed in vitro to one of the following three doses of irradiation: 0 Gy, n = 60; 1 Gy, n = 54; 4 Gy, n = 72. Non-irradiated control fragments (0 Gy) were placed into the Faxitron for 6.6 min without irradiation. For 1 Gy and 4 Gy irradiation was applied for 1.7 and 6.6 min, respectively. Grafts were then either immediately analyzed or subcutaneously implanted under the back skin of 39 nude mice and analyzed after 6.5 months.

Participants/materials setting methods: Post grafting, 133 testicular xenografts were retrieved. The body weight, serum testosterone level and seminal vesical weight of the host mice as well as the number and weight of retrieved grafts were determined. Larger grafts were used to evaluate both mRNA expression profiles and protein expression patterns. In total, 71 testicular fragments were used for morphometric and histological analysis while 68 fragments were analyzed for gene expression. For PCR arrays, M. fascicularis-specific primer sequences were employed. Irradiation-induced changes in the transcript levels of 34 marker genes were determined for each testicular graft. The effects of irradiation on peritubular myoid cells and Sertoli cells were confirmed by immunohistochemical analysis of chemokine (C-X-C motif) ligand type 11 (CXCL11), alpha smooth muscle actin (SMA) and chemokine (C-X-C motif) ligand type 12 (CXCL12).

Main results and the role of chance: The four testes gave rise to 106 xenografts, which were individually analyzed, limiting the role of chance despite using only two monkeys in the study. Prior to grafting, the two donors displayed spermatogonia as the most advanced germ cell type in 95% and 70% of seminiferous tubules, respectively, while remaining tubules contained SCO. No spermatocytes were encountered prior to grafting in either monkey. After 6.5 months, non-irradiated grafts displayed spermatocytes in 15.4% and 1.8% of seminiferous tubules indicating an induction of meiosis. Irradiation resulted in a complete absence of spermatocytes. The percentage of seminiferous tubules containing spermatogonia declined in a dose-dependent manner. In non-irradiated xenografts, ~40% of tubules contained spermatogonia. This proportion was reduced to 3.4% and 4.3% in the 1 Gy treated group and to 1.3% and 0.2% in 4 Gy irradiated grafts. A dose-dependent decline in mRNA levels of selected germ cell marker genes supported the morphologically detected loss of germ cells. Irradiation had no effect on CXCL12 transcript levels. At the protein level, CXCL12-positive Sertoli cells were most abundant in the 1 Gy group compared to the 4 Gy group (P < 0.05), indicating a potential role of CXCL12 during recovery of primate spermatogenesis. The most prominent radiation-evoked changes were for CXCL11, which was localized to smooth muscle cells of blood vessels and seminiferous tubules. Transcript levels declined in a dose-dependent manner in grafts from both monkeys (MM687: P < 0.01 (0 Gy versus 4 Gy), MM627: P < 0.05 (0 Gy versus 4 Gy), P < 0.001 (1 Gy versus 4 Gy)). CXCL11 patterns of protein expression revealed irradiation-dependent changes as well. That peritubular cells are affected by X-irradiation was substantiated by changes at the transcript level between 1 and 4 Gy exposed groups (P < 0.01) and at the protein level of SMA (P < 0.05, 0 Gy versus 4 Gy).

Large scale data: n/a.

Limitations, reasons for caution: The spermatogonial stem cell system in primates is remarkably different from rodents. Therefore, data from a non-human primate may be more relevant to man. However, species-specific differences amongst primates cannot be fully excluded and the use of only two donors may raise concerns toward the generalization of the findings. There may also be important differences across the prepubertal period (e.g. infancy, early childhood) that are not represented by the ages included in the present study.

Wider implications of the findings: This study is the first to indicate relevant testicular somatic cell responses following irradiation of prepubertal primate tissue. In addition to the well-known depletion of germ cells, the changes in Sertoli, and in particular peritubular myoid, cells may have important consequences for spermatogenic recovery. These novel findings should be taken into consideration when irradiation effects are assessed in tumor survivors.

Study funding and competing interest(s): Interdisciplinary Center for Clinical Research (IZKF) Münster (Schl2/001/13) and the Excellence Cluster 'Cells in Motion' at the University Münster. There are no conflicts of interest to declare.