Ground-based Simulated Microgravity Combined Carbon Ion Radiation On Reproductive Function Effects In Male Mice

Purpose: The tail suspension model combined carbon ion radiation was used tosimulate space environment, to determine the effect of a simulated space environmenton male mice reproductive function, and to explore potential mechanisms forprotecting the health and safety of astronauts and space travelers.Materials and Methods: The mice were first subjected to tail-suspended for7days,then were whole-body irradiated with carbon ion beam irradiation (CIR) to simulate aspace environment. Experiments were divided into a control group (CK), simulatedmicrogravity group (SUS), simulated microgravity combined CIR group (SUS+CIR),and CIR group (CIR). After irradiation, the serum testosterone and serum folliclestimulating hormone (FSH) levels were tested at6,12,18, and24h, respectively. TheJohnsen score judged the spermatogenic cell damage; the testicular malonaldehyde(MDA), superoxide dismutase (SOD), and intracellular reactive oxygen species (ROS)in testicular cells were determined. Epididymal sperm density, motility, viability,acrosome integrity, membrane integrity, DNA damage, abnormal sperm, and spermintracellular ROS levels were detected. The levels of expression of β-tubulin inepididymal sperm was detected by Western blot and the distribution of β-tubulinprotein in epididymal sperm was located by immunofluorescence. Apoptosis ofspermatogenic cells was detected by TUNEL staining and an annexin V-propidiumiodide assay, respectively. BrdU labeling combined with the expression of PCNA wasused to judge spermatogenic cell proliferation levels. The levels of UCH-L1,Actived-caspase-3, Bcl-2, Bax and p53expression were analyzed by Western blot, and immunohistochemistry was used to detect the distribution of UCH-L1,Actived-caspase-3, PCNA, and p53protein in testis sections.Results:1. At24h after irradiation, the serum testosterone level was lowest and FSHwas highest in each experimental group. SUS+CIR significantly reduced serumtestosterone and elevated serum FSH levels, while serum testosterone and FSH levelsin the SUS+CIR group were between the SUS and CIR groups, and this trend did notchange over time.2. At24h after irradiation, a significant decrease in the testesweight, testes ratio, spermatogenic cell damage, and antioxidant capacity, and asignificant change in testicular ROS was observed in the SUS group, respectively.3.At24h after irradiation, SUS+CIR significantly reduced epididymal sperm density,motility, viability, acrosome integrity, and membrane integrity, and significantlyincreased the epididymal sperm abnormal rate, DNA damage rate, and spermintracellular ROS levels. A decrease in sperm motility induced by SUS+CIR wasrelated to decreased β-tubulin expression.4. SUS+CIR can increase spermatogeniccell apoptosis, excessive apoptosis of spermatogenic cells temporarily stored in thecauda epididymidis, UCH-L1and PCNA, p53, Actived-caspase3, Bax, and Bcl-2protein has a relationship, and may be involved in mediating suppression ofspermatogenic cell proliferation and increased apoptosis.Conclusion:1. The SUS combined CIR disrupts spermatogenesis and steroidogenesisin the testes of mice; these processes may be related to the diminished testicularantioxidant capacity and spermatogenic cell damage. However, the CIR is moreserious than SUS+CIR in testicular damage and steroidogenesis dysfunction.2.SUS+CIR increased ROS in sperm is related with a decrease in sperm quality, andmay cause male infertility. SUS+CIR reduced β-tubulin expression, resulting indecreased sperm motility.3. Spermatogenic cell apoptosis was related withubiquitination pathway mediated by UCH-L1. UCH-L1can accumulate p53, activatecaspase3and bax, resulting in spermatogenic cell apoptosis. The mechanism of spermatogenic cell apoptosis and proliferation induced by SUS+CIR were associatedwith CIR, however, SUS+CIR is not a simple superposition of SUS and CIR.