Effects of paternal ionizing radiation exposure on fertility and offspring's health

Abstract

Purpose: The intergenerational effects of ionizing radiation remain controversial. Extensive insights have been revealed for DNA mutations and cancer incidence in progeny, yet many of these results were obtained by immediate post-radiation mating. However, conception at short times after radiation exposure is likely to be avoided. After a long period of fertility recovery, whether unexposed sperm derived from exposed spermatogonia would challenge the health of the offspring is not yet clearly demonstrated.

Methods: Ten-week-old C57BL/6J males underwent whole-body acute γ irradiation at 0 and 6.4 Gy. Testes and sperm were collected at different times after radiation to examine reproductive changes. The reproductive, metabolic, and neurodevelopmental parameters were measured in the offspring of controls and the offspring derived from irradiated undifferentiated spermatogonia.

Results: Paternal fertility was lost after acute 6.4 Gy γ radiation and recovered at 10-11 weeks post irradiation in mice. The reproductive, metabolic, and neurodevelopmental health of offspring born to irradiated undifferentiated spermatogonia were comparable to those of controls.

Conclusion: The male mice could have healthy offspring after recovery from the damage caused by ionizing radiation.

Keywords: fertility recovery; intergenerational effects; ionizing radiation; offspring; undifferentiated spermatogonia.

FIGURE 1

Male fertility underwent damage‐repair process after γ radiation. (A) Representative H&E images of testes in irradiated males and controls at different times after IR. Scale bar, 100 μm. (B) Comparison of the testes/body weight ratio, sperm concentration, sperm motility, and testosterone in irradiated males and controls at different times after IR. At each time point, n = 5 for each group. (C) Representative images of spermatozoa from irradiated males at 11 weeks pIR and the percentage of abnormal spermatozoa were shown. At least 1000 sperm were counted from five different mice in each group at each time point. (D) Testes of irradiated males and controls were obtained at different times after IR for western blotting and western blotting analysis of PLZF, PCNA, PARP, and RAD51. (E) Cumulative pregnancy rates for both groups at different times after IR. (F) Comparison of time to pregnancy in both groups. The average time to pregnancy (days) in 6.4 Gy mice was significantly longer than that in 0 Gy mice (n = 16 for 0 Gy group and n = 19 for 6.4 Gy group). (G) Litter size and number of female (male) pups per litter in fertility assays of F0 males (n = 16 for each group). Data are presented as means ± SD. A Student's t‐test (two‐tailed) was used for statistical analysis; *p value < 0.05, **p value < 0.01, ***p value < 0.001, and ****p value < 0.0001. ns, not significant.

FIGURE 2

F1 male offspring of irradiated males had normal spermiogenesis. (A) Growth curve of F1 males (n = 9 for each group). (B) Fertility assay in F1 males (n = 8 for each group). (C) Comparison of the testes/body weight ratio, sperm concentration, sperm motility, and testosterone in 12‐week‐old F1 males (n = 12 for each group). (D) Comparison of CASA parameters in 12‐week‐old F1 males. (E) IF staining of the germ cell marker DDX4 (red) in F1 male offspring at P12. Scale bar, 50 μm. (F) The expression levels of the genes associated with different testicular cells were determined in 12‐week‐old F1 males (n = 9 for each group). (G) Testes of F1 males at 12 and 36 weeks old were obtained for western blotting and western blotting analysis of PLZF, PCNA, PARP, and SOX9. Data are presented as means ± SD. A Student's t‐test (two‐tailed) was used for statistical analysis.

FIGURE 3

Reproductive system in F1 female offspring of irradiated males functioned without distinct defects. (A) Growth curve of F1 females (n = 9 for each group). (B) Fertility assay in F1 males (n = 8 for each group). (C) The levels of reproductive hormones in F1 female offspring (n = 5 for each group) at different ages. (D) Representative H&E images of ovaries in F1 female offspring at different ages. Scale bar, 200 μm. And comparison of the numbers of primordial follicles (PrFs), primary follicles (PFs), secondary follicles (SFs), and antral follicles (AFs) of mice (n = 5 for each group) at different ages. (E) Representative estrus cycles in 12‐week‐old F1 female offspring. (F) The percent time in cycle phases of 12‐week‐old F1 female offspring (n = 10 for each group). (G) Masson's trichrome staining and statistics of collagen volume fraction (%) in ovaries of 36‐week‐old F1 female offspring (n = 5 for each group). Left panel, scale bar, 200 μm. Right panel, scale bar, 100 μm. Data are presented as means ± SD. A Student's t‐test (two‐tailed) was used for statistical analysis.

FIGURE 4

F1 offspring of irradiated males had normal glucose metabolic health. (A) Comparison of body composition in F1 offspring (n = 9 for each group) at different ages. (B) GTT and ITT for F1 males (n = 9 for each group) at different ages. (C) GTT and ITT for F1 females (n = 9 for each group) at different ages. Data are presented as means ± SD. A Student's t‐test (two‐tailed) was used for statistical analysis.

FIGURE 5

F1 offspring of irradiated males had normal lipid metabolic health. (A) Lipid levels in 36‐week‐old F1 offspring (n = 5 for each group). (B) Representative PAS and oil red O staining of liver sections in F1 males. Scale bar, 100 μm. (C) Representative PAS and oil red O staining of liver sections in F1 females. Scale bar, 100 μm. (D) Representative H&E images of GAS, gWAT, and BAT in 36‐week‐old F1 males. Statistics of CSA in GAS and gWAT (n = 9 for each group). Scale bar, 100 μm. (E) Representative H&E images of GAS, gWAT, and BAT in 36‐week‐old F1 females. Statistics of CSA in GAS and gWAT (n = 9 for each group). Scale bar, 100 μm. Data are presented as means ± SD. A Student's t‐test (two‐tailed) was used for statistical analysis.

FIGURE 6

No apparent anomalies existed in neurodevelopment of F1 offspring. (A) OFT for F1 males (n = 9 for each group) at 3 weeks old. Left, representative movement trace images of the two groups. Right, comparison of distance, center duration, and the number of center entries in the OFT. (B) OFT for F1 females (n = 9 for each group) at 3 weeks old. Left, representative movement trace images of the two groups. Right, comparison of distance, center duration, and the number of center entries in the OFT. (C) Place discrimination index of F1 offspring (n = 9 for each group) at 8 and 12 weeks old in the OLT. (D) Representative movement trace images of NORT in F1 offspring at 8 and 12 weeks old. And the percentage of exploration time in the old and novel objects for NORT in F1 offspring (n = 9 for each group) at 8 and 12 weeks old. All data are shown as the mean ± SD. A Student's t‐test (two‐tailed) was used for statistical analysis.