Dose-Dependent Testicular Injury and Recovery after Total-Body Irradiation in Rhesus Monkeys

Abstract

Testicular injury is a well-documented acute effect of radiation exposure, though little is known about recovery years after irradiation, especially at higher doses. We examined the testes from 143 irradiated and control male rhesus monkeys, who were part of the Radiation Late Effects Cohort over a four-year period. Irradiated animals were exposed to doses ranging from 3.5 to 8.5 Gy of total-body irradiation. The testes were assessed using computed tomography (CT) volumetry, serum testosterone, and histology for deceased members of the cohort. Irradiated animals exhibited dose-dependent testicular atrophy as well as decreased serum testosterone during the winter breeding season when compared to age-matched unirradiated controls. No significant difference in summer testosterone levels was observed. Volumetric and histologic evidence of testicular recovery was present approximately three years postirradiation for animals who received ≤8 Gy. The study demonstrates dose-dependent testicular injury after total-body irradiation and provides evidence for volumetric and spermatogonial recovery even at lethal doses of total-body irradiation in rhesus monkeys.

FIG. 1.

Cohort demographics for 143 male rhesus monkeys. The Y-axis shows dose range from 0–8.5 Gy as well as LD dose groups. The X-axis represents age, with the dotted line beginning at the time of irradiation and the solid line when the animal arrived at WFSM. The bubbles represent relative total testicular volume, and indicate the age at which CT volumetry was performed. Numerical volumetric data for this figure are provided in Supplementary Table S1 (https://doi.org/10.1667/RADE-23-00008.1.S1).

FIG. 2.

Testicular volume was approximated as an ellipsoid: $volume=\left(\frac{\mathrm{\pi }}{6}\right)\mathrm{*}\left(a\mathrm{*}b\mathrm{*}c\right)$, where $a,b$, and $c$ are the measured diameters.

FIG. 3.

Percent of adult animals (≥7 years old at the time of CT scan) with atrophic testes defined as a total testis volume <10,000 μL. Occurrence of testicular atrophy increased exponentially with radiation dose. No unirradiated adults had a total testicular volume less than 10,000 μL. NOTE: this figure represents multiple annual measurements per animal, with some receiving four scans over the four-year measurement period. Total animals with scans ≥7 years old = 65, total CT scans on these 65 animals = 187.

FIG. 4.

CT measurements $(\mathrm{n}=187\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{n}\mathrm{s})$ of total testicular volume by radiation lethality dose group of adult animals (≥7 years old at the time of CT scan; $\mathrm{n}=65$ animals). $\mathrm{P}<0.0001$ when comparing unirradiated volumes to dose groups from LD10 to LD90+ using a one-way ANOVA Kruskal-Wallis test. $\mathrm{P}\le 0.01$ when comparing the unirradiated group to <LD10, and between the LD10–50 and LD50–90 groups. Lines represent the median and 95% confidence intervals.

FIG. 5.

Computed tomography volume measurements of total testicular volume versus age by radiation lethality dose group over a four-year period in 105 animals for a total of 326 measurements. Testicular volume exhibited an inverse dose-dependent effect, with those receiving the higher doses of irradiation having smaller testicular volume. Volume recovery evidenced by a positive slope with increasing age for LD groups that received less than an LD90 dose. Curves represent a logistic growth least squares best fit.

FIG. 6.

Plot showing mean seasonal testosterone concentrations for winter and summer seasons versus any irradiation dose in adult animals (>7 years old). A statistical difference was observed between the winter season unirradiated and irradiated groups, $\mathrm{P}=0.0009$ using a one-way ANOVA Kruskal-Wallis test. All other pair-wise comparisons were not statistically significant. Lines represent the median and 95% confidence intervals.

FIG. 7.

Mean winter testosterone concentrations by dose group. An irradiation inverse dose effect was observed in the LD50/90, $\mathrm{P}=0.0035$, and LD90 groups, $\mathrm{P}=0.0060$ using a one-way ANOVA Kruskal-Wallis test. Lines represent the median and 95% confidence intervals. NOTE: One mean testosterone measurement of 1,325 ng/dL from the unirradiated group lies outside the Y-axis range and is not shown but was included in the statistical analysis.

FIG. 8.

Percent animals with or without sperm present in the epididymis $(\mathrm{n}=58)$. The absence of sperm in the epididymis exhibited an increasing dose dependent effect, with animals who received ≥LD10/50 doses.

FIG. 9.

Histopathology of the seminiferous tubules and epididymis stained with hematoxylin and eosin. Panel A: Unirradiated, shows normal seminiferous tubules with ongoing spermatogenesis. Panel B: LD <10, demonstrates a multifocal pattern of epithelial degeneration, with many tubules still exhibiting normal spermatogenesis. Large coalescing areas of epithelial loss are shown in panel C (LD10/50), which lie adjacent to relatively normal clusters of tubules. Panel D: LD90+, demonstrates a complete loss of epithelium as well as tubule dilatation. The bottom two panels contrast normal epididymal ducts (panel E) filled with mature sperm with those from an animal which received an LD90+ dose of radiation (panel F). The irradiated animal’s ducts exhibit a complete lack of sperm and markedly reduced diameters.

FIG. 10.

Percent animals with or without sperm present in the epididymis $(\mathrm{n}=58)$. The presence of sperm was graded as yes/no. Animals that received higher doses were more likely to have a complete absence of sperm in their epididymal ducts.