Figure 1.
Consumption of probiotic microbes increases the weight of mouse testes.
a. Gross appearance of outbred Swiss male mice with oral Lactobacillus reuteri treatment at 12 months of age. Testes of L. reuteri-fed mice are larger compared to control mice. b–e. Results of statistical analyses of testes weight at different time-points after starting treatment, including the (b) 5, (c) 7, (d) 9 and (e) 12 months of age time-points. L. reuteri consistently increased testicular weight in both control- and new western-diet-fed mice compared to age- and diet-matched controls. Dietary supplementation with L. reuteri, but not with E coli K12, increased testicular weight in control-diet-fed mice at age 7 months when compared to age- and diet-matched controls (c). Numbers on the y-axis of bar graphs correspond to the mean±SEM of testes weight or the testicular weight; *p<0.05, **p<0.001.
Figure 2.
Dietary L. reuteri increases the cross-sectional profile area of seminiferous tubules and their germ cell population.
a. Representative histology of seminiferous tubules cross-sectioned profiles of Swiss mice at the age of 5 months. Increased testicular weight due to L. reuteri consumption in both control- and New Western diet (NWD)-fed mice co-existed with a histologically discernible increase of seminiferous tubule (ST) cross-sectional area. Hematoxylin and eosin. Bars = 100 µm. b. Histomorphometrical assessment of circular ST cross-sectioned profiles size in different time points. The effect of L. reuteri remains highly significant with the progression of age. Numbers on the y axis of bar graphs correspond to the mean±SEM of ST area size; *** p<0.0001. c. Stereological morphometric assessment shows that L. reuteri-fed mouse testes have significantly increased nuclear volumes of germ cells. Numbers on the y axis of bar graphs correspond to the mean±SEM of “Nuclear volume of germ cells per unit area” or the “absolute nuclear volume of germ cells”; *p<0.05, **p<0.001, ***p<0.0001.
Figure 3.
L. reuteri consumption increases the size of interstitial Leydig cell areas and Leydig cell numbers.
a. Representative histology of Swiss mice testes at the age of 12 months. Leydig cell areas in the testicular interstitium of probiotic-fed mice are of increased size compared to the corresponding areas of control mice. Hematoxylin and eosin. Bars = 100 µm b. Histomorphometrical counts of Leydig cell areas at different time-points revealed the statistical significance of the L. reuteri effect. Numbers on the y-axis of bar graphs correspond to the mean±SEM of Leydig area size. c. Point-counting stereology counts reveal that L. reuteri-fed mouse testes have significantly increased nuclear volumes and an increased number of Leydig cells per testis. Numbers on the y axis of bar graphs correspond to the mean±SEM of “Nuclear volume of germ cells per unit area” or the “absolute nuclear volume of germ cells”; *p<0.05, **p<0.001, ***p<0.0001.
Figure 4.
L. reuteri increases Leydig cell proliferation and serum testosterone levels.
a. At the age of 5 months a small number of proliferating (Ki-67+) Leydig cells did exist in the testicular interstitium (arrows). Proliferating Leydig cells were significantly more in probiotic-fed mice. DAB chromogen, Hematoxylin counterstain. Bars = 25 µm. The y-axis of the bar graph stands for the mean±SEM of Ki-67+ Leydig cells. b. Serum testosterone levels are significantly higher when consuming L. reuteri, as compared to their diet-matched controls. Numbers on the y-axis corresponds to the mean±SEM of serum testosterone level in ng/ml.; *p<0.05, **p<0.001.
Figure 5.
Dietary L. reuteri rescues mice from age-associated testicular atrophy.
a. Representative histology of Swiss mouse testes at the age of 12 months. The aged control mouse testes demonstrated atrophic seminiferous tubules. By contrast, L. reuteri-fed mice had uniformly normal ST epithelium with rare atrophic tubules. Hematoxylin and eosin. Bars = 100 µm. The y-axis in the bar graph stands for the mean±SEM of the atrophic/total ST ratio counted in each experimental group;**p<0.001. b. Ki-67-specific immunohistochemistry in the testes of 12-months-old mice. A side-by-side comparison of representative images from control and L. reuteri-fed mouse testes reveals that the probiotic organism counteracts the age-associated decrease of epithelial proliferation observed in control mice. DAB chromogen, Hematoxylin counterstain. Bars = 100 µm.
Figure 6.
Systemic depletion of IL-17 recapitulates reproductive fitness attributes of L. reuteri.
Compared to sham IgG-treated control mice, 12 month-old mice depleted of IL-17A had a. increased paired testicular weights; b. increased cross-sectional ST areas; c. increased germ cell nuclear volumes; d and e. increased interstitial Leydig cell areas. Hematoxylin and eosin. Bars = 100 µm; f. increased Leydig cell nuclear volumes and total number per testis and g. decreased ST atrophy. *p<0.05, **p<0.001, ***p<0.0001.