Abstract The decline of circulating testosterone levels in aging men is associated with adverse health effects. During studies of probiotic bacteria and obesity, we discovered that male mice routinely consuming purified lactic acid bacteria originally isolated from human milk had larger testicles and increased serum testosterone levels compared to their age-matched controls. Further investigation using microscopy-assisted histomorphometry of testicular tissue showed that mice consuming Lactobacillus reuteri in their drinking water had significantly increased seminiferous tubule cross-sectional profiles and increased spermatogenesis and Leydig cell numbers per testis when compared with matched diet counterparts This showed that criteria of gonadal aging were reduced after routinely consuming a purified microbe such as L. reuteri. We tested whether these features typical of sustained reproductive fitness may be due to anti-inflammatory properties of L. reuteri, and found that testicular mass and other indicators typical of old age were similarly restored to youthful levels using systemic administration of antibodies blocking pro-inflammatory cytokine interleukin-17A. This indicated that uncontrolled host inflammatory responses contributed to the testicular atrophy phenotype in aged mice. Reduced circulating testosterone levels have been implicated in many adverse effects; dietary L. reuteri or other probiotic supplementation may provide a viable natural approach to prevention of male hypogonadism, absent the controversy and side-effects of traditional therapies, and yield practical options for management of disorders typically associated with normal aging. These novel findings suggest a potential high impact for microbe therapy in public health by imparting hormonal and gonad features of reproductive fitness typical of much younger healthy individuals.

Citation: Poutahidis T, Springer A, Levkovich T, Qi P, Varian BJ, Lakritz JR, et al. (2014) Probiotic Microbes Sustain Youthful Serum Testosterone Levels and Testicular Size in Aging Mice. PLoS ONE 9(1): e84877. https://doi.org/10.1371/journal.pone.0084877 Editor: Stefan Schlatt, University Hospital of Münster, Germany Received: June 30, 2013; Accepted: November 20, 2013; Published: January 2, 2014 Copyright: © 2014 Poutahidis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grants P30-ES002109 (pilot project award to SEE and EJA), U01 CA164337 (to SEE and EJA), and RO1CA108854 (to SEE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The main cellular source of testosterone in male mammals is the Leydig cell, which resides in clusters within the testicular interstitium. Adult Leydig cell androgen production is regulated by luteinizing hormone-mediated signals, originating from the hypothalamus-pituitary gland axis [1]. In males, an inevitable reduction of circulating testosterone occurs with increasing age [2]–[4]. The mechanisms governing this phenomenon remain largely unknown. Studies in both humans and rodents, however, suggest that low testosterone is due to age-related lesions in testes rather than irregular luteinizing hormone metabolism [1], [5]–[7]. The reduction of testosterone has been implicated in many adverse effects of aging in men, including reduced spermatogenesis, libido and sexual function, increased body fat, decreased muscle and bone mass, low energy levels, fatigue, poor physical performance, depressed mood, and impaired cognitive dysfunction [8]–[11]. Whether low testosterone can be considered the cause of a defined clinical entity in elderly men or not has been a matter of debate [12]. Additionally, different names have been used to describe this condition (male menopause, andropause, partial androgen deficiency of the aging male and late-onset hypogonadism) and different criteria have been used to define it, hence the discrepancies of epidemiological studies during reporting on its prevalence [12]–[13]. Nonetheless, there is an unquestionable consensus in that age-related progressive morphological and functional alterations of Leydig cells lead to low testosterone levels that, in turn, reduce reproductive fitness and affect the quality of life of the aging male [1], [4]–[5], [9]–[10], [12]–[13]. Various dietary factors and diet-induced obesity have been shown to increase the risk for late onset male hypogonadism and low testosterone production in both humans and mice. Testosterone deficiency and metabolic diseases such as obesity appear to inter-digitate in complex cause-and-effect relationships [14]–[17]. Recently we have found that the dietary supplementation of aged mice with the probiotic bacterium Lactobacillus reuteri makes them appear to be younger than their matched untreated sibling mice, at least in part by inducing beneficial integumentary effects that manifest as luxuriant hair [18] and inhibition of diet-induced obesity [19]. Interestingly, both these effects were linked with a CD4+ regulatory-T cell and Il-10-associated systematic down-regulation of the pro-inflammatory cytokine Il-17 [18]–[19]. These results indicate that gut microbiota induce modulation of local gastrointestinal immunity resulting in systemic effects on the immune system which activate metabolic pathways that restore tissue homeostasis and overall health. Indeed, in another study we discovered that aged mice eating L. reuteri show accelerated healing of skin wounds, which depends upon the suppressive arm of the immune system and the up-regulation of the pituitary gland neuropeptide hormone oxytocin (manuscript submitted for publication). During all these studies we consistently observed that young and aged mice consuming purified L. reuteri organisms had particularly large testes and a dominant male behavior. Our previous studies and observations, taken together, led us to hypothesize that dietary L. reuteri may act to prevent age- and obesity-related testicular atrophy in mice. The results of the present study confirmed this hypothesis. The testes of probiotic-fed aged mice were rescued from both seminiferous tubule atrophy and interstitial Leydig cell area reduction typical of the normal aging process. Preservation of testicular architecture despite advanced age or high-fat diet coincided with remarkably high levels of circulating testosterone. The beneficial effects of probiotic consumption were recapitulated by the depletion of the pro-inflammatory cytokine Il-17. Given the aggravated controversy about the benefits and side-effect risks of testosterone replacement therapy [9]–[10], [13], [20], dietary L. reuteri may provide an alternative natural approach to the prevention of late-onset male hypogonadism.

Discussion While conducting diet studies involving probiotic microbe Lactobacillus reuteri we discovered that aging male animals had larger testes compared to their age-matched controls [18]–[19]. This led us to hypothesize that dietary L. reuteri supplementation may act to prevent age- and diet-related testicular atrophy in mice. Knowing that age-related hypogonadism has been linked with functional alterations of Leydig cells and low testosterone levels that reduce reproductive fitness and quality of life [1], [4]–[5], [9]–[10], [12]–[13], we examined testes of probiotic microbe-fed mice and found that they had less testicular atrophy coinciding with higher levels of circulating testosterone compared to their age-matched controls. Similar testicular health benefits were produced using systemic depletion of the pro-inflammatory cytokine Il-17 alone, implicating a chronic inflammatory pathway in hypogonadism. Based on these observations, we propose a model whereby probiotic bacteria modulate gastrointestinal immunity resulting in systemic effects on the immune system that activate metabolic pathways that restore tissue homeostasis and overall health. One specific aspect of this paradigm is reciprocal activities of pro-inflammatory Th-17 and anti-inflammatory Treg cells [21]. Along mucosal surfaces, anti-inflammatory cytokine Interleukin (IL)-10 facilitates immune tolerance [22] and recruitment of CD4+ Treg cells to skew host immunity away from pro-inflammatory IL-17. Feeding of L. reuteri organisms was previously shown to up-regulate IL-10 levels and reduce levels of IL-17 [19] serving to lower systemic inflammation. Conversely, insufficient levels of IL-10 may increase the risk for autoimmunity, obesity, and other inflammatory disease syndromes [23]–[27]. Indeed, many health disorders associated with Westernized life are believed due to insufficient IL-10 and improper host immune calibration resulting in uncontrollable inflammation [28]. Westernized diets are also low in vitamin D, a nutrient that when present normally works together with IL-10 to protect against inflammatory disorders [29]–[31] and even some types of cancer [32]. Physiological feedback loops apparently exist between microbes, host hormones, and immunity. The hormone testosterone has been shown to act directly through androgen receptors on CD4+ cells to increase IL-10 expression [33]. Leydig cells are the main cellular source of testosterone in male mammals that naturally decline during aging [2]–[4]. Although adult Leydig cell androgen production is regulated by LH-mediated signals originating from the hypothalamus-pituitary gland axis [1], studies in both humans and rodents suggest that hypogonadism is due to age-related lesions in testes rather than irregular LH metabolism [1], [5]–[7]. Further studies of LH and the hypothalamus-pituitary axis are underway to investigate this possibility. We investigated spermatogenesis and other features associated with reproductive fitness [8]–[11], seeing significant retention of testicular heath criteria after feeding of L. reuteri. We postulate that probiotic gut microbes function symbiotically with their mammalian hosts to impart immune homeostasis to maintain systemic and testicular health [34]–[35] despite suboptimal dietary conditions. Dietary factors and diet-induced obesity were previously shown to increase risk for age-associated male hypogonadism, reduced spermatogenesis, and low testosterone production in both humans and mice [2]–[4], [8]–[11], [14]–[17], phenotypic features that in this study were inhibited by oral probiotic therapy absent milk sugars, extra protein, or vitamin D supplied in yogurt. Similar beneficial effects of probiotic microbes on testosterone levels and sperm indices were reported in male mice that had been simultaneously supplemented with selenium [36]. Although neither of these studies analyze testicles of human males, probiotic yogurt did produce leaner physiques in mice [19] matching findings of a large epidemiological survey of human subjects when eating yogurt [37] [38]–[43]. Additional studies using human subjects are needed to assess translational potential. A proposed health-protective role for L. reuteri in mammalian host metabolism offers mutually beneficial gut symbiont-host relationship reflecting co-evolutionary relationships [44]. From an evolutionary perspective, we assert that lactic acid bacteria may have co-evolved with mammals exploiting testosterone to optimize mental, physical, and reproductive fitness. Higher serum testosterone levels compared to controls in our separate studies correlated with not only leaner physique but also increased muscle mass and higher activity levels in mice (data not shown). Benefits of this microbial synergy may extend beyond individual fitness to reproductive success, impacting a natural selection process favoring evolutionary success for the microbe and mammalian host. Our data suggest that the L. reuteri-associated prevention of age- and diet-related testicular atrophy correlates with increased numbers and size of Leydig cells. At the earliest time-point examined in the present study (age = 5months), we found no histological evidence of age-related testicular atrophy in mice. This matches the results of a previous study, which describes that the initial changes of testicular atrophy begin to occur in mice from the age of 6 moths onwards [7] and indicates that the trophic effect of L. reuteri on Leydig cells is a key event which precedes and prevents age-related changes in the testes of mice. This effect is reminiscent of earlier studies describing Leydig cell hyperplasia and/or hypertrophy in the mouse and the rat testis that were achievable by the administration of gonadotropins, including human chorionic gonadotropin, FSH and LH [45]–[49]. The similarity of the effects of hypopheseal hormones and L. reuteri administration on Leydig cells warrant further investigation, especially in the light of recent evidence for the effects of edible Lactobaccili in the hypothalamus and pituitary gland [50]–[51]. In human males as well as in Brown Norway rats, a strain that is often used to study the effects of aging in Leydig cells, the age-associated decrease of serum testosterone does not co-exist with decreased plasma luteinizing hormone (LH) concentrations. This indicates that sub-normal testosterone levels are due to testicular dysfunction rather than a disruption of the hypothalamus-pituitary axis. The primary gonadal deficit was found to result from compromised Leydig cell steroidogenesis, but not from their loss [1], [4]–[6]. Studies in other strains of rats as well as recent studies in humans, however, suggest that LH and the hypothalamic-pituitary level are important in male hypogonadism [1], [4]–[6], [52]. These discrepancies make it difficult to determine whether the L. reuteri-induced effects found in mice in the present study could be directly relevant to aging human males. The restoration of testosterone levels, if proven true as an L. reuteri-induced effect in aged humans, would directly benefit their health regardless to whether the level of the L. reuteri intervention in the hypothalamus-pituitary-testis axis [9], [11], [16], [20]. Taken together, our data indicate that probiotic organisms may offer practical options for management of disorders frequently associated with normal aging. Reduced circulating testosterone levels have been implicated in many adverse effects including reduced spermatogenesis, libido and sexual function, increased body fat, decreased muscle and bone mass, low energy levels, fatigue, poor physical performance, depressed mood, and impaired cognitive dysfunction [8]–[11]. Ultimately, dietary L. reuteri or other probiotic supplementation may provide an alternative natural approach to prevention of male hypogonadism, absent the controversy and side-effect risks of testosterone replacement therapy [9]–[10], [13], [20]. Such microbial immune system re-programming may ultimately target other diseases linked to low testosterone including increased body fat, decreased muscle and bone mass, weak physical and mental performance, and depressed mood [8]–[11] for more healthful longevity.

Experimental Procedures Animals Genetically outbred CD-1 mice (Charles River; Wilmington, MA) were housed and handled in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facilities with diets, experimental methods, and housing as specifically approved by the Institutional Animal Care and Use Committee. The MIT CAC (IACUC) specifically approved the studies as well as the housing and handling of these animals. Mice were bred in-house or purchased directly from the vendor to achieve experimental groups. Mice were housed in groups of four animals per cage, except for those animals specifically used in breeding experiments. The experimental design was to expose mice to diets starting at age = eight weeks, and then continue the treatment until euthanasia using carbon dioxide: 1) for 12 weeks to achieve the cohort collected at five months of age; 2) for 20 weeks to achieve the cohort collected at seven months of age; 3) for 28 weeks to achieve the cohort collected at nine months of age; and 4) for 40 weeks to achieve the cohort collected at 12 months of age. Each experiment included 5–10 animals per group with at least one replicate for each experiment. Necropsy procedures were performed in mid-afternoon to standardize collections relative to circadian rhythms. After humane euthanasia with CO 2 overdose, 1 ml of blood was collected using cardiac puncture method in unconscious animals. Body weights of whole mice or their isolated paired testes were recorded (Scout pro sp202; Ohaus Corporation, Pine Brook NJ) for comparisons between treatment groups. Special Diets for Animals Mice of 6–8 wks were placed on experimental diets: control AIN-76A (Harlan-Teklad Madison WI), and a Westernized diet with high fat and low fiber with substandard levels of Vitamin D (TD.96096; Harlan-Teklad) starting at 8 weeks of age until euthanasia at time points of five months of age, seven months of age, nine months of age, or 12 months of age. Separate groups of animals received a purified preparation of an anti-inflammatory strain of Lactobacillus reuteri ATCC PTA 6475 cultivated as described elsewhere [53] using a dosage of 3.5×105 organisms/mouse/day, or a sham E coli K12 3.5×105 organisms/mouse/day in drinking water. Drinking water was replaced twice weekly. Systemic Depletion of Interleukin-17A using Anti-cytokine Antibodies Mice at age 6 months or older were treated with intraperitoneal injection of anti-IL17A antibody (clone 17F3; BioXcel, Lebanon, NH) at 500 µg per mouse three times weekly for at least four weeks. Mice were then euthanized and compared with age-matched mice that received isotype-matched sham IgG antibody alone. Measurements of Serum Testosterone Terminal cardiac blood collections were performed upon euthanasia in mid-afternoon to normalize for Circadian rhythm effect on testosterone. RadioImmunoAssay (RIA) was performed using Coat-A-Count Total Testosterone RIA kit (Siemens Diagnostics CA) as performed by AniLytics Inc (Gaithersburg MD). Quantification of Sperm Quality After humane euthanasia using C0 2 , both testes were isolated to remove combined epididymal and vas tissues from a particular male mouse. Tissue was immediately placed into a plastic Petri dish and immersed in 1 ml FHM solution (Specialty medium, Millipore). Under dissecting microscope, sperm were mobilized from each epididymis and vas deferens using microdissection with a 16 G needle. The dish was placed to a CO 2 incubator at 37°C for 10 minutes to allow for complete sperm mobilization. Sperm were then suspended 1∶10 with FHM to evaluate sperm concentration using a hemocytometer (Fisher). Sperm concentration/ml was calculated using (Dilution factor)×(count number in 5 squares)×(0.05×106). Sperm activity was assessed as number of actively moving sperm/total number of sperm [54]–[56]. Histopathology and Immunohistochemistry For histologic evaluation, testes were fixed in neutral-buffered formalin, cut transversely in half, embedded in paraffin and cut at 4 µm or at 10 µm for stereology. Serial sections were stained with hematoxylin and eosin and immunohistochemistry (IHC). Rabbit monoclonal anti-Ki-67 (Cell Marque, Rocklin, CA) or polyclonal anti-cleaved caspase-3 (Cell Signaling, Beverly, MA) antibodies were used for IHC. Heat-induced antigen retrieval was performed with CC1 epitope retrieval solution (Ventana Medical Systems, Inc., Tucson, AZ) for ki-67 or with citrate buffer pH6 for caspase-3. Quantitative histomorphometry was done using planimetry-based morphometry for the assessment of seminiferous tubules (ST) cross-sectional areas, the Leydig cell areas and nuclear diameters and for total ST profiles and atrophic/total ST profile measurements. A stereology-based point-counting morphometry method [57] was applied for determining germ and Leydig cell nuclear volumes. Six to eight histological sections from each mouse testes were randomly selected after step-sectioning and used for the morphometric counts. Images taken at ×2 magnification were used for ST cross-sectional areas and for total ST and atrophic/total ST measurements. ×10 magnification images were used to measure Leydig area size and for germ and Leydig cell nuclear volume stereology, ×40 for ki67+ Leydig cell counts and ×60 for determining Leydig cell nuclear diameters. Images were analyzed using the Image J image processing and analysis program (NIH, Bethesda, MD). For ST cross-sectional areas, for total ST and atrophic/total ST and for ki67+ Leydig cell counts, depending on the experimental group size, 1–3 images were randomly selected from each mouse to achieve a total number of 25 images per group. Total ST profiles, atrophic ST profiles and ki-67+ Leydig cells were counted in each image. For ST cross-sectional area planimetry, the largest 4–5 perfectly circular in shape ST profiles found in each image were subscribed and each ones' area was automatically measured in pixels using the ImageJ “measure” command. A total number of at least 100 ST cross-section area values was achieved per group. For Leydig cell area planimetry, the 3 images containing the largest proportion of Leydig cell areas from each mouse were used. Depending on the group size 1–3 images were randomly selected from each mouse to achieve a total number of 25 images to work with per group. The multiple Leydig cell areas in each image were subscribed, excluding vessels or “empty” spaces, and measured. The total Leydig cell area per image was recorded. For Leydig cell nuclear diameter measurements, 5 images from randomly selected areas of each mouse testis were captured using the ×60 high power magnification lens. Depending on the experimental group size, the diameter of 10–20 circular Leydig cell nuclear profiles were measured to achieve 200 nuclear size values per treatment group. For germ and Leydig cell point-counting, seven mice were randomly selected from each experimental group. Ten ×10 images were randomly captured from each mouse testis. Specifically, from each one of the 6–8 histological sections available for that assessment, the first 1 to 2 fields found to contain target cells after a random movement of the microscope stage with a closed field diaphragm were captured (simple random sampling)”. A grid with 130 points was super-imposed in each image using the “grid” plug-in of the ImageJ software. The number of points co-localizing with germ or Leydig cell nuclei was counted in each image. The mean count of nuclei-positive points found in the 10 images was determined for each mouse. The ratio of the mean “nuclei-positive points”/130, determined the “Nuclear volume of cells per unit area” (×10 figure) for a given mouse. The product of “Nuclear volume per unit area” of a mouse testis multiplied by the testis weight (“fresh” testis weight) determined the “absolute nuclear volume of cells” in each mouse testis. The quotient “Nuclear volume of cells per unit area”/Testis weight expressed as percentage was the “Relative percentage of nuclear volume of cells” of each testis. In order to calculate the total number of Leydig cells per testis, the mean nuclear diameter of 100 randomly selected circular profiles of Leydig cells was determined for each testis of the mice used for stereometry (n = 7 per group). The mean diameter in pixels was converted to µm and the weight ( = volume) of each testis was expressed in µm3. Then the Floderus equation N V = N A /(T+D−2 h) was used to transform the “Nuclear volume of cells per unit area” (N A ). T is the section thickness, D is the mean Leydig cell diameter and h is the height of the smallest nuclear profile found in the section. The latter was arbitrarily set at the 0.1× mean nuclear diameter [58]. The total number of Leydig cells per unit area (N V ) of each testis was multiplied by the testis volume (assuming that each testis volume equals to its weight) to determine the total number of Leydig cells per testis. Statistical analyses. The Mann-Whitney U test was used for body and testes weights and histomorphometry. A p-value <0.05 was statistically significant.

Supporting Information Figure S1. L. reuteri increases testicular weight/body weight ratio. Dietary supplementation with L. reuteri, increased testicular weight-body weight ratio when compared to age- and diet-matched controls. A similar effect was observed with the neutralization of IL17. Numbers on the y-axis of bar graphs correspond to the mean±SEM of the testicular weight/body weight ratio. *p<0.05, **p<0.001, ***p<0.0001. https://doi.org/10.1371/journal.pone.0084877.s001 (TIF) Figure S2. Effects of L. reuteri on Leydig cells. a. L. reuteri-fed mouse testes and mice undergoing depletion of IL-17 have a significantly increased Leydig cell nuclear volume per size unit area and b. increased relative percentage of Leydig cell nuclear volume compared to control mice. Numbers on the y axis of bar graphs correspond to the mean±SEM of “relative percentage of Leydig cell nuclear volume”;. c. In a similar manner, the same mice show an increased Leydig cell nuclear diameter compare to their age-, diet- and treatment-matched controls. Numbers on the y axis of bar graphs correspond to the mean±SEM of Leydig cell nuclear diameter. *p<0.05, **p<0.001, ***p<0.0001 https://doi.org/10.1371/journal.pone.0084877.s002 (TIF) Figure S3. Dietary L. reuteri increases spermatogenesis in mice. L. reuteri has a beneficial effect in sperm concentration and sperm activity of 12-month-old outbred Swiss mice. *p<0.05, **p<0.001. https://doi.org/10.1371/journal.pone.0084877.s003 (TIF)

Acknowledgments We thank James Versalovic for the gift of ATCC 6475 Lactobacillus reuteri, and special thanks to James G. Fox for encouragement and support.

Author Contributions Conceived and designed the experiments: TP EJA SEE. Performed the experiments: AS TL TP PQ BJV YMI JRL SEE. Analyzed the data: TP PQ AC EJA SEE. Contributed reagents/materials/analysis tools: TP PQ EJA SEE. Wrote the paper: TP AS EJA SEE.