Probiotic organisms have been identified in most human organs, including the male reproductive organs such as seminal plasma and testis [1,2]. Several scientific reports suggest that gut microbiota may play a significant role in male reproductive health. As a potential therapeutic approach, supplementation with targeted probiotics could serve as a complementary strategy to support overall reproductive well-being (R). Although the interaction between the gut microbiota—a diverse community of microorganisms residing in the gut—and Leydig cells has not been extensively studied, there is emerging evidence that the gut microbiota regulates both testosterone concentrations and the development of Leydig cells [3]. For example, certain bacterial species in the gut have been shown to affect the expression of genes encoding enzymes involved in testosterone synthesis. Nonetheless, further research is required to elucidate the relationship between the gut microbiota and Leydig cells.

Leydig cells are specialized cells located in the testis that secrete testosterone, which plays crucial roles in male sexual development and reproductive function [4]. The testosterone produced by Leydig cells induces extra-testicular androgenic and anabolic effects, serving as the key hormonal determinant of the male phenotype. Thus, Leydig cells are essential in supporting male reproductive functions. However, the connection between probiotics and the endocrine system in male reproductive physiology remains insufficiently explored. Although only a limited number of studies have addressed the effects of specific probiotics on Leydig cells, recent reports indicate that the gut microbiota does influence Leydig cell function, particularly with regard to testosterone production.

Several studies have indicated that probiotics exert therapeutic effects on male infertility [5]. Therefore, probiotics have the potential for clinical application to improve male fertility, including increasing sperm number and motility. The endocrine regulatory effects of probiotics on the male reproductive system have also been more clearly elucidated in recent research.

In the present study, we aimed to clarify the mechanisms underlying the effects of probiotics on the endocrine function of Leydig cells. To achieve this, we analyzed the effects of several probiotic bacterial strains on endocrine signaling pathways in TM3 cells in vitro.

Ethical approval was not applicable to this study as it did not involve human participants or animal experiments.

In the present study, we demonstrated that probiotics improve the endocrine function of TM3 cells, a cell line derived from mouse Leydig cells. Commensal organisms originating from the human gut increased cell proliferation, expression of genes and proteins involved in steroidogenesis and mitochondrial regulation, as well as mitochondrial function in TM3 cells. L. rhamnosus BST0042, L. fermentum BST0117, and B. longum subsp. longum BST0143 enhanced the proliferation of TM3 cells, increased the OCR, and upregulated expression of StAR and HSD-3β, both of which are key mediators of steroidogenesis. These findings suggest that specific probiotics positively affect indices related to male reproduction and may be useful in the treatment of male hypogonadism.

Leydig cells serve as the principal endocrine support cells in testicular tissue. Maintaining a sufficient population of Leydig cells is critical for homeostasis and the maintenance of spermatogenesis in the testis. Our findings show that probiotics such as L. rhamnosus BST0042 and B. longum subsp. longum BST0143 promote the proliferation of Leydig cells. This is consistent with previous reports that endogenous microbiota regulate intestinal stem cell activities and cell fate decisions [6]. Furthermore, probiotics have been previously reported to affect cell proliferation and death. In a previous in vivo study, probiotic-treated mice exhibited a higher number of intestinal stem cells than mice with normal microbiota [7-9].

Steroidogenesis in Leydig cells produces testosterone, which is essential for male reproductive function. The regulation of testicular steroidogenesis occurs at several levels. Initially, StAR proteins mediate cholesterol transport from outside mitochondria to the inner mitochondrial membrane. Cholesterol is then converted to pregnenolone by CYP11A1, after which pregnenolone is metabolized by enzymes in the smooth endoplasmic reticulum, including 3β-HSD, CYP17A1, and 17β-HSD [10]. Our study revealed that probiotics significantly upregulate genes encoding these steroidogenic enzymes. Probiotics markedly increased StAR expression, and both L. rhamnosus BST0042 and B. longum subsp. longum BST0143 increased HSD-3β protein expression in TM3 cells.

Gut probiotics also affect serum cholesterol concentration through metabolism of cholesterol to coprostanol. Previous studies have engineered probiotics to express the intestinal steroid metabolism A (IsmA) gene, as part of multidisciplinary approaches involving de novo gene assembly, metabolomics, and biochemical knowledge. These IsmA+ gut bacteria have been shown to reduce fecal and serum cholesterol concentrations in humans [10]. In addition, other probiotic strains, such as B. longum and Bifidobacterium bifidum, upregulate oxidative phosphorylation in white adipose tissue by increasing bile acid signaling, thereby protecting against diet-induced metabolic conditions such as obesity and hepatic steatosis [11]. However, the components or products of probiotic strains that affect steroidogenesis in Leydig cells remain to be identified. No direct link has yet been established between StAR and the effects of probiotics. Our results indicate that these probiotics increase steroidogenesis, which in turn may improve spermatogenesis in the testis, as the steroid hormone testosterone is essential for the production of mature sperm from germ cells during spermatogenesis.

Lipid metabolism and steroidogenesis are closely integrated in mitochondria and are both required for testosterone production in Leydig cells [12]. Mitochondria are also responsible for generating energy through the catabolism of carbohydrates, amino acids, lipids, and nucleotides [11]. Probiotics generate energy and are therefore indirectly linked to host metabolism. The observation that probiotic treatment increases mitochondrial respiration highlights the importance of mitochondrial function and homeostasis in Leydig cells. In this study, we demonstrated that probiotics significantly increased the basal mitochondrial respiration rate of Leydig cells. Excessively high OCR is generally associated with proton leakage, which results in the production of reactive oxygen species [13]. Therefore, our findings that probiotics increased OCR, proton leakage, and energy production suggest that these bacteria help preserve mitochondrial function (Figure 2). Probiotic-induced enhancements of energy generation and metabolism may support spermatogenesis, given that this process requires a substantial supply of mitochondria-derived energy. We propose that probiotics improve male fertility by increasing mitochondrial energy metabolism.

To better understand the effects of probiotics on mitochondrial function in Leydig cells, we examined the expression and activation of several mitochondrial proteins that are crucial for adaptation to metabolic stress and energy balance in mammals, including mTOR, AMPK, and SIRT1. Mitochondria play several essential roles, such as in the synthesis of amino acids, lipids, and nucleotides, and in promoting cell growth via nutrient metabolism [14]. Proteins such as AMPK (a serine/threonine kinase), mTOR, and the SIRT family are major regulators of mitochondrial metabolism [15]. These proteins are associated with multiple metabolic signaling pathways that underpin mitochondrial function [14]. AMPK is activated in response to low ATP levels and increases cellular energy metabolism [16]. mTOR regulates synthesis of proteins involved in cellular homeostasis, growth, and survival [17] and also controls the assembly of microtubular proteins and cell proliferation. SIRT proteins, as nicotinamide adenine dinucleotide+-dependent histone deacetylases, modulate the acetylation of specific proteins and thereby play pivotal roles in cell physiology and pathology [18]. AMPK serves as a key energy sensor in most cell types and regulates various physiological responses, including autophagy, lipid metabolism, and protein synthesis [19]. Our data show that the p-AMPK/AMPK ratio is elevated in probiotic-treated TM3 cells. Another study reported that AMPK activation improves stem cell function through interactions with SIRT1/forkhead box O and inhibition of mTOR complex 1 (mTORC1) [20]. mTOR regulates several processes in eukaryotic cells, including growth and metabolism in response to environmental stimuli, such as nutrients and growth factors [19]. mTOR is a kinase that regulates a major eukaryotic signaling network and plays a fundamental role in cellular and organismal physiology; however, some aspects of its function remain unclear. It exists as two subtypes, mTOR1 and mTOR2, which have different roles and downstream signaling pathways. Inhibition of mTORC1 has been shown to induce autophagy, preserve the stem cell pool, and improve tissue function, thus holding potential clinical relevance [21]. AMPK and mTORC1 often exert antagonistic effects, for example, on autophagy and apoptosis [22]. SIRT1, the best-studied member of the SIRT family, has a broad range of molecular targets [23] and has been implicated as an important mediator of host-probiotic interactions. It plays roles in numerous cellular processes, including gene transcription, mitochondrial biogenesis, insulin secretion, and glucose and lipid metabolism. Collectively, these findings suggest that probiotics activate AMPK, mTOR, and SIRT1, which likely underpins their positive effects on lipid metabolism and mitochondrial energy production (Figure 6). Thus, probiotics exert beneficial effects on both the energy status and endocrine activity of TM3 cells through AMPK, mTOR, and SIRT1 signaling.

The primary function of Leydig cells is to secrete testosterone, supporting spermatogenesis and male reproductive capacity. Our results indicate that probiotic treatment enhances both steroidogenesis and mitochondrial function in Leydig cells, thereby promoting testosterone production and overall male reproductive health. Nonetheless, several limitations remain. The effects of probiotic administration on testosterone production have not previously been investigated in detail. The role of probiotics and other signaling molecules in male hypogonadism requires further study. Despite these limitations, our findings highlight the potential impact of probiotics on testosterone synthesis and offer new insights into the management of male hypogonadism.

The probiotics Lactobacillus rhamnosus, Lactobacillus fermentum, and B. longum subsp. longum have been observed to influence both steroidogenesis and mitochondrial activity in Leydig cells. Specifically, these strains increase the OCR by modulating longevity-related factors such as mTOR and SIRT1, as well as proteins involved in steroidogenesis, including StAR and HSD-3β. Taken together, these findings suggest a potential role for these probiotics in supporting male reproductive health; however, further research is warranted to determine their therapeutic applications in conditions such as male hypogonadism.