Selenoproteins and Total Selenium in Testicles
Selenoprotein extraction protocols from mammalian tissues requires high efficiency, avoiding interferences and protein degradation during the procedure. For total protein extraction we used here the CelLytic™ MT extraction reagent for total protein extraction, which contains of a low concentration of a dialyzable mild detergent for minimal interference with protein interactions and biological activity 33. Selenoproteins obtained from mice testicles for each group of study were clearly identified in the typical mass flow chromatogram (Fig. 2). These chromatograms represent the mass of Se (µg) along time (min), and thus they do not reflect the abundance of different selenoproteins, but the Se accounted from each one. Making the conversion of chromatograms peaks to selenoproteins abundance, it must be taken into consideration that mice and human SELENOP contains 10 selenocysteine molecules (C3H7NO2Se) 34, while GPx has 4 gram atoms of Se per mole 35. In addition, we quantified SeAlb, which is not a “real selenoprotein”, but a protein that incorporates Se postraslationally in the form of selenomethionine (SeMet) 36. In adition, the relative concentration of selenoproteins (as Se) in testicles is SELENOP > GPx + unretained (unr) ~ SeAlb (Fig. 2). It is noteworthy that GPx elutes in the void of the column and this peak should be assigned to GPx and other unretained selenoproteins (they have not been identified in mice testicles by organic mass spectrometry and will be the object of further works).
Significant changes in the selenoproteins and total Se concentrations were observed between groups (Fig. 3 and Table S1). The total Se content in testicles is affected by Se-supplementation. In conventional mice fed with Se-supplemented diet (C-Se group), the total Se content were similar to control group, and there not statistical differences between groups. In contrast, mice fed Se-supplemented diet after microbiota depletion (Abx + Se group) presented the highest concentration of Se in testicles, showing statistical differences with the control group (C, 1.24-fold↑, p = 0.01), with the Se-supplemented conventional mice (C-Se, 1.19-fold↑, p = 0.005) and with the microbiota depleted mice fed rodent diet (Abx, 1.15-fold↑, p = 0.01).
Regarding selenoproteins, the concentration of SELENOP and SeAlb present the highest concentration in testicles of conventional mice fed Se-supplemented diet, while the concentration of GPx + unr was the lowest in this group. Thus, Se supplementation increases the concentration of SELENOP (1.61-fold↑, p = 0.000) and SeAlb (1.30-fold↑, p = 0.013) in the testicles of conventional mice (C-Se vs C), while decreases the concentration of GPx + unr (2.32-fold↓, p = 0.000). SeAlb concentration in testicles was significantly different when comparing Abx + Se vs C (1.54-fold↓, p = 0.017). Se-supplementation of microbiota depleted mice (Abx + Se vs C-Se) increased the concentration of GPx + unr in testicles (2.22-fold↑, p = 0.000) and decreased that of SELENOP (1.85-fold↓, p = 0.000) and SeAlb (2.00-fold↓, p = 0.000). As previously commented, PHGPx/GPx4 (most abundant) and SELENOP have been previously identified in testicles along with other proteins with unknown function or less abundant . However, to our knowledge, this is the first identification of SeAlb in testicles, probably due to the methodologies previously used (transcriptomics and enzymatic activities 37,38 or a lack of information about its presence and role in testicles. Testosterone is produced by the Leydig cell and secreted into the interstitial fluid from where it is taken up by the Sertoli or diffuses into the interstitial capillaries to bind to albumin for transport through the body. If the presence of Se in albumin favors the testosterone binding and distribution to other organs and tissues, is an interesting issue to be addressed in further studies. In addition, the absolute quantification of selenoproteins in testicles has not been reported before. Moreover, the selenoproteins determined in this work accounts for the highest Se content linked to proteins in testicles as can be concluded from the mass flow chromatogram (Fig. 2). Selenometabolites (SeO42-, SeO32-, SeMet, SeCys, Selenomethylselenocysteine (SeMetSeCys)) that elutes between GPx and SELENOP were under the detection limits in all the analyzed samples (LD = 0.5 ng Se g-1).
Thus, we suggest that Se-supplementation in conventional mice influences the selenoprotome, but not the total concentration of Se in testicles (Fig. 3). Indeed, SELENOP and SeAlb patterns are parallel, as they both increased their concentration after Se-supplementation. However, the concentration of GPx + unr decreased the concentration in testicles in Se-Supplemented conventional mice. Nevertheless, Se-supplementation of microbiota depleted mice (Abx + Se vs Abx) has not effect on the testicular selenoproteome, but the total concentration of Se is significantly increased. These findings may suggest that the effect of Se-supplementation on the selenoproteome of testicles could be influenced and mediated by microbiota although the exact mechanisms remains unknown.
Se supplementation have proven the beneficial effects of this element in male fertility reproduction and testicular damage 23,39–41.The main function of SELENOP is the transport and distribution of Se to other tissues, but also possess antioxidant action and it is involved in Se homeostasis 42, while GPx family are antioxidant selenoproteins 43 and SeAlb is a Se transporter 44. In testicles, SELENOP is located in the Leydig cells 45 and can influence the sperm quality and hence, the male fertility 38,46. PHGPx/GPx4 has also been related with sperm midpiece, mitochondrial sheath and sperm chromatin condensation .
In a previous work, the expression levels of GPx and SELENOP in testicles of mice were not affected with a dietary Se deficiency or selenomethionine excess 47, however, in rats with Se deficiency the expression levels of SELENOP were decreased 37. Other authors reported that Se-supplementation sharply increase the activity of testicular SELENOP 46 and, in studies of co-exposure to Cd + Se, Se ameliorates the effects of Cd by increasing SelP and GPX4 gene expression 48. The results obtained from studies of supplementation of Se in human prostate adenocarcinoma cells (F-9 and Du-145 cells) revealed an increase in the mRNA expression levels on the glutathione peroxidases GPX1, GPX2 and GPX3, SelS and SEP15, while some selenoproteins located in testes, such as SelW and SelV changes slightly and the TRXR3 selenoprotein decreased sharply 49.
To sum up, our results suggest that the use of antibiotics (Abx) could affect the ability of the host to incorporate Se into SELENOP, which could influence, as we have mentioned, testicular activity and reproductive function and are in a good agreement with the above results.
Metals and metalloids are very important in biology since around one-third of all proteins in the human body require a metal cofactor for functionality 50. Metallomics can be defined as the research field that elucidates the identification, distribution, dynamics, role and impact of metals and metalloids in biological systems 51,52. The methodology for a metallomic analysis usually involve the use of an inductively coupled plasma mass spectrometer (ICP-MS) hyphenated to high performance liquid chromatography (HPLC), gas chromatography (GC-MS) or capillary electrophoresis (CE)) using the heteroelement (an atom different to C, H, N, O or F, e.g. Se) in the biomolecule as a “tag” (heteroatom-tagged proteomics) 53. Thus, this approach, is more sensitive than the typical proteomic approaches that involve tryptic digestion and further analysis of peptides that are usually difficult to separate and it has not been previously applied for the absolute quantification of selenoproteins in testicular tissue 54. Other techniques such as UV-Vis spectrophotometry allow determining the total content of proteins or their activities, but not the absolute quantification of specific proteins.
Influence of Selenium Supplementation on Testicular Metal Homeostasis
The concentration of toxic and essential metals (Al, V, Cr, Mn, Fe, Co, Cu, Zn, As, Mo Cd, Sb, Tl, and Pb) has been determined in testicles of mice of the different groups to evaluate the metal homeostasis. The results are presented in Table 1. The concentrations of Cd, Sb, Tl and Pb in testicles were below the limit of detection (0.02-0.05 ng g-1) in all mice groups.
The statistical analysis showed numerous differences in the concentration of metals between groups. The significant differences for each comparison are summarized in Table S2. Se-supplementation of conventional mice (C-Se vs C) increased the levels of Cr (p=0.004) and As (p=0.008) and decreased the levels of Fe (p=0.000), Cu (p=0.003) and Zn (p=0.020) in testicles. The apparent paradox of Se increasing the As concentration in testicles but contributing to As detoxification can be explained by the ability of Se to additionally increase the concentration of betaine in testicles, which sequester As in the non-toxic form of arsenobetaine 55. The toxicity of Cr is determined by its chemical form, Cr(III) being essential while Cr(VI) is carcinogenic 56. The effect of Se on metal homeostasis in testicles was different after microbiota depletion. In fact, the concentration of Cr (p=0.023), Cu (p=0.013) and As (p=0.009) were lower in the Abx+Se group when compared with control group, while the concentration of Zn was higher (p=0.013). However, when Abx+Se is compared with C-Se, the levels of Fe (p=0.000), Co (p=0.028), Zn (p=0.004) and Mo (p=0.024) increased and the levels of Al (p=0.012), Cr (p=0.001) and As (p=0.003) decreased. Finally, Se-supplementation in microbiota depleted mice (Abx+Se vs Abx) reduces the concentrations of Al (p=0.031) and Mo (p=0.003) in testicles, whereas, the concentration of Fe (p=0.002) and Zn (p=0.005) augmented. The cytosol of most eukaryotic cells contains the enzyme superoxide dismutase (SOD), which contains Cu and Zn. After exposure to antibiotics, tissues are subjected to oxidative stress, which probably led to an increase of SOD and therefore, increased Zn levels 57,58.
Essential metals like Mn, Cu and Zn are crucial for maintaining male reproductive functions, since they are involved in spermatogenesis and sperm motility 59,60. Moreover, their interaction with toxic elements (As, Cd, Hg, Pb, etc) could change the toxicity of these metals 61. The synergistic/antagonistic interactions between elements through metal traffic and homeostasis in the different organs and tissues have been reported 62. The antagonistic role of Se has been proven with toxic elements such as Hg 63, Cd 64 or As 65 and with organic pollutants 66.
Selenoproteins and Gut Microbiota
It is well-known that gut microbiota play important roles in host health, modulating physiological, immunological and metabolic functions, but also participate in the regulation of hormones related to reproductive functions 16 through the hypothalamic-pituitary-testicular axis 67. Recently, the studies about the effects of dietary and supplemented Se on gut microbiota is now growing attention, but, the interplay between testicular selenoproteins and microbiota have not been reported before. As detailed elsewhere 28, Se-supplementation shape the gut microbiota composition as well as the effect of the antibiotics treatment (Figure S3). In brief, Se supplemented groups showed an increase in the members from the Lachnospiraceae and Ruminococcaceae families as well as Christensenellaceae family and Lactobacillus genus.
Regarding the microbial richness (Chao1 index) and diversity (Shannon index) indexes were associated to selenoproteins in testicles despite the impact of Abx on the microbial composition. In control group, higher microbial diversity (R=0.67, p≤0.05) and richness (R=0.71, p≤0.05) were associated to higher levels of SeAlb. In the Se-supplemented group, higher microbial diversity was also associated to SeAlb (R=0.81, p≤0.05), but not with microbial richness. Furthermore, the relations between SeAlb with microbial diversity disappear after the Abx treatment; with one exception: in the Abx+Se group, Chao index showed a positive association with SELENOP (R=0.64, p≤0.05). These data suggest the potential implication of the microbiota on the specific selenoproteins in mice testis. To further explore the gut microbiota-testicular selenoproteome interplay, specific association at genus level in each groups were determined (Figure 4). The associations at phylum and family levels were detailed in Tables S3-S6.
As shown in Figure 4, an elevated number of correlations between selenoproteins in testicles and microbiota composition appeared after Se-supplementation of conventional mice (C-Se) and/or microbiota depleted mice (Abx+Se). It is also noteworthy that the correlations between total Se and SELENOP with microbiota were very similar in control mice, and always in the opposite sense than those of GPx+unr. Moreover, this behavior was dependent on the group/treatment. As previously concluded, these findings may suggest that the effect of Se-supplementation on the selenoproteome of testicles is influenced by microbiota. In the control group, higher total Se concentration was positively linked to several groups from the Ruminococcaceae and Lachnospiraceae families as well as Butyricoccus genus, all of them known as short chain fatty acids (SCFA) producers, and having a beneficial impact on intestinal homeostasis and promoting health benefits. Furthermore, members from the Lachnospiraceae and Ruminococcaceae families were also found to be associated with the testicular functions 68. Indeed, after the supplementation with Se, some SCFA producers such as Faecalibacterium genus as well as Lachnospiraceae_UCG001 were related to SELENOP and Ruminococceae_UCG009 to SeAlb. Moreover, in the Abx group these correlations were lost and most of the relations between microbiota and selenoproteome components were found with the Total Se and SeAlb (Tables S4-S7). However, in the Abx+Se group, several new associations between all components of selenoproteome were observed, including the positive associations of the mentioned families such as Lachnospiraceae groups and Ruminococcus_1 with Total Se and SeAlb (Figure 4). It is noteworthy that in the control group these bacteria correlated positively with total Se, while in Se-supplemented mice groups they correlated with specific selenoproteins such as Mucispirillim. This observation may indicate that Se supplementation aids specific functions such as transport (SeAlb and SELENOP) or sperm quality and male fertility (SELENOP) 38,46.
Higher relative abundance of Mucispirillum genus was associated to higher total Se content in the control group. However, after Se-supplementation, higher relative abundance of Mucispirillum genus was associated to lower GPx+unr and higher SeAlb concentrations in testicular tissue of conventional mice. A decrease in the level of this genus in mice fed with supranutritional Se has been previously reported 69. Other authors indicated numerous associations between bacterial taxa and testicular functions, but specially showed that Mucispirillum were positively correlated with testosterone and sperm activity 68. Escherichia/Shigella has been related with sex hormones in the reproductive endocrine system 70. This genus correlated positively with SELENOP and negatively with total Se in conventional mice after Se supplementation, while correlated positively with SeAlb in Abx group. After Se supplementation of this group (Abx+Se), Escherichia/Shigella correlated positively with GPx+unr and SELENOP, and negatively with SeAlb.
In summary, Se-supplementation has an impact on the selenoproteome and mineral homeostasis in testis and also, on the gut microbiota, suggesting a pivotal key interplay between Se-microbiota and reproductive health. The metallomic analytical approach, based on the quantification of selenoproteins using an atomic spectrometric detector such as ICP-MS coupled to HPLC, allowed for the first time the absolute quantification of the selenoproteins containing most of the bonded Se in testicles as well as the novel identification of SeAlb in testicles. Our data indicate that Se supplementation of conventional mice did not changed the total content of Se in testicles, but significantly changed the selenoproteome profile. Moreover, the opposite situation was observed in microbiota depleted mice suggesting that the effect of Se-supplementation on the selenoproteome of testicles could be influenced by microbiota. Specific associations between Selenoproteins in testicles and gut microbiota composition and diversity have been observed being some of them related with sperm activity and sex hormones, demonstrating the interplay of Se supplementation with microbiota and the impact on reproductive health. More studies are needed to ascertain the mechanisms behind the Se-microbiota-reproductive health.