Selenium Supplementation Influences Mice Testicular Selenoproteins Driven By Gut Microbiota

doi:10.21203/rs.3.rs-923352/v1

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Selenium Supplementation Influences Mice Testicular Selenoproteins Driven By Gut Microbiota

https://doi.org/10.21203/rs.3.rs-923352/v1

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published 10 Mar, 2022

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Selenium is a well-known essential element with important roles in human reproductive health mainly due to its antioxidant character. This study aimed to investigate the potential role of selenoproteins on the gut microbiota-reproductive health. A new assay for the absolute quantification of selenoproteins in testicular tissue based on two dimensional chromatography with inductively coupled plasma mass spectrometry was performed for the first time. Gut microbiota profile was obtained by 16S rRNA gene sequencing. Numerous associations were found between testicular selenoproteins and gut microbiota (e.g. Mucispirillum, related with sperm activity and testosterone, was associated to glutathione peroxidase (GPx) and selenoalbumin (SeAlb), while Escherichia/Shigella, related to sex hormones, correlated with GPx, selenoprotein P (SelP) and SeAlb). The effects of Se-supplementation on testicular selenoproteins only occurs in conventional mice, suggesting a potential selenoproteins-microbiota interplay that underlies in testicular function. The selenoproteins GPx and SelP have been quantified for the first time in testicles, including the novel identification of SeAlb, a protein with nonspecifically incorporated Se. These findings demonstrates the significant impact of Se-supplementation on gut microbiota and reproductive health. In addition, the analytical methodology applied for selenoproteins quantification in testicular tissue opens new possibilities to evaluate their role on the gut microbiota-reproductive health axis.

Chemical Biology

General Microbiology

selenoproteins

gut microbiota

testicular

SeAlb

Selenium (Se) is an essential trace element with important roles in the immune function, the metabolism of thyroid hormones ¹ and cancer chemoprevention ². Se deficiency has been related to heart failure, nutritional myodegeneration (white muscle disease) ³ and Keshan disease ⁴ among other pathologies. Se dietary supplementation leads to the formation of specific selenoproteins, in which Se occupies the active centre, hence influencing the redox-regulated genes and helping the cell to cope with reactive oxygen species (ROS) into less reactive molecules ⁵. Se is also important in reproductive health ⁶, being essential for gonadal development, gametogenesis and fertilization ⁷, probably as a result of its ability to modulate the antioxidant defence mechanisms and the redox sensitive pathways. There is a clear evidence that a deficiency of Se and selenoproteins can lead to several reproductive health and obstetric complications as well as infertility, preeclampsia, miscarriage, preterm labour, foetal growth restriction, gestational diabetes and obstetric cholestasis ⁶. Mammalian Se-containing proteins can be divided into three groups: (i) proteins containing non-specifically incorporated Se, in which sulfur is replaced by Se in amino acids like methionine (SeMet) (e.g. selenoalbumin (SeAlb), not considered as a “real” selenoprotein), (ii) specific Se-binding proteins (e.g. Se-binding protein 1, SBP1), in which Se is tightly associated with a cysteine (Cys) residue in the peptide, but not as a component of selenocysteine (SeCys) and (iii) specific selenocysteine-containing selenoproteins (e.g. selenoprotein P (SELENOP)) ⁸. The role of Se in mammal spermatogenesis is mainly mediated by two selenoproteins, namely phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4) related to sperm quality and male fertility and SELENOP, a plasma protein required for Se supply to the gonads used as a reservoir of Se ⁹. Other selenoproteins transcripts (~ 10-fold lower than PHGPx and the majority of them with unknown function) have also been identified in male gonads (Thioredoxin/Glutathione Reductase (TGR), selenoprotein V, selenoprotein W, selenoprotein K, selenoproteins 15 and selenoprotein S) ⁹.

Recent studies have pointed out the potential role of dietary Se in shaping the gut microbiota, and then, exerting effects on host metabolism and immunity ^10–13. Diet is considered a key regulator of gut microbiota with effects at local and systemic levels ^14,15 and also, on reproductive hormones levels ¹⁶. Several authors pointed out that high-fat diet induced gut dysbiosis can affect the systems health even causing neurological disorders ^17,18 and spermatogenesis impairments ¹⁹. Recent studies suggest the impact of gut microbiota on fertility and reproductive health in both, males and females ^20–22. However, little is known about the mechanisms underlying the shifts in the gut microbiota across reproductive state. An increase in dietary Se intake has also been involved in enhancing the antioxidant GPX activity, thereby improving male fertility ²³. The encouraging results in the last years suggest that the combination of Se with other essential micronutrients could improve the reproductive efficiency in males ²³. However, till date, there is not sufficient and consistent finding to draw solid conclusions. Indeed, previous studies about selenoproteins in testicles applied non quantitative methods such as transcriptomics or enzymatic assays ⁹.

In this scenario, the aim of this work is to investigate the potential role of selenoproteins in the gut microbiota-reproductive health axis. For this purpose, the absolute quantification of selenoproteins by intact protein analysis will be performed using a metallomic approach based on hyphenated inductively coupled plasma mass spectrometry (ICP-MS), a methodology reported for serum, plasma ²⁴ and liver ²⁵. To this end, mice testicular selenoproteome has been determined after Se-supplementation of conventional mice and mice with microbiota depleted by antibiotics. The total metal content in testicles has been also measured to evaluate the possible impact of Se supplementation and microbiota on the homeostasis of elements in testicles and their traffic.

Preliminary Observations and Histopathology Evaluation

This study analyzed the impact/effect of a Se-enriched diet on selenoproteins, total Se concentration and metal homeostasis in the testes on 8 weeks-old male Mus musculus mice as well as the relationships of these parameters with the gut microbiota composition. To study the influence of the gut microbiota, half of the mice received a cocktail of antibiotics (200 mg·kg^-1 per body weight (bw) of ampicillin, neomycin and metronidazole, 100 mg·kg^-1 bw of vancomycin and 2 mg kg^-1 bw of amphotericin B)²⁶ for a week, and later, they were fed with a regular or a Se-supplemented diet for two additional weeks. The Se-enriched diet provided the mice with a daily intake of 120 µg kg^-1 bw, three times the regular mouse intake of Se ²⁷. This is a nontoxic dose of Se, but able to modify some biological parameters ²⁸. The animals showed no external evidence of illness or discomfort and all survived the treatment. No differences were found between the body weights of the mice at the end of the treatment. However, pretreatment with Abx caused a decrease, albeit statistically non-significant, in the testicular weight of mice (Figure S2).

Studies in animal models clearly demonstrate the deleterious effects of antibiotics on testicular function ²⁹. The antibiotic cocktail we used here to deplete the intestinal microbiota included the aminoglycoside neomycin, an antibiotic that adversely affects spermatogenesis by cessation of meiosis at the level of primary spermatocytes ³⁰. Our histopathological study confirms this effect (Fig. 1), since we found a greater number of spermatogonia, the diploid undifferentiated germ cells, in the seminiferous tubules of the Abx group. Although the differences with the control group were not statistically significant, these results suggest a possible arrest of the cell division processes that convert spermatogonia into spermatozoa. The Abx group histological samples also showed alteration of the normal structure of the seminiferous tubes with mild and punctual degenerative changes at the level of the basement membrane. This morphological layout is essential for the process of spermatogenesis since it is the structural and hormonal support of the spermatogonia in the different stages of the seminiferous epithelial cycle ³¹ and abnormal basement membrane structures have been associated to spermatogenesis ³². Although no significant decrease in the number of sperm per seminiferous tube was observed in any sample, our data suggests that Abx treatment might alter male fertility. The intake of a Se supplement after pretreatment with Abx (Abx-Se group) did not completely prevent the effect of Abx on the meiotic process but prevented the basement membrane abnormalities observed in the Abx group. These results suggest that Se supplementation probably improves the fertility of mice, in agreement with previous reports ⁹.

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 [8]. 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 (SeO₄^2-, SeO₃^2-, 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 [8].

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.

Animal Experimental Design

After three days of acclimation period, forty mice (male Mus musculus BALB/c, 8 weeks old) were randomly divided in two groups, one receiving water and the other, receiving water with a mixture of antibiotics (ampicillin 1%, metronidazole 1%, neomycin 1%, vancomycin 0.5%) and an antifungal (amphotericin B, 10 mg/L) for a week. After this pretreatment time, half of the mice of each group (n = 10) were fed for two additional weeks (treatment period) with the same regular diet used in the previous days and the other half, with a Se-enriched diet (0.65 mg/kg of sodium selenite). The four groups, C (control), C-Se (Se-enriched diet during the treatment); Abx (Antibiotics in the water during pretreatment) and Abx + Se (Antibiotics in the water during pretreatment and Se-enriched diet during the treatment) were caged in pairs, with free access to water and food, which were changed every other day. Figure S1 summarize the design of the experiment. At the end of the experiment, mice were anesthetized (isoflurane) and sacrificed by cervical dislocation, and organs were immediately removed, cleaned in 0.9% NaCl (w/w) solution, cryo-homogenized in liquid nitrogen and stored at -80ºC until the analysis.

Ethics statement

The experimental procedures were carried out at the Animal Experimentation Service of the University of Cordoba (SAEX-UCO), after approval by the bioethics committee of the university and the regional government (Code Num. 02-01-2019-001), in accordance with current European Union regulations. Furthermore, the study was carried out in compliance with the ARRIVE Guideline.

Histopathological evaluation

Testicle tissues were fixed in 10 % neutral buffered formalin for 24 hours and then embedded in paraffin wax. Four-micrometer-thick paraffin serial sections were obtained using a rotary microtome (SAKURA Tissue Tek Accu Cut SRM 119 200) and stained with hematoxylin-eosin according to routine protocols. Photomicrographs were obtained with a Nikon Eclipse E400 photomicroscope at 400 magnifications.

Speciation of Selenoproteins in Mice Testicles

To isolate selenoproteins, testicles were cryo-homogenized with mortar and pestle in the presence of liquid nitrogen. Selenoproteins were extracted using the CelLytic^™ MT extraction reagent (Sigma-Aldrich, Steinheim, Germany) (3 mL·g^-1) into a glass/teflon homogenizer at a temperature of 4ºC. Protease inhibitor cocktail (Sigma-Aldrich, Steinheim, Germany) was added to CelLytic MT reagent to avoid protein degradation. Then, the mixture was centrifuged at 15500 g for 20 min at 4°C. The supernatant was collected and filtered through low protein absorption Iso-Disc poly(vinylidene difluoride) filters (PVDF, 25 mm diameter, 0.45 µm pore size). A preconcentration step is necessary due to the low concentration of selenospecies. To this end, the extracts were completely evaporated employing a nitrogen stream and re-dissolved in 0.1 mL of MilliQ water before the analysis.

Selenoproteins were separated from the obtained extracts using a previously described method ⁷¹. Briefly, the chromatographic separation of GPx, SELENOP and SeAlb were performed in an ultra-high performance liquid chromatograph (model 1260 Infinity Quaternary LC, Agilent Technologies) using two size exclusion columns (5 ml HiTrap ®Desalting Columns, GE Healthcare, Uppsala, Sweden) and two different affinity chromatography columns (AFC, GE Healthcare, Uppsala, Sweden) with stationary phases of heparine-sepharose column (HEP-HP) and blue-sepharose column (BLUE-HP). The HEP-HP retains only SELENOP, while BLUE-HP retains SELENOP and SeAlb. Column switching method allows the simultaneous separation of selenoproteins and selenometabolites: GPx and selenometabolites elute when the column switching system is in the position 1 (0–20 min), while SELENOP and SeAlb are retained in the AFC columns. The valve switches to position 2 (20–24 min) to elute the SELENOP, and then returns to position 1 for the elution of SeAlb. A polyether ether ketone (PEEK) tubing (30 cm x 0.6 mm i.d.) and a T-connector were used to connect the eluent of the chromatograph to the Micromist nebulizer (Glass Expansion, Switzerland) of the triple quadrupole inductively coupled plasma mass spectrometer (ICP-QqQ-MS, model Agilent 8800 Triple Quad, Agilent Technologies, Tokyo, Japan) (2D-SEC-SEC-AFxAF-ICP-MS). ⁷⁴Se (Cambridge Isotope Laboratories, Andover, MA, USA) was also introduced via T-connector into the system for isotope dilution analysis. The absolute quantification of selenoproteins by 2D-SEC-AF-SUID-ICP-MS was carried out using the operational conditions summarized in Table S7.

Total Elements Determination in Mice Testicles

For total elemental analysis, testicular tissue from mice of each group were pooled, and approximately, 0.1000 g of sample were digested in a microwave reaction system MARS 6 (CEM Corporation, Matthewa, NC, USA) with a mixture of nitric acid and hydrogen peroxide (4:1, v/v). The mineralization was carried out from room temperature ramping to 160 ºC in 15 min and then maintained at 400 W for 40 minutes. Then, the samples were 5-fold diluted in 5% HNO₃ containing 100 µg L^− 1 of rhodium, and filtered using 0.45 µm PTFE syringe filters before the analysis by ICP-QqQ-MS. The operational conditions for ICP-QqQ-MS are listed in Table S7. The validation of the methodology was carried out using a fish protein certified reference material for trace element DORM-4 (National Research Council of Canada) (Table S8).

Gut Microbiota Analysis

Fecal samples were collected from colon and immediately frozen in liquid nitrogen. DNA from fecal samples (approx. 100 mg) was obtained with the Master-Pure DNA extraction Kit (Epicentre, Madison, WI, US) following the manufacturer’s instructions. Specific modifications were included as described elsewhere ⁷². DNA concentration (Qubit® 2.0 Fluorometer, Life Technology, Carlsbad, CA, US) was measured. Specific 16S rRNA amplicon (V3-V4 variable region of the 16S rRNA gene) was amplified and sequenced following Illumina protocols. Briefly, a multiplexing step was conducted by the NextEra Index Kit (Illumina, San Diego, CA, United States) and amplicons were checked with a Bioanalyzer DNA 1000 chip (Agilent Technologies, Santa Clara, CA, United States). Libraries were sequenced (2x300bp paired-end run, MiSeq Reagent kit v3) on a MiSeq-Illumina platform (FISABIO sequencing service, Valencia, Spain). Controls (DNA extraction procedure and libraries amplification) were included. DADA2 pipeline was used to achieve quality filtering, sequence joining and chimera removal ⁷³. Taxonomy assignment was performed using Silva v132 database ^74,75. Sample with less than 1000 reads as well as specific Taxa present in less than 0.01% and those present in less than 3 times in at least 20% of the samples were filtered and removed from the analysis. Furthermore, sequences classified as Chloroplast and Cyanobacteria were filtered from the final dataset as they are associated with potential contaminants.

Statistical Analysis

Statistical analysis was performed using Minitab16 Statistical Software (State College, PA, USA) and STATISTICA 8 Software. First of all, Anderson-Darling normality test was used to determine whether data are not normally distributed. Differences between groups were tested using Kruskal-Wallis test (non-parametric statistics) and one-way ANOVA (parametric statistics). Spearman correlation test was performed for correlation analysis between gut microbiota abundance and selenoproteins concentrations. The level of p < 0.05 was considered statistically significant. Heatmaps were generated in R Project software (version 4.0.2) (R Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). For the microbiota analyses, total sum normalization (TSS) for the statistical analysis, multivariate test and data mining were performed with Calypso web platform v. 8.56 ⁷⁶. Alpha- diversity metrics (Chao1 and Shannon indexes) and beta diversity analysis (based on Bray Curtis distance) were obtained. Briefly, Permutational multivariate analysis of variance (ADONIS) and Redundancy Discriminant Analysis (RDA) were obtained. Relative abundance (%) differences between groups at different taxonomical levels were tested using Krustal-Wallis test with False discovery test rate (FDR) for multiple test correction. Alpha diversity indexes were obtained after a rarefaction to 93525 sequences (minimum number of reads per sample). The level of statistical significance for all tests was fixed to p < 0.05.

Acknowledgements

This work was supported by the projects PG2018-096608-B-C21 from the Spanish Ministry of Economy and Competitiveness (MINECO) and UHU-1256905 from the FEDER Andalusian Operative Program 2014-2020 (Ministry of Economy, Knowledge, Business and Universities, Regional Government of Andalusia, Spain). S.R.A. thanks the Spanish Ministry of Economy and Competitiveness for a PhD scholarship (BES-2016-076364). The authors are grateful to FEDER (European Community) for financial support, Grant UNHU13-1E-1611. The authors would like to acknowledge the support from The Ramón Areces Foundation (ref. CIVP19A5918).

Author contributions

S.R.A: Formal analysis, Investigation, Data Curation, Writing-Original Draft, Writing – Review & Editing, Visualization. M.S.R.: Formal Analysis, Investigation, Data Curation, Software, Writing - Review & Editing, Visualization. M.C.C.: Conceptualization, Methodology, Resources, Supervision, Writing- Reviewing and Editing. F.N.R.: Conceptualization, Investigation, Writing - Review & Editing. N.A.: Conceptualization, Methodology, Data Curation, Writing - Original Draft, Writing - Review & Editing. T.G.B.: Conceptualization, Methodology, Resources, Writing - Original Draft, Writing - Review & Editing, Visualization, Supervision, Funding acquisition.

Competing interests

The authors declare that there are no conflicts of interest.

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Table 1. Concentration of elements (ng·g^-1) expressed as mean ± SD (n=3) in mice testicles from the different groups.

Element	Control	C-Se	Abx	Abx + Se
Al	1337 ± 380	1239 ± 31	1196 ± 75	829 ± 54
V	9.3 ± 7.3	6.9 ± 0.2	4.5 ± 1.3	6.4 ± 0.2
Cr	65 ± 8	161 ± 4	33 ± 0.2	20 ± 6
Mn	467 ± 16	470 ± 3	448 ± 10	463 ± 1
Fe	31843 ±147	25557 ± 44	28281 ± 266	32312 ± 96
Co	17.1 ± 6.7	13.8 ± 1.7	16.3 ± 1.7	21.6 ± 0.8
Cu	1702 ± 17	1429 ± 12	1426 ± 16	1482 ± 30
Zn	3815 ± 31	3662 ± 2	3723 ± 1	4143 ± 42
As	15.4 ± 1.6	40.2 ± 2.7	2.9 ± 1.1	2.4 ± 0.7
Mo	63 ± 4	54 ± 3	130 ± 4	68 ± 1

No competing interests reported.

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Selenium Supplementation Influences Mice Testicular Selenoproteins Driven By Gut Microbiota

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Selenoproteins and Total Selenium in Testicles

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