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Vitamin B12 Prevents Cimetidine-Induced Androgenic Failure and Damage to Sperm Quality in Rats

Vitamin B12 Prevents Cimetidine-Induced Androgenic Failure and Damage to Sperm Quality in Rats ORIGINAL RESEARCH published: 10 July 2019 doi: 10.3389/fendo.2019.00309 Vitamin B Prevents Cimetidine-Induced Androgenic Failure and Damage to Sperm Quality in Rats 1 1 1 Flávia Luciana Beltrame , Fabiane de Santi , Vanessa Vendramini , 1 1 2 Regina Elizabeth Lourenço Cabral , Sandra Maria Miraglia , Paulo Sérgio Cerri and Estela Sasso-Cerri * 1 2 Department of Morphology and Genetics, Federal University of São Paulo (UNIFESP/EPM), São Paulo, Brazil, Laboratory of Histology and Embryology, Department of Morphology, Dental School – São Paulo State University (UNESP/FOAr), Araraquara, Brazil Cimetidine, used as an anti-ulcer and adjuvant treatment in cancer therapy, causes disorders in the male reproductive tract, including steroidogenesis. However, its effect on sperm quality and male fertility has been poorly addressed. Since vitamin B has demonstrated to recover spermatogonia number and sperm concentration in cimetidine-treated rats, we evaluated the impact of cimetidine on sperm quality and fertility potential and whether vitamin B is able to prevent the harmful effect of Edited by: Marc Yeste, this drug on steroidogenesis and sperm parameters. Adult male rats were treated University of Girona, Spain for 52 consecutive days as follows: cimetidine group (100 mg/kg of cimetidine), Reviewed by: cimetidine/vitamin B group (100 mg/kg of cimetidine + 3 μg vitamin B ), vitamin 12 12 Ricardo Daniel Moreno, B group (3 μg vitamin B ) and control group (saline). Serum testosterone levels Pontifical Catholic University of Chile, 12 12 Chile and immunofluorescence associated to western blot for detection of 17β-HSD6 were John Even Schjenken, performed. Sperm morphology and motility, mitochondrial activity, acrosome integrity, University of Adelaide, Australia DNA integrity by Comet assay, lipid peroxidation as well as fertility potential were *Correspondence: Estela Sasso-Cerri analyzed in all groups. Apoptotic spermatids were also evaluated by caspase-3 [email protected] immunohistochemistry. In the cimetidine-treated animals, reduced serum testosterone levels, weak 17β-HSD6 levels and impaired spermiogenesis were observed. Low sperm Specialty section: This article was submitted to motility and mitochondrial activity were associated with high percentage of sperm Reproduction, tail abnormalities, and the percentage of spermatozoa with damaged acrosome and a section of the journal DNA fragmentation increased. MDA levels were normal in all groups, indicating that Frontiers in Endocrinology the cimetidine-induced changes are associated to androgenic failure. In conclusion, Received: 17 December 2018 Accepted: 30 April 2019 despite the fertility potential of rats was unaffected by the treatment, the sperm Published: 10 July 2019 quality was significantly impaired. Therefore, considering a possible sperm-mediated Citation: transgenerational inheritance, the long term offspring health needs to be investigated. Beltrame FL, de Santi F, Vendramini V, Cabral REL, Miraglia SM, Cerri PS and The administration of vitamin B to male rats prevents the androgenic failure and Sasso-Cerri E (2019) Vitamin B counteracts the damage inflicted by cimetidine upon sperm quality, indicating that this Prevents Cimetidine-Induced vitamin may be used as a therapeutic agent to maintain the androgenic status and the Androgenic Failure and Damage to Sperm Quality in Rats. sperm quality in patients exposed to androgen disrupters. Front. Endocrinol. 10:309. doi: 10.3389/fendo.2019.00309 Keywords: cimetidine, vitamin B , sperm quality, spermiogenesis, androgenic dysfunction, DNA damage Frontiers in Endocrinology | www.frontiersin.org 1 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter INTRODUCTION Cimetidine has exerted an androgenic disrupter effect in testes (7, 9–12), vas deferens (13) and epididymis (8). Over the years, pharmacological research has made increasing However, the effect of this drug on the sperm quality and contributions in the area of reproductive medicine. Medications fertility potential needs to be clarified. In this study, we can negatively impact male reproduction, causing changes evaluated the impact of cimetidine on the sperm quality in sperm quality, infertility, and adverse progeny outcomes. parameters and fertility potential. Since vitamin B has Endocrine disrupters, for example, affect the testes, disrupting prevented the harmful effect of cimetidine on spermatogenesis, Leydig and/or Sertoli cells function and spermatogenesis. These we also evaluated whether this vitamin is able to prevent drugs can also harm the epididymal function and sperm the antiandrogenic effect of cimetidine on steroidogenesis and maturation (1). sperm parameters. Cimetidine, an anti-ulcer drug widely prescribed over the past 30 years, is an antagonist of histamine H receptors MATERIALS AND METHODS (2). Nowadays, cimetidine has also been used as an adjuvant therapy in some types of cancer treatments, by exerting Animals and Experimental Groups immunomodulatory and antiangiogenic effects (3, 4). However, One-hundred-day-old male (n = 56) and ninety-day-old female it has been reported in men that this drug causes adverse (n = 55) Holtzman rats were maintained in polypropylene cages effects related to antiandrogenic action, such as: loss of libido, under 12-h light/12-h dark cycle at a controlled temperature (23 impotence, gynecomastia, changes in the serum testosterone ± 2 C), with water and food provided ad libitum. levels and decrease in sperm concentration (5, 6). In rodents, The male rats were distributed into four groups: cimetidine cimetidine causes reduction in the serum testosterone levels (7, 8) (CMTG; n = 14), cimetidine/vitamin B (CMT/B G; n = 14), 12 12 associated to Leydig cell apoptosis and reduced steroidogenesis vitamin B (B G; n = 14) and control group (CG; n = 14). The 12 12 (7). This androgenic dysfunction has been associated with animals from CMTG received daily intraperitoneal injections germ cell loss and Sertoli cell apoptosis (9–12). The treatment (ip) of 100 mg of cimetidine (Hycimet , 300 mg—Hypofarma, has also induced epididymal androgenic dysfunction due to MG, Brazil) per kg. This dosage does not cause systemic toxicity reduction of SHBG levels and impairment in the epithelial and was based on the therapeutic dose range of cimetidine, AR nuclear translocation (8). Structural changes caused by usually administered to humans. Allometric extrapolation of the 3/4 cimetidine have also been reported in adult rat vas deferens dosage was performed to rats by the exponent 0.75 (BW (13). Some studies have demonstrated the impact of cimetidine scaling), taking into account body mass and basal metabolic on sperm count and motility (12, 14, 15), but the effect of this rate (26). drug on other sperm parameters and male fertility has not yet The animals from CMT/B G received ip injections been addressed. containing a solution of 100 mg/kg of cimetidine and 3 μg Vitamin B , or cobalamin, is an essential nutrient found of vitamin B -cianocobalamin (Citoneurin , 5,000 μg—Merck in foods of animal origin, including dairy products, and S.A., Brazil). This dosage of vitamin was determined from a has been useful for the treatment of peripheral nerve preliminary evaluation of the amount of daily vitamin B injury (16) and neuropathic pain (17). This vitamin also intake with the food by the animals at this age (11). The animals plays an important role in DNA synthesis and cell division from B G received only vitamin (3 μg/day) and the control (18), being an essential nutrient for the maintenance animals received saline solution, corresponding to the volume of testicular function and normal fertility [revised by of CMTG. All animals received the treatment for 52 consecutive Banihani (19)]; the therapeutic effects of this vitamin have days, a period that corresponds to a complete spermatogenic been demonstrated in rats with damaged spermatogenesis cycle in rats (27), and that induce testicular changes (7, 11, 28). and sperm parameters (20, 21). Vitamin B is able to 12 According to studies using rodents treated with other drugs, recover the seminiferous epithelium of animals treated with this period corresponds to a long term treatment in rats (29). It cimetidine (11), increasing the number of spermatogonia and is important to emphasize that, clinically, patients with gastric spermatocytes, and ameliorating the sperm concentration and/or colon cancer may be treated with cimetidine for up to 2 (12). A potential effect of vitamin B on the spermatogenic 12 years (30). recovery has also been demonstrated in animals receiving After the treatment, the animals were anesthetized with 80 co-administration of the antineoplastic busulphan and mg/Kg of ketamine hydrochloride (Francotar; Virbac Brazil vitamin B (22, 23). Ind. Com. Ltda) and 8 mg/Kg of xylazine hydrochloride For years, traditional methods for evaluation of (Virbaxyl; Virbac Brazil Ind. Com. Ltda). Blood samples semen quality, such as sperm count and morphology, were collected via cardiac puncture with BD Vacutainer have been usually used to assess male fertility potential Blood Collection Tubes (SSTII Plus, BD Biosciences) for and conception (24). However, additional tests have serum testosterone measurement. Testicular fragments been developed to provide more information on the were collected for histological analysis or frozen at −80 C fertilizing potential of spermatozoa since DNA damage for western blot and lipid peroxidation analysis. Sperm and changes in packaging of sperm chromatin may samples were collected from cauda epididymis for the affect the expression of paternal genes and the embryonic analysis of motility, morphology, mitochondrial activity, development (25). acrosome and DNA integrity, and TBARS (Thiobarbituric Frontiers in Endocrinology | www.frontiersin.org 2 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter Acid Reactive Substances) test. After sperm collection, reagent (Life Technologies, Calrsbad, USA) for 30 min at the animals were euthanized by overdose of anesthesia. room temperature. The nuclear staining was performed with The other 24 males and the female rats were used for DAPI (Molecular Probes by Life Technologies; Carlsbad, fertility test. California, USA). The analyses were performed using a fluorescent microscope DM400 B LED, a camera DFC-550 Serum Testosterone Measurement and an Image Analysis System LAS4 (Leica Microsystems, Serum testosterone levels were determined by Wetzlar, Germany). chemiluminescence immunoassay by using Access 2 Sections used as negative controls were incubated with non- Immunoassay System (Beckman Coulter, CA, USA) and immune serum in place of primary antibodies. the Access Testosterone Immunoassay kit (Beckman Coulter, CA, USA). The antibody was specific for testosterone (≤2% 17β-HSD6 Immunofluorescent Area cross-reactivity) and the analytical sensitivity was 10 ng/dL. The analysis of the immunofluorescence was performed The analyses were performed at São Lucas Clinical and using a DFC 550 Camera (Leica) attached to a BM4000 Microbiological Laboratory (Araraquara, SP, Brazil). B LED microscope (Leica), and the Leica Application Suite software (LAS 4.3, Leica).The measurement of Histological Procedures 17β-HSD6 immunoexpression was performed in two non- The testes were removed and fixed for 48 h at room temperature serial testicular sections per animal from the CG, B G, in 4% freshly prepared paraformaldehyde (MERK, Germany) CMTG and CMT/B G (n = 5/group). In the sections, buffered with 0.1M sodium phosphate (pH 7.4). Subsequently, the 17β-HSD6 immunofluorescence was measured in the testes were dehydrated in graded ethanol and embedded a standardized interstitial tissue area of 120,000 μm in glycol methacrylate (Historesin Embedding Kit, Jung, per animal at x220, and the immunofluorescent area Germany) or paraffin. The historesin sections (3 μm) per μm of interstitial tissue was obtained. All the were stained with Gill’s Hematoxylin and Eosin (H.E) parameters of the software, including threshold adjustment for morphological analyses. The paraffin sections (6 μm and color range—hue, saturation and intensity (LAS thick) were adhered to silanized slides and subjected to 4.3, Leica) were calibrated and standardized for each the immunohistochemistry reaction for detection of activated image analyzed. caspase-3 and immunofluorescence reaction for detection of 17β- hydroxysteroid dehydrogenase 6 (17β-HSD6), enzyme involved Western Blot Assay in steroidogenesis. Since 17β-HSD is a specific enzyme present only in Leydig cells (LC), fragments of the whole testes (containing interstitial Immunohistochemistry and tissue/LC and seminiferous tubules) was assessed for detection Immunofluorescence Reactions of the levels of this enzyme by western blot. Protein extraction Paraffin testicular sections adhered to silanized slides were was performed using lysis buffer [50 mM Tris pH 8.0, 150 mM immersed in 0.001 M sodium citrate buffer (pH 6.0) and NaCl, 1 mM EDTA, 10% glycerol, 1%Triton X-100, 1 mM maintained at 90 C in a microwave oven for antigen recovery. phenylmethylsulfonyl fluoride (PMSF)], containing 5 ng/mL For the immunohistochemistry reaction, the slides were of the following protease inhibitors: Pepstatin, Leupeptin, submitted to inactivation of endogenous peroxidase with 9% Aprotinin, Antipain, and Chymostatin (Sigma-Aldrich, St. Louis, hydrogen peroxide. The sections were incubated in 2% BSA to USA; P8340). After tissue homogenization using Polytron PT block non-specific binding and washed in phosphate-buffered 1600E (Kinematica, Luzernerstrasse, Switzerland), the samples saline (PBS; pH 7.4). The slides were incubated overnight were centrifuged at 8,944 × g (20 min, 4 C), and the supernatant in a humidified chamber at 4 C with the following primary was collected. Bradford assay (Sigma-Aldrich, St. Louis, USA; antibodies: rabbit anti-activated caspase-3 antibody (Abcam; B6916) was performed to determine protein concentration. Cambridge, UK; ab-4051), diluted 1:100 (7); and rabbit anti- Protein samples were separated in 10% SDS-PAGE and 17β-HSD6 antibody (Santa Cruz Biotechnology Inc., Dallas, transferred to a nitrocellulose membrane (GE Healthcare, Little USA; sc-101878), at a concentration of 2 μg/ml, according Chalfont, UK). The membranes were treated with 5% non- to Beltrame et al. (7) and Sasso-Cerri et al. (23). The fat dry milk diluted in PBS/T (PBS/0.05% Tween 20) for sections incubated with anti-activated caspase-3 antibody were 1 h for nonspecific blocking and incubated overnight at 4 C washed in PBS and incubated at room temperature with with rabbit anti-17β-HSD6 polyclonal antibody (0.5 μg/mL; Labeled StreptAvidin-Biotin Kit (Universal Dako LSAB, Dako Santa Cruz Biotechnology Inc., Dallas, USA; sc-101878). The Inc., Carpinteria, CA, USA), and the reaction was revealed membranes were washed in PBS/T and incubated for 1 h at with 3,3 -diaminobenzidine (DAB, Sigma-Aldrich, USA). After room temperature with HRP conjugated anti-rabbit secondary washing in tap water, the sections were counterstained with antibody (2.2 μg/mL; Sigma-Aldrich, St. Louis, USA). The Carazzi’s hematoxylin. The images were captured using a reactions were detected using enhanced chemiluminescence DP-71 Olympus camera attached to an Olympus BX-51 system (ECL). The membranes were incubated with stripping light microscope. The sections incubated with anti-17β-HSD6 buffer (1,5% glycine, 0,1% SDS, 1% Tween-20) for 10 min at room antibody were washed in PBS and incubated with Alexa temperature, washed in PBS and reprobed with rabbit anti-actin R R Fluor 594 goat anti-rabbit IgG antibody ReadyProbes antibody (0.09 μg/mL; Sigma-Aldrich, USA) for positive control. Frontiers in Endocrinology | www.frontiersin.org 3 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter R ◦ For all the groups, the assays were reproduced in triplicate. 150 μL of PNA-Alexa Fluor 488 (20 μg/mL), at 37 C for The optical density of protein bands was analyzed using ImageJ 30 min. After the incubation, 100 μL of mixture was smeared (version 1.50i) according to the NIH instructions for Gel analysis. on glass slide for microscopy and analyzed under a fluorescent For background subtraction, the baseline of each peak was microscope DM400 B LED (Leica, Germany). Two hundred defined and the area under the curve was measured. The protein spermatozoa were analyzed at ×545 and classified as intact levels were normalized to actin. acrosome (spermatozoa exhibiting intense and moderate bright fluorescence in the acrosome region) or damaged acrosome Sperm Morphology and Motility (spermatozoa presenting weak, irregular, or absent fluorescence The epididymis was dissected, and a small cut was made in the in the acrosome region). cauda epididymis with a razor blade. An aliquot (3 μl) of sperm was placed in 4 mL of distilled water to immobilize spermatozoa. Lipid Peroxidation in Testicular A sample drop was smeared onto a slide and stained by Shorr Homogenate and Epididymal Sperm method. The images of two hundred spermatozoa were captured (Thiobarbituric Acid Reactive Substances by a DP-71 Olympus camera attached to a light microscopy Test—TBARS) (BX-51, Olympus, Japan) and a software Image-Pro Express 6.0 The levels of lipid peroxidation in the testis and epididymal (Olympus). Spermatozoa showing sperm head and tail alterations sperm were assessed by the dosage of malondialdehyde (MDA) were quantified, at x1380, according to Filler (31) and Miranda- used as an oxidative stress marker. The TBARS assay is based on Spooner et al. (32). the reaction of MDA with thiobarbituric acid (TBA) to form a 1:2 For the motility analysis, the same cauda epididymis was adduct (39). placed into a petri dish containing Hank’s Balanced Salt Solution The MDA content in testicular homogenate was determined (HBSS) medium, supplemented with 0.2% bovine serum albumin by a chemical colorimetric method using a MDA-TBA assay (BSA), and incubated at 37 C. Subsequently, 50 μL were smeared kit [Lipid Peroxidation (MDA) Assay Kit; Sigma-Aldrich , onto a slide and evaluated under light microscopy at ×1,000. MO, USA]. The assay was performed in accordance with the Two-hundred spermatozoa were evaluated and the ratio of manufacturer’s instructions and the absorbance was measured motile sperm to the total number of sperm was obtained. The at 532 nm (Synergy H1 Hybrid Multi-Mode Microplate Reader; motile sperm were classified according to the type of movement, BioTek Instruments, Inc., VT, USA). The concentrations of based on the World Health Organization category (33, 34): fast MDA in each sample was estimated from a standard curve and progressive, slow progressive and nonprogressive. expressed as malondialdehyde nmol/g tissue. For the detection of lipid peroxidation in epididymal sperm, Mitochondrial Activity the protocol was modified from Aitken et al. (40) and Gomez The sperm samples were collected and prepared as described et al. (41). In order to make sensitive measurements of MDA previously (35). The mitochondrial activity was evaluated on spermatozoa, the stimulation of a lipid peroxidation cascade according to the method proposed by Hrudka (36) and as with a ferrous ion promoter was necessary. For this assay, 250 described by Mendes et al. (37), with minor modifications. A μL sperm suspension at a concentration of 40 × 10 cells/mL 50 μL semen aliquot was incubated with 200 μL of a medium ′ R were incubated with 125 μL ferrous sulfate (4 mM) and 125 μL containing 3–3 Diaminobenzidine (DAB, Sigma-Aldrich , MO, ascorbic acid (20 mM) at 37 C for 2 h. After cooling the samples USA) at 1 mg/mL of phosphate-buffered saline (PBS) (0.15 M, in an ice bath, 250 μL of this reaction mixture was supplemented pH 7.2) at 37 C, in a dark room, for 1 h. Following incubation, with 500 μL 10% chilled trichloroacetic acid (TCA) and the samples were smeared onto a slide and fixed in 10% centrifuged at 18,000 × g (15 min, 15 C). Two-hundred μL formaldehyde (prepared from paraformaldehyde). The images of the supernatant was added to a mixture containing 8.1% were captured using a DP-71 Olympus camera attached to a sodium dodecyl sulfate (SDS), 20% acetic acid solution (pH 3.5), light microscope (Olympus, BX-51, Tokyo, Japan) and a software 0.8% aqueous solution of TBA (cleared with 0.05N NaOH) and Image-Pro Express 6.0 (Olympus). Two-hundred spermatozoa distilled water (42). The reaction mixture was incubated at 95 C were analyzed at ×1,380. The spermatozoa were evaluated and for 1 h, cooled in ice bath and pipetted into a 96 well black- classified according to the mid-piece stain, and the rate of plate for analysis. The fluorescence intensity was measured on a cytochemical activity (RCA) was obtained. fluorescence multiwell plate reader (Synergy H1 Hybrid Multi- Mode Microplate Reader; BioTek Instruments, Inc., VT, USA) Acrosome Integrity with excitation wavelength at 515 nm and emission at 553 nm. The acrosome integrity was assessed by a staining method with The results were estimated from a standard curve generated Peanut Agglutinin (PNA), according to Varisli et al. (38), with by incubating serial dilutions of a MDA Standard Solution few modifications. To determine the acrosome integrity, PNA (Sigma-Aldrich , MO, USA) and expressed as malondialdehyde Lectin from Arachis hypogaea (peanut) conjugated with Alexa R R nmol/10 spermatozoa. Fluor 488 (Molecular Probes , Inc., OR, USA) was used. This lectin is specific for terminal β-galactose, present in the acrosome. Sperm samples were collected and stored as described previously Comet Assay by Vendramini et al. (35). For the analysis, the samples were Sperm DNA damage were analyzed by the alkaline Comet assay, thawed and 100 μL were incubated in dark tubes containing as described by Vendramini et al. (35), with minor modifications. Frontiers in Endocrinology | www.frontiersin.org 4 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter Samples containing spermatozoa stored at −80 C were area, optical density of protein bands, percentage of motile thawed at 37 C for 2 min in a water bath and diluted in spermatozoa, rate of cytochemical activity, acrosome integrity, prewarmed 0.5% low melting point (LMP) agarose (Sigma- levels of lipid peroxidation in the testis and epididymal sperm, Aldrich , MO, USA). This solution was placed onto slides parameters of Comet assay, and the following fertility parameters: precoated with 1% normal melting point agarose (Agargen ; corpora lutea, implantations and live fetuses number, and fetus Madrid, Spain) and the slides were stored at 4 C for 15 min. To and placental weights) and to the two-way ANOVA (sperm avoid further damage to the sperm DNA, the next steps were morphology, quality of sperm movement and classification of performed in the dark. The slides were covered with chilled lysis mitochondrial activity) were followed by Tukey’s post-hoc test. buffer, containing dithiothreitol at 40 mmol/L (Sigma-Aldrich , Non-parametric data (fertility potential, pre- and post- MO, USA), and incubated for 60 min at 4 C. Subsequently, a implantation losses and resorptions number) were compared second lysis buffer, prewarmed (37 C) and containing proteinase by Kruskal-Wallis test (followed by Dunn’s post-hoc test when K at 100 mg/mL (Sigma-Aldrich , MO, USA), was used to necessary). The significance level considered for all tests cover the slides for 2.5 h in a 37 C incubator. The slides were was p < 0.05. then gently washed in chilled distilled H O, covered with freshly prepared alkaline solution (pH 12.1) for 45 min and placed into a RESULTS horizontal electrophoresis box (Bio-Rad Laboratórios Brasil Ltda; Testicular Histology and 17β-HSD6 São Paulo, Brazil) filled with Tris/borate/EDTA (TBE) buffer, and submitted to electrophoresis at 30 V for 10 min. Immunoexpression For the analysis, the slides were stained with ethidium In the animals from CG and B G, the seminiferous tubules bromide and observed under a fluorescent microscope DM400 showed normal aspect, with germ cells organized in concentric B LED (Leica, Germany) containing a camera DFC-550 (Leica, layers and absence of germ cells in the tubular lumen Germany). One hundred comets were analyzed per animal by (Figures 1A,B,E). On the other hand, seminiferous tubules using LUCIA Comet Assay Analysis v. 7.30 software (Prague, at androgen-dependent stages (VII-VIII) showing epithelial CZ). The parameters evaluated included: tail DNA percentage, disorganization and detached germ cells (mainly spermatids) tail length, tail moment (tail length × tail DNA %/100) and Olive in the tubular lumen were found in CMTG (Figures 1C,H). moment (tail DNA % × the distance between the centers of mass Depletion of round spermatids, round spermatids with abnormal of head and tail regions). features and elongated spermatids abnormally distributed in the epithelium were observed (Figures 1F–H). Caspase-3-positive Fertility Evaluation spermatids were also found in CMTG (Figures 1I,J), but not Twenty-four male rats (n = 6/group) were mated overnight in CG (1K). Although tubules with sloughed germ cells in with two nulliparous females in natural proestrous/estrous. The the lumen were also found in CMT/B G, the seminiferous following morning, the presence of spermatozoa was checked in epithelium histoarchitecture was preserved in the animals from the vaginal smears, and the day of sperm detection in the vaginal this group (Figure 1D). smear was considered gestational day 1 (GD 1). On the 20th day The immunoexpression of 17β-HSD6 in the Leydig cells of of gestation (GD 20), the females were euthanized, and uterus and the animals from CMTG reduced significantly when compared ovaries were collected. The number of corpora lutea, implants, to CG, B G and CMT/B G (Figures 2A–D, F). However, 12 12 resorptions and live fetuses was recorded, and the following in the cimetidine-treated animals supplemented with vitamin parameters were calculated: (1) fertility potential (number of B (CMT/B G), the immunofluorescence was similar to CG 12 12 implantation sites/number of corpora lutea × 100); (2) rate and B G (Figures 2D,F). The analysis by western blot (tudo of pre-implantation loss (number of corpora lutea–number of minúsculo), including the optical density analysis, confirms the implantations/number of corpora lutea × 100); and (3) rate of results obtained by immunofluorescence (Figures 2G,H). post-implantation loss (number of implantations–number of live fetuses/number of implantation × 100) (35, 43). The weights of Serum Testosterone Levels live fetuses and placenta were also obtained. As shown in Figure 2E, serum testosterone levels decreased significantly in CMTG when compared with CG (51%) and Statistical Analysis B G (55%). A significant increase (2 fold) was observed in Statistical analysis of data was performed using the GraphPad the CMT/B G when compared with CMTG. No statistical Prism 6.0 software (GraphPad Software, CA, USA). All data difference was detected among the animals from CG, B G and were checked for normal distribution using the D’Agostino- CMT/B G groups. Pearson normality test. The homogeneity of variance was tested Sperm Morphology and Motility using the Bartlett’s or Brown-Forsythe tests. Normally distributed data were presented as mean ± SD. Non-normally distributed The percentage of normal spermatozoa (Figure 3A) decreased data were logarithmically transformed. In case of persistent non- significantly in CMTG (Figure 3H). In this group, the percentage normality, non-parametric test was used and the data were of spermatozoa with abnormal tail (broken, bent, or coiled— presented as median and interquartile intervals. Figures 3B–D) increased significantly when compared with CG The parametric variables submitted to the one-way ANOVA and B G animals. On the other hand, the CMT/B G showed 12 12 (testosterone measurement, 17β-HSD6 immunofluorescent a significant reduction of abnormal spermatozoa (with tail Frontiers in Endocrinology | www.frontiersin.org 5 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 1 | Photomicrographs of seminiferous tubules stained by H.E (A–H) and submitted to immunohistochemistry reaction for detection of activated-caspase-3 (I–K). In (A,B), the seminiferous tubule sections show normal epithelial integrity (EP) and absence of germ cells in the tubular lumen (L). In (C), damaged seminiferous tubules show epithelial disorganization (EP) and detached germ cells filling the tubular lumen (asterisk). In (D), except for the presence of sloughed germ cells filling the tubular lumen (asterisk), the seminiferous tubules (EP) show normal integrity similarly to CG and B . IT, interstitial tissue. In (E–H), portions of seminiferous epithelium at androgen-dependent stages of rats from CG and CMTG under high magnification. In (E), note the normal integrity of the seminiferous epithelium, in which spermatocytes (Sp), round (St) and elongate (white arrowheads) spermatids are organized in concentric layers. Residual bodies are also observed (black arrowheads). In (F), the disorganized epithelium shows round (St) and elongated (arrowheads) spermatids abnormally distributed. In (G,H), the epithelium shows lack of spermatids (asterisks) and abnormal spermatids with condensed chromatin, suggesting apoptosis (thin arrows). Residual bodies are observed (arrowheads). In (H), numerous sloughed round spermatids are seen in the tubular lumen (thick arrows). In (I,J), portions of tubules at androgen-dependent stages show caspase-3 immunolabeled (brown-yellow color) spermatids (thin arrows), confirming apoptosis. In K (control group), immunolabeled germ cells are not found. Bars: 37 μm (A–D), 18 μm (E–H), 7 μm (I–K). Frontiers in Endocrinology | www.frontiersin.org 6 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 2 | (A–D) Photomicrographs of testicular sections submitted to immunofluorescence for detection of 17β-HSD6. In CG, B G and CMT/B G (A,B,D), the 12 12 Leydig cells show strong immunofluorescence (arrows) in comparison with CMTG (C). Bars: 36 μm. In (E), the testosterone level in CMTG is significantly reduced when compared to the other groups (Values expressed as mean ± SD. One-way ANOVA followed by Tukey’s test; a 6= b; p < 0.05; n = 8). In (F), the 17β-HSD6 immunofluorescent area per μm of interstitial tissue of animals from CMTG is significantly reduced when compared to the other groups (Values expressed as mean ± SD. One-way ANOVA followed by Tukey’s test; a 6= b; p < 0.05; n = 5). In (G,H), Western blot analysis of 17β-HSD6 levels in testicular extracts. Strong bands at 35 kDa, corresponding to the 17β-HSD6 molecular weight, are detected in the CG, B G and CMT/B G while a weak band is detected in CMTG. Actin signal is 12 12 observed in all groups. 17β-HSD6 optical density (OD) shows high 17β-HSD6 levels in the CG, B G and CMT/B G and low 17β-HSD6 levels in the CMTG (Values 12 12 expressed as mean ± SD. One-way ANOVA followed by Tukey’s test; a 6= b; p < 0.05; n = 3). Frontiers in Endocrinology | www.frontiersin.org 7 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 3 | (A–G) Representative photomicrographs of spermatozoa classified according to the morphology. In (A), a normal spermatozoon. In (B–D), spermatozoa with abnormal tail (broken, bent, or coiled). In (E–G), spermatozoa with abnormal head (without characteristic curvature or isolated). Bars: 7 μm. In (H), percentage of normal spermatozoa and sperm head and tail morphological abnormalities. (Values expressed as mean ± SD. Two-way ANOVA followed by Tukey’s test; a 6= b; p <0.05; n = 8). alterations) in comparison with the CMTG (Figure 3H). The movement (Figure 4B). It is important to emphasize that in the percentage of spermatozoa with head morphological alterations animals that received vitamin only (B G), the percentage of fast (without typical curvature or isolated—Figures 3E–G) was progressive spermatozoa was higher whereas the percentage of similar among the groups (Figure 3H). non-progressive spermatozoa was lower in comparison with the The percentage of motile spermatozoa in CMTG decreased other groups (Figure 4B). significantly in comparison with the other groups (Figure 4A). According to the quality of movement, a significant reduction in Mitochondrial Activity the percentage of spermatozoa with fast progressive movement In CMTG, while the percentage of Class I spermatozoa was observed in CMTG, when compared with the other groups (Figure 5A) was low, Class II spermatozoa (Figure 5B) was (Figure 4B). On the other hand, the animals from CMT/B G 12 higher than the other groups (Figure 5E), resulting in a low showed a significant increase in the percentage of spermatozoa rate of cytochemical activity (RCA) (Figure 5F). Otherwise, with fast progressive movement in comparison with CMTG, and CMT/B G showed a significant improvement of this qualitative decrease in the percentage of spermatozoa with slow progressive parameter in comparison with CMTG (Figures 5E,F). There was Frontiers in Endocrinology | www.frontiersin.org 8 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 4 | Percentage of motile spermatozoa (A) and quality of sperm movement (B) in animals from CG, B G, CMTG and CMT/B G. Values expressed as mean 12 12 ± SD. (A) One-way ANOVA followed by Tukey’s test (a 6= b; p < 0.05; n = 8). (B) Two-way ANOVA followed by Tukey’s test (a 6= b 6= c; p < 0.05; n = 8). no statistical difference among the groups in the percentage of Comet Assay Classes III and IV spermatozoa (Figures 5C–E). As shown in Table 1, the parameters evaluated by Comet assay (percentage of fragmented DNA in tail, tail length, tail moment and Olive moment) were significantly elevated in the Acrosome Integrity spermatozoa of the animals from CMTG when compared with According to the analysis of acrosome integrity (intact or the control and B groups. However, the supplementation of damaged), the animals from the CMTG showed a higher number the treated animals with vitamin B decreased significantly the of spermatozoa with damaged acrosome in comparison with percentage of fragmented DNA in tail when compared with the other groups. However, in CMT/B G, the percentage of CMTG. No statistical difference was detected among CG, B G spermatozoa with intact acrosome was significantly higher than and CMT/B G. CMTG (Figures 6A–C). Lipid Peroxidation Analysis (TBARS Test) Fertility Evaluation No significant difference was detected in the levels of MDA either The treatment with cimetidine did not alter the parameters of in the testicular homogenate or in the epididymal sperm among reproductive outcome (number of corpora lutea, implantation, the groups (Figures 7A,B). resorptions, fetuses, and weights of placenta and fetuses) in Frontiers in Endocrinology | www.frontiersin.org 9 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 5 | (A–D) Classification of the mitochondrial activity. (A) Class I spermatozoon, when the mid-piece was totally stained; (B) class II spermatozoon, when most of the mid-piece was stained; (C) class III spermatozoon, when a part of mid-piece was stained; and (D) class IV spermatozoon, when the mid-piece was not stained. Bars: 7 μm In (E), number of spermatozoa per classes of mitochondrial activity (Values expressed as mean ± SD. Two-way ANOVA followed by Tukey’s test; a 6= b 6= c; p < 0.05; n = 6). In (F), rate of cytochemical activity (RCA) (Values expressed as mean ± SD. One-way ANOVA followed by Tukey’s test; a 6= b; p < 0.05; n = 6). Frontiers in Endocrinology | www.frontiersin.org 10 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter with low or absent motility (45), and indicate failure in the spermiogenic process. Moreover, alterations in sperm motility may be related to a reduction in the expression of exclusive genes of spermatids, involved in the development of flagellar structures, such as the mitochondrial sheath of mid-piece, and required for the glycolytic enzymes function (46). Thus, the reduction of motile spermatozoa caused by cimetidine is related to the structural flagellar changes and low mitochondrial activity. Decrease in sperm motility has also been reported in rats treated with the antiandrogenic flutamide (47). Cimetidine exerts antiandrogenic action, competing with testosterone for the androgen receptor (AR) in prostate and kidney cells (48, 49). In the epididymis, this drug reduces the stromal SHBG levels and impairs the epithelial AR nuclear translocation (8). In the present study, serum testosterone levels reduced significantly in CMTG, confirming previous findings (7, 8). Since testosterone is essential for the completion of spermiogenesis (50), the deficient androgenization induced by cimetidine may be the main cause of the flagellar abnormalities and reduction of motile spermatozoa (Figure 8). This hypothesis is reinforced by the presence of acrosomal changes in CMTG, since the acrosome formation is also an androgen-dependent process (50). It is important to emphasize that the rats received cimetidine for 52 days, but the period of spermatogenesis and the transit time taken from the testis to the cauda epididymis lasts around 65 days; thus, the spermatozoa evaluated in this study (from epididymis) were exposed to the treatment from the 14th day of spermatogenesis (at pre-leptotene primary spermatocyte stage) (revised by Hermo FIGURE 6 | (A,B) Representative photomicrographs of spermatozoa classified according to the acrosome integrity. In (A), spermatozoa with intact acrosome; et al. (51)). Thus, these cells were exposed to cimetidine along in (B), spermatozoa with damaged acrosome. Bars: 7 μm. (C) Percentage of meiosis I and II (for 23 days) and throughout spermiogenesis spermatozoa with intact acrosome in the animals from CG, B G, CMTG and (for 15 more days), carrying and accumulating the cimetidine- CMT/B G. (Values expressed as mean ± SD. One-way ANOVA followed by induced changes for 38 days, plus the period of transit from the Tukey’s test; a 6= b; p < 0.05; n = 6). testis to cauda epididymis (14 days), totaling 52 days (period of treatment). Therefore, it is not surprising to find abnormal comparison with the other groups. The fertility potential was also spermatozoa in the epididymis in view of the long period by similar among the groups (Table 2). which these germ cells were exposed to this androgen disrupter. Although low levels of ROS (reactive oxygen species) are required for normal sperm function, capacitation, and acrosome DISCUSSION reaction, elevated levels of ROS may induce DNA fragmentation In previous studies, we have demonstrated that cimetidine and apoptosis in the germ cells and spermatozoa (52). In the impairs the testicular structure and steroidogenesis (7, 9– current study, no difference was observed in the levels of MDA 11, 28, 44), causing low testosterone bioavailability and either in the testis or in the epididymal sperm, indicating that epididymal androgenic dysfunction (8). Therefore, we evaluated the cimetidine-induced changes in the spermatozoa are not the impact of this androgen disrupter on sperm quality and due to increased LPO. Besides ROS, several other factors may male fertility, and whether vitamin B supplementation induce DNA fragmentation, such as: deficiency of chromatin is able to prevent the sperm parameters changes induced recombination and packaging during spermatogenesis, aborted by cimetidine. We also tested the hypothesis that the apoptosis of germ cells, variations in the expression pattern spermatogenic improvement induced by this vitamin of spermatic protamine and incomplete chromatin remodeling can be associated with its effect on steroidogenesis. The during spermiogenesis (53). Since androgenic dysfunction leads results showed that cimetidine causes significant changes to chromatin packaging errors during spermiogenesis and loss in the sperm quality. However, testosterone levels and of the protamine levels in the testis and spermatozoa (54), the steroidogenic activity were unchanged in the vitamin B increased levels of DNA damage induced by cimetidine may be supplemented rats, while the sperm quality was improved by the due to deficient testicular androgenization. supplementation (Figure 8). Although the cimetidine-treated animals showed 15% of A significant increase in the flagellum abnormalities, sperm DNA damage, studies have demonstrated that the cut- especially tail looping or bending, was observed in spermatozoa off point that indicate low fertility in men by alkaline Comet of CMTG. These changes have been reported in spermatozoa assay is ≥45% of sperm DNA fragmentation (55). Therefore, Frontiers in Endocrinology | www.frontiersin.org 11 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 7 | Malondialdehyde (MDA) levels in testis (A) and epididymal sperm (B) of rats from CG, B G, CMTG and CMT/B G. (Values expressed as mean ± SD. 12 12 One-way ANOVA. n = 6). TABLE 1 | Analysis of sperm DNA fragmentation assessed by comet assay in rats offspring health. As revised by Spadafora (59), the nucleus from CG, B G, CMTG and CMT/B G (n = 6). 12 12 of epididymal spermatozoa may internalize RNA-containing nanovesicles released from the paternal somatic tissues. The Tail DNA Tail length Tail moment Olive moment RNAs are amplified and the cDNA copies are transmitted to (%) (μm) embryos during fertilization. Thus, not only haploid genomes a a a a CG 7.3 ± 3.1 4.7 ± 2.0 0.39 ± 0.37 0.62 ± 0.34 are delivered from germ cells during fertilization, but also the a a a a B G 7.4 ± 2.2 4.0 ± 0.8 0.32 ± 0.14 0.58 ± 0.21 epigenomes, which can be susceptible to numerous factors, b b b b CMTG 15.6 ± 3.1 7.3 ± 1.2 1.16 ± 0.34 1.26 ± 0.24 including nutritional and harmful substances. Spermatozoa a a,b a a CMT/B G 9.3 ± 0.5 5.9 ± 1.2 0.57 ± 0.12 0.84 ± 0.08 carrying drug-induced DNA damage may cause aberrant epigenetic programing in early embryos and contribute to Values expressed as mean ± SD (One-way ANOVA followed by Tukey’s test). instabilities later in development (35, 60). Considering that The different superscript letters mean differences among the groups: a 6= b; (p < 0.05). cimetidine-treated rats presented a significant decrease in DNA integrity assessed by the Comet assay, it is recommended that further investigations are carried out on the epigenetic stability decreased semen quality is not necessarily associated with and the future consequences to the progeny fathered following impaired fertility (1). In the present study, no significant cimetidine exposure. reduction in embryo implantation rate or embryo loss was Vitamin B exerts a potential role in spermatogenesis (12, noted, confirming that cimetidine treatment was not able to 12 21–23), and sperm parameters in humans (61) and animals impair the fertility potential. According to Zenick and Clegg, (12, 20, 21, 62), and has been a therapeutic agent used for (56), the production of spermatozoa in rodents far exceeds the the treatment of male infertility, especially oligozoospermia necessary quantity for fertility, and spermatogenesis must be or asthenozoospermia (63). In previous studies, we have decreased by as much as 90% in rodents so that the progeny is demonstrated beneficial effects of vitamin B on the sperm affected. Moreover, rats submitted to androgen deprivation after 12 concentration and spermatogenesis of cimetidine-treated rats complete destruction and removal of Leydig cells by using ethane (11, 12). A similar effect has also been demonstrated in the dimethane sulphonate (EDS) have maintained normal sperm seminiferous epithelium of animals receiving co-administration output and fertility for 8–10 weeks (57). In a previous study, we of the antineoplastic busulphan and vitamin B (22, 23). verified a significant reduction (45%) in the sperm concentration 12 However, a preventive effect of vitamin on the sperm exposed to of cimetidine-treated rats; thus, the remaining concentration these drugs has not yet been reported. in these animals was about 60 million of spermatozoa/mL In this study, the percentage of motile spermatozoa, the (12), whose 75% are motile and 60% are morphologically mitochondrial activity, the percentage of sperm with normal normal, according to the present study, indicating that these morphology, intact acrosomes, and normal DNA integrity cells were able to fertilize the oocytes. Even if the fertilizing was higher in the animals from CMT/B G than in CMTG. spermatozoon carries some DNA damage, it is known that 12 The improvement in the acrosome, sperm tail morphology the oocyte is able to repair, to some extent, the damage after and motility indicates a beneficial effect of this vitamin fertilization (58). on spermiogenesis (Figure 8). A protective effect of vitamin In spite of fertility potential was not affected by the treatment, B on the integrity of the sperm membrane, acrosome sperm quality was significantly impaired by cimetidine treatment. (62) and motility (64, 65) has also been demonstrated in Therefore, we cannot exclude a possible impact of the treatment, other studies. via paternal transgenerational inheritance, on the long term Frontiers in Endocrinology | www.frontiersin.org 12 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter TABLE 2 | Fertility parameters of the females (n = 12) naturally mated with male rats (n = 6) from CG, B G, CMTG and CMT/B G. 12 12 Parameters CG B G CMTG CMT/B G 12 12 (n = 12) (n = 12) (n = 12) (n = 12) Fertility potential (%) 96.65 (91.85–100.0) 96.45 (91.7–100.0) 87.5 (79.28–92.9) 93.8 (85.13–100.0) Pre-implantation loss (%) 3.35 (0.0–8.15) 3.55 (0.0–8.3) 12.5 (7.1–20.75) 6.3 (0.0–14.88) Post-implantation loss (%) 0.0 (0.0–0.0) 0.0 (0.0–8.5) 7.4 (0.0–13.0) 2.95 (0.0–7.1) Corpora lutea number 13.8 ± 1.4 13.4 ± 1.6 13.9 ± 1.7 14.8 ± 1.7 Implantation number 13.2 ± 1.2 12.8 ± 1.7 12.0 ± 2.0 13.3 ± 2.8 Resorption number 0.0 (0.0–0.0) 0.0 (0.0–1.0) 1.0 (0.0–1.75) 0.5 (0.0–1.0) Number of live fetuses 12.9 ± 1.0 12.3 ± 1.6 11.2 ± 1.6 12.2 ± 2.9 Fetus weight (g) 2.29 ± 0.16 2.53 ± 0.41 2.63 ± 0.41 2.44 ± 0.19 Placental weight (g) 0.50 ± 0.07 0.49 ± 0.09 0.56 ± 0.05 0.54 ± 0.06 Values expressed as median and interquartile intervals (Kruskal-Wallis test). Values expressed as mean ± SD (One-way ANOVA). FIGURE 8 | Representative scheme of the mechanism of action of cimetidine (A) and the preventive effect of vitamin B supplementation during the treatment (B) in the testes and sperm quality. In (A), cimetidine impairs LC steroidogenic activity, reducing 17β-HSD6 and the serum T levels. The low androgenic supply to the seminiferous epithelium impairs spermiogenesis and, subsequently, sperm quality, including: morphology, motility, mitochondrial activity as well as acrosome and DNA integrity. In (B), the supplementation of cimetidine-treated animals with vitamin B is able to prevent the harmful effect of cimetidine on the LC, maintaining the normal T levels, tubular androgenization and spermatogenic process (spermiogenesis), avoiding damage to sperm parameters. In CMT/B G, testosterone levels were similar to CG, activity as well as the serum testosterone at normal levels in and this result was consonant with the strong 17β-HSD6 rats following treatment with an androgen disrupter (Figure 8). immunofluorescence in the Leydig cells and the high levels Further studies are necessary to clarify the mechanism by which of this enzyme in the testicular extracts in the supplemented this vitamin maintains steroidogenesis. animals, confirming the preventive effect of vitamin B against the antiandrogenic effect of cimetidine. Adult Leydig cells express CONCLUSION high levels of a transmembrane protein amnionless (66, 67). This protein participates in the vitamin B transport and absorption Although the fertility potential was not impaired by cimetidine in testes, suggesting that the steroidogenic activity seems to in rodents, this drug induced significant sperm quality changes, depend on the transport of vitamin B in Leydig cells (66). including DNA fragmentation. Thus, considering a possible Except for a study in which rats fed with diet containing high paternal epigenetic and transgenerational inheritance, further levels of vitamin B showed high serum testosterone levels 12 studies focusing on the impact of cimetidine on the long term (68), studies showing preventive effect of vitamin B against offspring health are necessary. The concomitant beneficial effect androgenic disruption are inexistent in the literature. Our results of vitamin B either in the androgenic maintenance or in showed that vitamin B is able to maintain the steroidogenic the sperm parameters confirms that the cimetidine-induced Frontiers in Endocrinology | www.frontiersin.org 13 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter androgenic failure is the main cause of spermatic changes, and in fertility evaluation. FdS carried out the mitochondrial points to a potential preventive role of vitamin B on the Leydig activity analysis and western blot. FB and PC carried out the cell steroidogenesis. Therefore, vitamin B may be a potential immunohistochemistry reaction. VV, SM, and PC contributed therapeutic agent for the maintenance of sperm quality and the in the critical analysis of results, statistical analysis and in androgenic status of patients exposed to androgen disrupters. the revision of the manuscript. FB and ES-C examined and selected the images and participated in the manuscript design. All the authors read and approved the final version of ETHICS STATEMENT the manuscript. The protocol regarding the treatment of the animals used in this study was approved by the Ethical Committee for Animal FUNDING Research of São Paulo Federal University-UNIFESP/EPM, Brazil (CEUA n 7950060514) and by Ethics Committee of Dental This work was supported by São Paulo Research Foundation- School—São Paulo State University (UNESP), Araraquara/SP, FAPESP (grant numbers: 2012/23845-3; 2013/25322-0; Brazil (CEUA n 28/2014). 2019/11525-3) and CNPq. AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS ES-C coordinated the study. SM and VV contributed to the We thank Luis Antônio Potenza and Pedro Sérgio Simões for methodological design of the experiment. FB carried out the technical assistance; the Laboratory of Experimental Pathology treatment of animals. FB, FdS, and ES-C collected and carried and Biomaterials of Dental School-São Paulo State University out the histological processing. RC cooperated in the processing (FOAr/UNESP) for the assistance in spectrophotometric of sperm samples. FB carried out the sperm analysis (motility analysis; and Sérgio Valladão, from São Lucas Clinical and morphology, acrosome integrity, and comet assay), TBA and Microbiological Laboratory (Araraquara, SP), for the hormonal measurement and the assistance in the sperm test, fertility evaluation and immunofluorescence reaction. VV cooperated in comet assay. FdS, ES-C, and VV cooperated motility analysis. REFERENCES 11. Beltrame FL, Caneguim BH, Miraglia SM, Cerri PS, Sasso-Cerri E. Vitamin B12 supplement exerts a beneficial effect on the seminiferous 1. Drobnis EZ, Nangia AK. Male reproductive functions disrupted epithelium of cimetidine-treated rats. Cells Tissues Organs. (2011) 193:184–94. by pharmacological agents. Adv Exp Med Biol. 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Sasso-Cerri. This is an open-access article distributed under the terms of the Creative 66. Oh YS, Park HY, Gye MC. Expression of amnionless in mouse Commons Attribution License (CC BY). The use, distribution or reproduction in testes and Leydig cells. Andrologia. (2012) 44(Suppl. 1):383–9. other forums is permitted, provided the original author(s) and the copyright owner(s) doi: 10.1111/j.1439-0272.2011.01195.x are credited and that the original publication in this journal is cited, in accordance 67. Oh YS, Seo JT, Ahn HS, Gye MC. Expression of cubilin in mouse testes and with accepted academic practice. No use, distribution or reproduction is permitted Leydig cells. Andrologia. (2016) 48:325–32. doi: 10.1111/and.12450 which does not comply with these terms. Frontiers in Endocrinology | www.frontiersin.org 16 July 2019 | Volume 10 | Article 309 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Endocrinology Unpaywall

Vitamin B12 Prevents Cimetidine-Induced Androgenic Failure and Damage to Sperm Quality in Rats

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ORIGINAL RESEARCH published: 10 July 2019 doi: 10.3389/fendo.2019.00309 Vitamin B Prevents Cimetidine-Induced Androgenic Failure and Damage to Sperm Quality in Rats 1 1 1 Flávia Luciana Beltrame , Fabiane de Santi , Vanessa Vendramini , 1 1 2 Regina Elizabeth Lourenço Cabral , Sandra Maria Miraglia , Paulo Sérgio Cerri and Estela Sasso-Cerri * 1 2 Department of Morphology and Genetics, Federal University of São Paulo (UNIFESP/EPM), São Paulo, Brazil, Laboratory of Histology and Embryology, Department of Morphology, Dental School – São Paulo State University (UNESP/FOAr), Araraquara, Brazil Cimetidine, used as an anti-ulcer and adjuvant treatment in cancer therapy, causes disorders in the male reproductive tract, including steroidogenesis. However, its effect on sperm quality and male fertility has been poorly addressed. Since vitamin B has demonstrated to recover spermatogonia number and sperm concentration in cimetidine-treated rats, we evaluated the impact of cimetidine on sperm quality and fertility potential and whether vitamin B is able to prevent the harmful effect of Edited by: Marc Yeste, this drug on steroidogenesis and sperm parameters. Adult male rats were treated University of Girona, Spain for 52 consecutive days as follows: cimetidine group (100 mg/kg of cimetidine), Reviewed by: cimetidine/vitamin B group (100 mg/kg of cimetidine + 3 μg vitamin B ), vitamin 12 12 Ricardo Daniel Moreno, B group (3 μg vitamin B ) and control group (saline). Serum testosterone levels Pontifical Catholic University of Chile, 12 12 Chile and immunofluorescence associated to western blot for detection of 17β-HSD6 were John Even Schjenken, performed. Sperm morphology and motility, mitochondrial activity, acrosome integrity, University of Adelaide, Australia DNA integrity by Comet assay, lipid peroxidation as well as fertility potential were *Correspondence: Estela Sasso-Cerri analyzed in all groups. Apoptotic spermatids were also evaluated by caspase-3 [email protected] immunohistochemistry. In the cimetidine-treated animals, reduced serum testosterone levels, weak 17β-HSD6 levels and impaired spermiogenesis were observed. Low sperm Specialty section: This article was submitted to motility and mitochondrial activity were associated with high percentage of sperm Reproduction, tail abnormalities, and the percentage of spermatozoa with damaged acrosome and a section of the journal DNA fragmentation increased. MDA levels were normal in all groups, indicating that Frontiers in Endocrinology the cimetidine-induced changes are associated to androgenic failure. In conclusion, Received: 17 December 2018 Accepted: 30 April 2019 despite the fertility potential of rats was unaffected by the treatment, the sperm Published: 10 July 2019 quality was significantly impaired. Therefore, considering a possible sperm-mediated Citation: transgenerational inheritance, the long term offspring health needs to be investigated. Beltrame FL, de Santi F, Vendramini V, Cabral REL, Miraglia SM, Cerri PS and The administration of vitamin B to male rats prevents the androgenic failure and Sasso-Cerri E (2019) Vitamin B counteracts the damage inflicted by cimetidine upon sperm quality, indicating that this Prevents Cimetidine-Induced vitamin may be used as a therapeutic agent to maintain the androgenic status and the Androgenic Failure and Damage to Sperm Quality in Rats. sperm quality in patients exposed to androgen disrupters. Front. Endocrinol. 10:309. doi: 10.3389/fendo.2019.00309 Keywords: cimetidine, vitamin B , sperm quality, spermiogenesis, androgenic dysfunction, DNA damage Frontiers in Endocrinology | www.frontiersin.org 1 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter INTRODUCTION Cimetidine has exerted an androgenic disrupter effect in testes (7, 9–12), vas deferens (13) and epididymis (8). Over the years, pharmacological research has made increasing However, the effect of this drug on the sperm quality and contributions in the area of reproductive medicine. Medications fertility potential needs to be clarified. In this study, we can negatively impact male reproduction, causing changes evaluated the impact of cimetidine on the sperm quality in sperm quality, infertility, and adverse progeny outcomes. parameters and fertility potential. Since vitamin B has Endocrine disrupters, for example, affect the testes, disrupting prevented the harmful effect of cimetidine on spermatogenesis, Leydig and/or Sertoli cells function and spermatogenesis. These we also evaluated whether this vitamin is able to prevent drugs can also harm the epididymal function and sperm the antiandrogenic effect of cimetidine on steroidogenesis and maturation (1). sperm parameters. Cimetidine, an anti-ulcer drug widely prescribed over the past 30 years, is an antagonist of histamine H receptors MATERIALS AND METHODS (2). Nowadays, cimetidine has also been used as an adjuvant therapy in some types of cancer treatments, by exerting Animals and Experimental Groups immunomodulatory and antiangiogenic effects (3, 4). However, One-hundred-day-old male (n = 56) and ninety-day-old female it has been reported in men that this drug causes adverse (n = 55) Holtzman rats were maintained in polypropylene cages effects related to antiandrogenic action, such as: loss of libido, under 12-h light/12-h dark cycle at a controlled temperature (23 impotence, gynecomastia, changes in the serum testosterone ± 2 C), with water and food provided ad libitum. levels and decrease in sperm concentration (5, 6). In rodents, The male rats were distributed into four groups: cimetidine cimetidine causes reduction in the serum testosterone levels (7, 8) (CMTG; n = 14), cimetidine/vitamin B (CMT/B G; n = 14), 12 12 associated to Leydig cell apoptosis and reduced steroidogenesis vitamin B (B G; n = 14) and control group (CG; n = 14). The 12 12 (7). This androgenic dysfunction has been associated with animals from CMTG received daily intraperitoneal injections germ cell loss and Sertoli cell apoptosis (9–12). The treatment (ip) of 100 mg of cimetidine (Hycimet , 300 mg—Hypofarma, has also induced epididymal androgenic dysfunction due to MG, Brazil) per kg. This dosage does not cause systemic toxicity reduction of SHBG levels and impairment in the epithelial and was based on the therapeutic dose range of cimetidine, AR nuclear translocation (8). Structural changes caused by usually administered to humans. Allometric extrapolation of the 3/4 cimetidine have also been reported in adult rat vas deferens dosage was performed to rats by the exponent 0.75 (BW (13). Some studies have demonstrated the impact of cimetidine scaling), taking into account body mass and basal metabolic on sperm count and motility (12, 14, 15), but the effect of this rate (26). drug on other sperm parameters and male fertility has not yet The animals from CMT/B G received ip injections been addressed. containing a solution of 100 mg/kg of cimetidine and 3 μg Vitamin B , or cobalamin, is an essential nutrient found of vitamin B -cianocobalamin (Citoneurin , 5,000 μg—Merck in foods of animal origin, including dairy products, and S.A., Brazil). This dosage of vitamin was determined from a has been useful for the treatment of peripheral nerve preliminary evaluation of the amount of daily vitamin B injury (16) and neuropathic pain (17). This vitamin also intake with the food by the animals at this age (11). The animals plays an important role in DNA synthesis and cell division from B G received only vitamin (3 μg/day) and the control (18), being an essential nutrient for the maintenance animals received saline solution, corresponding to the volume of testicular function and normal fertility [revised by of CMTG. All animals received the treatment for 52 consecutive Banihani (19)]; the therapeutic effects of this vitamin have days, a period that corresponds to a complete spermatogenic been demonstrated in rats with damaged spermatogenesis cycle in rats (27), and that induce testicular changes (7, 11, 28). and sperm parameters (20, 21). Vitamin B is able to 12 According to studies using rodents treated with other drugs, recover the seminiferous epithelium of animals treated with this period corresponds to a long term treatment in rats (29). It cimetidine (11), increasing the number of spermatogonia and is important to emphasize that, clinically, patients with gastric spermatocytes, and ameliorating the sperm concentration and/or colon cancer may be treated with cimetidine for up to 2 (12). A potential effect of vitamin B on the spermatogenic 12 years (30). recovery has also been demonstrated in animals receiving After the treatment, the animals were anesthetized with 80 co-administration of the antineoplastic busulphan and mg/Kg of ketamine hydrochloride (Francotar; Virbac Brazil vitamin B (22, 23). Ind. Com. Ltda) and 8 mg/Kg of xylazine hydrochloride For years, traditional methods for evaluation of (Virbaxyl; Virbac Brazil Ind. Com. Ltda). Blood samples semen quality, such as sperm count and morphology, were collected via cardiac puncture with BD Vacutainer have been usually used to assess male fertility potential Blood Collection Tubes (SSTII Plus, BD Biosciences) for and conception (24). However, additional tests have serum testosterone measurement. Testicular fragments been developed to provide more information on the were collected for histological analysis or frozen at −80 C fertilizing potential of spermatozoa since DNA damage for western blot and lipid peroxidation analysis. Sperm and changes in packaging of sperm chromatin may samples were collected from cauda epididymis for the affect the expression of paternal genes and the embryonic analysis of motility, morphology, mitochondrial activity, development (25). acrosome and DNA integrity, and TBARS (Thiobarbituric Frontiers in Endocrinology | www.frontiersin.org 2 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter Acid Reactive Substances) test. After sperm collection, reagent (Life Technologies, Calrsbad, USA) for 30 min at the animals were euthanized by overdose of anesthesia. room temperature. The nuclear staining was performed with The other 24 males and the female rats were used for DAPI (Molecular Probes by Life Technologies; Carlsbad, fertility test. California, USA). The analyses were performed using a fluorescent microscope DM400 B LED, a camera DFC-550 Serum Testosterone Measurement and an Image Analysis System LAS4 (Leica Microsystems, Serum testosterone levels were determined by Wetzlar, Germany). chemiluminescence immunoassay by using Access 2 Sections used as negative controls were incubated with non- Immunoassay System (Beckman Coulter, CA, USA) and immune serum in place of primary antibodies. the Access Testosterone Immunoassay kit (Beckman Coulter, CA, USA). The antibody was specific for testosterone (≤2% 17β-HSD6 Immunofluorescent Area cross-reactivity) and the analytical sensitivity was 10 ng/dL. The analysis of the immunofluorescence was performed The analyses were performed at São Lucas Clinical and using a DFC 550 Camera (Leica) attached to a BM4000 Microbiological Laboratory (Araraquara, SP, Brazil). B LED microscope (Leica), and the Leica Application Suite software (LAS 4.3, Leica).The measurement of Histological Procedures 17β-HSD6 immunoexpression was performed in two non- The testes were removed and fixed for 48 h at room temperature serial testicular sections per animal from the CG, B G, in 4% freshly prepared paraformaldehyde (MERK, Germany) CMTG and CMT/B G (n = 5/group). In the sections, buffered with 0.1M sodium phosphate (pH 7.4). Subsequently, the 17β-HSD6 immunofluorescence was measured in the testes were dehydrated in graded ethanol and embedded a standardized interstitial tissue area of 120,000 μm in glycol methacrylate (Historesin Embedding Kit, Jung, per animal at x220, and the immunofluorescent area Germany) or paraffin. The historesin sections (3 μm) per μm of interstitial tissue was obtained. All the were stained with Gill’s Hematoxylin and Eosin (H.E) parameters of the software, including threshold adjustment for morphological analyses. The paraffin sections (6 μm and color range—hue, saturation and intensity (LAS thick) were adhered to silanized slides and subjected to 4.3, Leica) were calibrated and standardized for each the immunohistochemistry reaction for detection of activated image analyzed. caspase-3 and immunofluorescence reaction for detection of 17β- hydroxysteroid dehydrogenase 6 (17β-HSD6), enzyme involved Western Blot Assay in steroidogenesis. Since 17β-HSD is a specific enzyme present only in Leydig cells (LC), fragments of the whole testes (containing interstitial Immunohistochemistry and tissue/LC and seminiferous tubules) was assessed for detection Immunofluorescence Reactions of the levels of this enzyme by western blot. Protein extraction Paraffin testicular sections adhered to silanized slides were was performed using lysis buffer [50 mM Tris pH 8.0, 150 mM immersed in 0.001 M sodium citrate buffer (pH 6.0) and NaCl, 1 mM EDTA, 10% glycerol, 1%Triton X-100, 1 mM maintained at 90 C in a microwave oven for antigen recovery. phenylmethylsulfonyl fluoride (PMSF)], containing 5 ng/mL For the immunohistochemistry reaction, the slides were of the following protease inhibitors: Pepstatin, Leupeptin, submitted to inactivation of endogenous peroxidase with 9% Aprotinin, Antipain, and Chymostatin (Sigma-Aldrich, St. Louis, hydrogen peroxide. The sections were incubated in 2% BSA to USA; P8340). After tissue homogenization using Polytron PT block non-specific binding and washed in phosphate-buffered 1600E (Kinematica, Luzernerstrasse, Switzerland), the samples saline (PBS; pH 7.4). The slides were incubated overnight were centrifuged at 8,944 × g (20 min, 4 C), and the supernatant in a humidified chamber at 4 C with the following primary was collected. Bradford assay (Sigma-Aldrich, St. Louis, USA; antibodies: rabbit anti-activated caspase-3 antibody (Abcam; B6916) was performed to determine protein concentration. Cambridge, UK; ab-4051), diluted 1:100 (7); and rabbit anti- Protein samples were separated in 10% SDS-PAGE and 17β-HSD6 antibody (Santa Cruz Biotechnology Inc., Dallas, transferred to a nitrocellulose membrane (GE Healthcare, Little USA; sc-101878), at a concentration of 2 μg/ml, according Chalfont, UK). The membranes were treated with 5% non- to Beltrame et al. (7) and Sasso-Cerri et al. (23). The fat dry milk diluted in PBS/T (PBS/0.05% Tween 20) for sections incubated with anti-activated caspase-3 antibody were 1 h for nonspecific blocking and incubated overnight at 4 C washed in PBS and incubated at room temperature with with rabbit anti-17β-HSD6 polyclonal antibody (0.5 μg/mL; Labeled StreptAvidin-Biotin Kit (Universal Dako LSAB, Dako Santa Cruz Biotechnology Inc., Dallas, USA; sc-101878). The Inc., Carpinteria, CA, USA), and the reaction was revealed membranes were washed in PBS/T and incubated for 1 h at with 3,3 -diaminobenzidine (DAB, Sigma-Aldrich, USA). After room temperature with HRP conjugated anti-rabbit secondary washing in tap water, the sections were counterstained with antibody (2.2 μg/mL; Sigma-Aldrich, St. Louis, USA). The Carazzi’s hematoxylin. The images were captured using a reactions were detected using enhanced chemiluminescence DP-71 Olympus camera attached to an Olympus BX-51 system (ECL). The membranes were incubated with stripping light microscope. The sections incubated with anti-17β-HSD6 buffer (1,5% glycine, 0,1% SDS, 1% Tween-20) for 10 min at room antibody were washed in PBS and incubated with Alexa temperature, washed in PBS and reprobed with rabbit anti-actin R R Fluor 594 goat anti-rabbit IgG antibody ReadyProbes antibody (0.09 μg/mL; Sigma-Aldrich, USA) for positive control. Frontiers in Endocrinology | www.frontiersin.org 3 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter R ◦ For all the groups, the assays were reproduced in triplicate. 150 μL of PNA-Alexa Fluor 488 (20 μg/mL), at 37 C for The optical density of protein bands was analyzed using ImageJ 30 min. After the incubation, 100 μL of mixture was smeared (version 1.50i) according to the NIH instructions for Gel analysis. on glass slide for microscopy and analyzed under a fluorescent For background subtraction, the baseline of each peak was microscope DM400 B LED (Leica, Germany). Two hundred defined and the area under the curve was measured. The protein spermatozoa were analyzed at ×545 and classified as intact levels were normalized to actin. acrosome (spermatozoa exhibiting intense and moderate bright fluorescence in the acrosome region) or damaged acrosome Sperm Morphology and Motility (spermatozoa presenting weak, irregular, or absent fluorescence The epididymis was dissected, and a small cut was made in the in the acrosome region). cauda epididymis with a razor blade. An aliquot (3 μl) of sperm was placed in 4 mL of distilled water to immobilize spermatozoa. Lipid Peroxidation in Testicular A sample drop was smeared onto a slide and stained by Shorr Homogenate and Epididymal Sperm method. The images of two hundred spermatozoa were captured (Thiobarbituric Acid Reactive Substances by a DP-71 Olympus camera attached to a light microscopy Test—TBARS) (BX-51, Olympus, Japan) and a software Image-Pro Express 6.0 The levels of lipid peroxidation in the testis and epididymal (Olympus). Spermatozoa showing sperm head and tail alterations sperm were assessed by the dosage of malondialdehyde (MDA) were quantified, at x1380, according to Filler (31) and Miranda- used as an oxidative stress marker. The TBARS assay is based on Spooner et al. (32). the reaction of MDA with thiobarbituric acid (TBA) to form a 1:2 For the motility analysis, the same cauda epididymis was adduct (39). placed into a petri dish containing Hank’s Balanced Salt Solution The MDA content in testicular homogenate was determined (HBSS) medium, supplemented with 0.2% bovine serum albumin by a chemical colorimetric method using a MDA-TBA assay (BSA), and incubated at 37 C. Subsequently, 50 μL were smeared kit [Lipid Peroxidation (MDA) Assay Kit; Sigma-Aldrich , onto a slide and evaluated under light microscopy at ×1,000. MO, USA]. The assay was performed in accordance with the Two-hundred spermatozoa were evaluated and the ratio of manufacturer’s instructions and the absorbance was measured motile sperm to the total number of sperm was obtained. The at 532 nm (Synergy H1 Hybrid Multi-Mode Microplate Reader; motile sperm were classified according to the type of movement, BioTek Instruments, Inc., VT, USA). The concentrations of based on the World Health Organization category (33, 34): fast MDA in each sample was estimated from a standard curve and progressive, slow progressive and nonprogressive. expressed as malondialdehyde nmol/g tissue. For the detection of lipid peroxidation in epididymal sperm, Mitochondrial Activity the protocol was modified from Aitken et al. (40) and Gomez The sperm samples were collected and prepared as described et al. (41). In order to make sensitive measurements of MDA previously (35). The mitochondrial activity was evaluated on spermatozoa, the stimulation of a lipid peroxidation cascade according to the method proposed by Hrudka (36) and as with a ferrous ion promoter was necessary. For this assay, 250 described by Mendes et al. (37), with minor modifications. A μL sperm suspension at a concentration of 40 × 10 cells/mL 50 μL semen aliquot was incubated with 200 μL of a medium ′ R were incubated with 125 μL ferrous sulfate (4 mM) and 125 μL containing 3–3 Diaminobenzidine (DAB, Sigma-Aldrich , MO, ascorbic acid (20 mM) at 37 C for 2 h. After cooling the samples USA) at 1 mg/mL of phosphate-buffered saline (PBS) (0.15 M, in an ice bath, 250 μL of this reaction mixture was supplemented pH 7.2) at 37 C, in a dark room, for 1 h. Following incubation, with 500 μL 10% chilled trichloroacetic acid (TCA) and the samples were smeared onto a slide and fixed in 10% centrifuged at 18,000 × g (15 min, 15 C). Two-hundred μL formaldehyde (prepared from paraformaldehyde). The images of the supernatant was added to a mixture containing 8.1% were captured using a DP-71 Olympus camera attached to a sodium dodecyl sulfate (SDS), 20% acetic acid solution (pH 3.5), light microscope (Olympus, BX-51, Tokyo, Japan) and a software 0.8% aqueous solution of TBA (cleared with 0.05N NaOH) and Image-Pro Express 6.0 (Olympus). Two-hundred spermatozoa distilled water (42). The reaction mixture was incubated at 95 C were analyzed at ×1,380. The spermatozoa were evaluated and for 1 h, cooled in ice bath and pipetted into a 96 well black- classified according to the mid-piece stain, and the rate of plate for analysis. The fluorescence intensity was measured on a cytochemical activity (RCA) was obtained. fluorescence multiwell plate reader (Synergy H1 Hybrid Multi- Mode Microplate Reader; BioTek Instruments, Inc., VT, USA) Acrosome Integrity with excitation wavelength at 515 nm and emission at 553 nm. The acrosome integrity was assessed by a staining method with The results were estimated from a standard curve generated Peanut Agglutinin (PNA), according to Varisli et al. (38), with by incubating serial dilutions of a MDA Standard Solution few modifications. To determine the acrosome integrity, PNA (Sigma-Aldrich , MO, USA) and expressed as malondialdehyde Lectin from Arachis hypogaea (peanut) conjugated with Alexa R R nmol/10 spermatozoa. Fluor 488 (Molecular Probes , Inc., OR, USA) was used. This lectin is specific for terminal β-galactose, present in the acrosome. Sperm samples were collected and stored as described previously Comet Assay by Vendramini et al. (35). For the analysis, the samples were Sperm DNA damage were analyzed by the alkaline Comet assay, thawed and 100 μL were incubated in dark tubes containing as described by Vendramini et al. (35), with minor modifications. Frontiers in Endocrinology | www.frontiersin.org 4 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter Samples containing spermatozoa stored at −80 C were area, optical density of protein bands, percentage of motile thawed at 37 C for 2 min in a water bath and diluted in spermatozoa, rate of cytochemical activity, acrosome integrity, prewarmed 0.5% low melting point (LMP) agarose (Sigma- levels of lipid peroxidation in the testis and epididymal sperm, Aldrich , MO, USA). This solution was placed onto slides parameters of Comet assay, and the following fertility parameters: precoated with 1% normal melting point agarose (Agargen ; corpora lutea, implantations and live fetuses number, and fetus Madrid, Spain) and the slides were stored at 4 C for 15 min. To and placental weights) and to the two-way ANOVA (sperm avoid further damage to the sperm DNA, the next steps were morphology, quality of sperm movement and classification of performed in the dark. The slides were covered with chilled lysis mitochondrial activity) were followed by Tukey’s post-hoc test. buffer, containing dithiothreitol at 40 mmol/L (Sigma-Aldrich , Non-parametric data (fertility potential, pre- and post- MO, USA), and incubated for 60 min at 4 C. Subsequently, a implantation losses and resorptions number) were compared second lysis buffer, prewarmed (37 C) and containing proteinase by Kruskal-Wallis test (followed by Dunn’s post-hoc test when K at 100 mg/mL (Sigma-Aldrich , MO, USA), was used to necessary). The significance level considered for all tests cover the slides for 2.5 h in a 37 C incubator. The slides were was p < 0.05. then gently washed in chilled distilled H O, covered with freshly prepared alkaline solution (pH 12.1) for 45 min and placed into a RESULTS horizontal electrophoresis box (Bio-Rad Laboratórios Brasil Ltda; Testicular Histology and 17β-HSD6 São Paulo, Brazil) filled with Tris/borate/EDTA (TBE) buffer, and submitted to electrophoresis at 30 V for 10 min. Immunoexpression For the analysis, the slides were stained with ethidium In the animals from CG and B G, the seminiferous tubules bromide and observed under a fluorescent microscope DM400 showed normal aspect, with germ cells organized in concentric B LED (Leica, Germany) containing a camera DFC-550 (Leica, layers and absence of germ cells in the tubular lumen Germany). One hundred comets were analyzed per animal by (Figures 1A,B,E). On the other hand, seminiferous tubules using LUCIA Comet Assay Analysis v. 7.30 software (Prague, at androgen-dependent stages (VII-VIII) showing epithelial CZ). The parameters evaluated included: tail DNA percentage, disorganization and detached germ cells (mainly spermatids) tail length, tail moment (tail length × tail DNA %/100) and Olive in the tubular lumen were found in CMTG (Figures 1C,H). moment (tail DNA % × the distance between the centers of mass Depletion of round spermatids, round spermatids with abnormal of head and tail regions). features and elongated spermatids abnormally distributed in the epithelium were observed (Figures 1F–H). Caspase-3-positive Fertility Evaluation spermatids were also found in CMTG (Figures 1I,J), but not Twenty-four male rats (n = 6/group) were mated overnight in CG (1K). Although tubules with sloughed germ cells in with two nulliparous females in natural proestrous/estrous. The the lumen were also found in CMT/B G, the seminiferous following morning, the presence of spermatozoa was checked in epithelium histoarchitecture was preserved in the animals from the vaginal smears, and the day of sperm detection in the vaginal this group (Figure 1D). smear was considered gestational day 1 (GD 1). On the 20th day The immunoexpression of 17β-HSD6 in the Leydig cells of of gestation (GD 20), the females were euthanized, and uterus and the animals from CMTG reduced significantly when compared ovaries were collected. The number of corpora lutea, implants, to CG, B G and CMT/B G (Figures 2A–D, F). However, 12 12 resorptions and live fetuses was recorded, and the following in the cimetidine-treated animals supplemented with vitamin parameters were calculated: (1) fertility potential (number of B (CMT/B G), the immunofluorescence was similar to CG 12 12 implantation sites/number of corpora lutea × 100); (2) rate and B G (Figures 2D,F). The analysis by western blot (tudo of pre-implantation loss (number of corpora lutea–number of minúsculo), including the optical density analysis, confirms the implantations/number of corpora lutea × 100); and (3) rate of results obtained by immunofluorescence (Figures 2G,H). post-implantation loss (number of implantations–number of live fetuses/number of implantation × 100) (35, 43). The weights of Serum Testosterone Levels live fetuses and placenta were also obtained. As shown in Figure 2E, serum testosterone levels decreased significantly in CMTG when compared with CG (51%) and Statistical Analysis B G (55%). A significant increase (2 fold) was observed in Statistical analysis of data was performed using the GraphPad the CMT/B G when compared with CMTG. No statistical Prism 6.0 software (GraphPad Software, CA, USA). All data difference was detected among the animals from CG, B G and were checked for normal distribution using the D’Agostino- CMT/B G groups. Pearson normality test. The homogeneity of variance was tested Sperm Morphology and Motility using the Bartlett’s or Brown-Forsythe tests. Normally distributed data were presented as mean ± SD. Non-normally distributed The percentage of normal spermatozoa (Figure 3A) decreased data were logarithmically transformed. In case of persistent non- significantly in CMTG (Figure 3H). In this group, the percentage normality, non-parametric test was used and the data were of spermatozoa with abnormal tail (broken, bent, or coiled— presented as median and interquartile intervals. Figures 3B–D) increased significantly when compared with CG The parametric variables submitted to the one-way ANOVA and B G animals. On the other hand, the CMT/B G showed 12 12 (testosterone measurement, 17β-HSD6 immunofluorescent a significant reduction of abnormal spermatozoa (with tail Frontiers in Endocrinology | www.frontiersin.org 5 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 1 | Photomicrographs of seminiferous tubules stained by H.E (A–H) and submitted to immunohistochemistry reaction for detection of activated-caspase-3 (I–K). In (A,B), the seminiferous tubule sections show normal epithelial integrity (EP) and absence of germ cells in the tubular lumen (L). In (C), damaged seminiferous tubules show epithelial disorganization (EP) and detached germ cells filling the tubular lumen (asterisk). In (D), except for the presence of sloughed germ cells filling the tubular lumen (asterisk), the seminiferous tubules (EP) show normal integrity similarly to CG and B . IT, interstitial tissue. In (E–H), portions of seminiferous epithelium at androgen-dependent stages of rats from CG and CMTG under high magnification. In (E), note the normal integrity of the seminiferous epithelium, in which spermatocytes (Sp), round (St) and elongate (white arrowheads) spermatids are organized in concentric layers. Residual bodies are also observed (black arrowheads). In (F), the disorganized epithelium shows round (St) and elongated (arrowheads) spermatids abnormally distributed. In (G,H), the epithelium shows lack of spermatids (asterisks) and abnormal spermatids with condensed chromatin, suggesting apoptosis (thin arrows). Residual bodies are observed (arrowheads). In (H), numerous sloughed round spermatids are seen in the tubular lumen (thick arrows). In (I,J), portions of tubules at androgen-dependent stages show caspase-3 immunolabeled (brown-yellow color) spermatids (thin arrows), confirming apoptosis. In K (control group), immunolabeled germ cells are not found. Bars: 37 μm (A–D), 18 μm (E–H), 7 μm (I–K). Frontiers in Endocrinology | www.frontiersin.org 6 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 2 | (A–D) Photomicrographs of testicular sections submitted to immunofluorescence for detection of 17β-HSD6. In CG, B G and CMT/B G (A,B,D), the 12 12 Leydig cells show strong immunofluorescence (arrows) in comparison with CMTG (C). Bars: 36 μm. In (E), the testosterone level in CMTG is significantly reduced when compared to the other groups (Values expressed as mean ± SD. One-way ANOVA followed by Tukey’s test; a 6= b; p < 0.05; n = 8). In (F), the 17β-HSD6 immunofluorescent area per μm of interstitial tissue of animals from CMTG is significantly reduced when compared to the other groups (Values expressed as mean ± SD. One-way ANOVA followed by Tukey’s test; a 6= b; p < 0.05; n = 5). In (G,H), Western blot analysis of 17β-HSD6 levels in testicular extracts. Strong bands at 35 kDa, corresponding to the 17β-HSD6 molecular weight, are detected in the CG, B G and CMT/B G while a weak band is detected in CMTG. Actin signal is 12 12 observed in all groups. 17β-HSD6 optical density (OD) shows high 17β-HSD6 levels in the CG, B G and CMT/B G and low 17β-HSD6 levels in the CMTG (Values 12 12 expressed as mean ± SD. One-way ANOVA followed by Tukey’s test; a 6= b; p < 0.05; n = 3). Frontiers in Endocrinology | www.frontiersin.org 7 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 3 | (A–G) Representative photomicrographs of spermatozoa classified according to the morphology. In (A), a normal spermatozoon. In (B–D), spermatozoa with abnormal tail (broken, bent, or coiled). In (E–G), spermatozoa with abnormal head (without characteristic curvature or isolated). Bars: 7 μm. In (H), percentage of normal spermatozoa and sperm head and tail morphological abnormalities. (Values expressed as mean ± SD. Two-way ANOVA followed by Tukey’s test; a 6= b; p <0.05; n = 8). alterations) in comparison with the CMTG (Figure 3H). The movement (Figure 4B). It is important to emphasize that in the percentage of spermatozoa with head morphological alterations animals that received vitamin only (B G), the percentage of fast (without typical curvature or isolated—Figures 3E–G) was progressive spermatozoa was higher whereas the percentage of similar among the groups (Figure 3H). non-progressive spermatozoa was lower in comparison with the The percentage of motile spermatozoa in CMTG decreased other groups (Figure 4B). significantly in comparison with the other groups (Figure 4A). According to the quality of movement, a significant reduction in Mitochondrial Activity the percentage of spermatozoa with fast progressive movement In CMTG, while the percentage of Class I spermatozoa was observed in CMTG, when compared with the other groups (Figure 5A) was low, Class II spermatozoa (Figure 5B) was (Figure 4B). On the other hand, the animals from CMT/B G 12 higher than the other groups (Figure 5E), resulting in a low showed a significant increase in the percentage of spermatozoa rate of cytochemical activity (RCA) (Figure 5F). Otherwise, with fast progressive movement in comparison with CMTG, and CMT/B G showed a significant improvement of this qualitative decrease in the percentage of spermatozoa with slow progressive parameter in comparison with CMTG (Figures 5E,F). There was Frontiers in Endocrinology | www.frontiersin.org 8 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 4 | Percentage of motile spermatozoa (A) and quality of sperm movement (B) in animals from CG, B G, CMTG and CMT/B G. Values expressed as mean 12 12 ± SD. (A) One-way ANOVA followed by Tukey’s test (a 6= b; p < 0.05; n = 8). (B) Two-way ANOVA followed by Tukey’s test (a 6= b 6= c; p < 0.05; n = 8). no statistical difference among the groups in the percentage of Comet Assay Classes III and IV spermatozoa (Figures 5C–E). As shown in Table 1, the parameters evaluated by Comet assay (percentage of fragmented DNA in tail, tail length, tail moment and Olive moment) were significantly elevated in the Acrosome Integrity spermatozoa of the animals from CMTG when compared with According to the analysis of acrosome integrity (intact or the control and B groups. However, the supplementation of damaged), the animals from the CMTG showed a higher number the treated animals with vitamin B decreased significantly the of spermatozoa with damaged acrosome in comparison with percentage of fragmented DNA in tail when compared with the other groups. However, in CMT/B G, the percentage of CMTG. No statistical difference was detected among CG, B G spermatozoa with intact acrosome was significantly higher than and CMT/B G. CMTG (Figures 6A–C). Lipid Peroxidation Analysis (TBARS Test) Fertility Evaluation No significant difference was detected in the levels of MDA either The treatment with cimetidine did not alter the parameters of in the testicular homogenate or in the epididymal sperm among reproductive outcome (number of corpora lutea, implantation, the groups (Figures 7A,B). resorptions, fetuses, and weights of placenta and fetuses) in Frontiers in Endocrinology | www.frontiersin.org 9 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 5 | (A–D) Classification of the mitochondrial activity. (A) Class I spermatozoon, when the mid-piece was totally stained; (B) class II spermatozoon, when most of the mid-piece was stained; (C) class III spermatozoon, when a part of mid-piece was stained; and (D) class IV spermatozoon, when the mid-piece was not stained. Bars: 7 μm In (E), number of spermatozoa per classes of mitochondrial activity (Values expressed as mean ± SD. Two-way ANOVA followed by Tukey’s test; a 6= b 6= c; p < 0.05; n = 6). In (F), rate of cytochemical activity (RCA) (Values expressed as mean ± SD. One-way ANOVA followed by Tukey’s test; a 6= b; p < 0.05; n = 6). Frontiers in Endocrinology | www.frontiersin.org 10 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter with low or absent motility (45), and indicate failure in the spermiogenic process. Moreover, alterations in sperm motility may be related to a reduction in the expression of exclusive genes of spermatids, involved in the development of flagellar structures, such as the mitochondrial sheath of mid-piece, and required for the glycolytic enzymes function (46). Thus, the reduction of motile spermatozoa caused by cimetidine is related to the structural flagellar changes and low mitochondrial activity. Decrease in sperm motility has also been reported in rats treated with the antiandrogenic flutamide (47). Cimetidine exerts antiandrogenic action, competing with testosterone for the androgen receptor (AR) in prostate and kidney cells (48, 49). In the epididymis, this drug reduces the stromal SHBG levels and impairs the epithelial AR nuclear translocation (8). In the present study, serum testosterone levels reduced significantly in CMTG, confirming previous findings (7, 8). Since testosterone is essential for the completion of spermiogenesis (50), the deficient androgenization induced by cimetidine may be the main cause of the flagellar abnormalities and reduction of motile spermatozoa (Figure 8). This hypothesis is reinforced by the presence of acrosomal changes in CMTG, since the acrosome formation is also an androgen-dependent process (50). It is important to emphasize that the rats received cimetidine for 52 days, but the period of spermatogenesis and the transit time taken from the testis to the cauda epididymis lasts around 65 days; thus, the spermatozoa evaluated in this study (from epididymis) were exposed to the treatment from the 14th day of spermatogenesis (at pre-leptotene primary spermatocyte stage) (revised by Hermo FIGURE 6 | (A,B) Representative photomicrographs of spermatozoa classified according to the acrosome integrity. In (A), spermatozoa with intact acrosome; et al. (51)). Thus, these cells were exposed to cimetidine along in (B), spermatozoa with damaged acrosome. Bars: 7 μm. (C) Percentage of meiosis I and II (for 23 days) and throughout spermiogenesis spermatozoa with intact acrosome in the animals from CG, B G, CMTG and (for 15 more days), carrying and accumulating the cimetidine- CMT/B G. (Values expressed as mean ± SD. One-way ANOVA followed by induced changes for 38 days, plus the period of transit from the Tukey’s test; a 6= b; p < 0.05; n = 6). testis to cauda epididymis (14 days), totaling 52 days (period of treatment). Therefore, it is not surprising to find abnormal comparison with the other groups. The fertility potential was also spermatozoa in the epididymis in view of the long period by similar among the groups (Table 2). which these germ cells were exposed to this androgen disrupter. Although low levels of ROS (reactive oxygen species) are required for normal sperm function, capacitation, and acrosome DISCUSSION reaction, elevated levels of ROS may induce DNA fragmentation In previous studies, we have demonstrated that cimetidine and apoptosis in the germ cells and spermatozoa (52). In the impairs the testicular structure and steroidogenesis (7, 9– current study, no difference was observed in the levels of MDA 11, 28, 44), causing low testosterone bioavailability and either in the testis or in the epididymal sperm, indicating that epididymal androgenic dysfunction (8). Therefore, we evaluated the cimetidine-induced changes in the spermatozoa are not the impact of this androgen disrupter on sperm quality and due to increased LPO. Besides ROS, several other factors may male fertility, and whether vitamin B supplementation induce DNA fragmentation, such as: deficiency of chromatin is able to prevent the sperm parameters changes induced recombination and packaging during spermatogenesis, aborted by cimetidine. We also tested the hypothesis that the apoptosis of germ cells, variations in the expression pattern spermatogenic improvement induced by this vitamin of spermatic protamine and incomplete chromatin remodeling can be associated with its effect on steroidogenesis. The during spermiogenesis (53). Since androgenic dysfunction leads results showed that cimetidine causes significant changes to chromatin packaging errors during spermiogenesis and loss in the sperm quality. However, testosterone levels and of the protamine levels in the testis and spermatozoa (54), the steroidogenic activity were unchanged in the vitamin B increased levels of DNA damage induced by cimetidine may be supplemented rats, while the sperm quality was improved by the due to deficient testicular androgenization. supplementation (Figure 8). Although the cimetidine-treated animals showed 15% of A significant increase in the flagellum abnormalities, sperm DNA damage, studies have demonstrated that the cut- especially tail looping or bending, was observed in spermatozoa off point that indicate low fertility in men by alkaline Comet of CMTG. These changes have been reported in spermatozoa assay is ≥45% of sperm DNA fragmentation (55). Therefore, Frontiers in Endocrinology | www.frontiersin.org 11 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter FIGURE 7 | Malondialdehyde (MDA) levels in testis (A) and epididymal sperm (B) of rats from CG, B G, CMTG and CMT/B G. (Values expressed as mean ± SD. 12 12 One-way ANOVA. n = 6). TABLE 1 | Analysis of sperm DNA fragmentation assessed by comet assay in rats offspring health. As revised by Spadafora (59), the nucleus from CG, B G, CMTG and CMT/B G (n = 6). 12 12 of epididymal spermatozoa may internalize RNA-containing nanovesicles released from the paternal somatic tissues. The Tail DNA Tail length Tail moment Olive moment RNAs are amplified and the cDNA copies are transmitted to (%) (μm) embryos during fertilization. Thus, not only haploid genomes a a a a CG 7.3 ± 3.1 4.7 ± 2.0 0.39 ± 0.37 0.62 ± 0.34 are delivered from germ cells during fertilization, but also the a a a a B G 7.4 ± 2.2 4.0 ± 0.8 0.32 ± 0.14 0.58 ± 0.21 epigenomes, which can be susceptible to numerous factors, b b b b CMTG 15.6 ± 3.1 7.3 ± 1.2 1.16 ± 0.34 1.26 ± 0.24 including nutritional and harmful substances. Spermatozoa a a,b a a CMT/B G 9.3 ± 0.5 5.9 ± 1.2 0.57 ± 0.12 0.84 ± 0.08 carrying drug-induced DNA damage may cause aberrant epigenetic programing in early embryos and contribute to Values expressed as mean ± SD (One-way ANOVA followed by Tukey’s test). instabilities later in development (35, 60). Considering that The different superscript letters mean differences among the groups: a 6= b; (p < 0.05). cimetidine-treated rats presented a significant decrease in DNA integrity assessed by the Comet assay, it is recommended that further investigations are carried out on the epigenetic stability decreased semen quality is not necessarily associated with and the future consequences to the progeny fathered following impaired fertility (1). In the present study, no significant cimetidine exposure. reduction in embryo implantation rate or embryo loss was Vitamin B exerts a potential role in spermatogenesis (12, noted, confirming that cimetidine treatment was not able to 12 21–23), and sperm parameters in humans (61) and animals impair the fertility potential. According to Zenick and Clegg, (12, 20, 21, 62), and has been a therapeutic agent used for (56), the production of spermatozoa in rodents far exceeds the the treatment of male infertility, especially oligozoospermia necessary quantity for fertility, and spermatogenesis must be or asthenozoospermia (63). In previous studies, we have decreased by as much as 90% in rodents so that the progeny is demonstrated beneficial effects of vitamin B on the sperm affected. Moreover, rats submitted to androgen deprivation after 12 concentration and spermatogenesis of cimetidine-treated rats complete destruction and removal of Leydig cells by using ethane (11, 12). A similar effect has also been demonstrated in the dimethane sulphonate (EDS) have maintained normal sperm seminiferous epithelium of animals receiving co-administration output and fertility for 8–10 weeks (57). In a previous study, we of the antineoplastic busulphan and vitamin B (22, 23). verified a significant reduction (45%) in the sperm concentration 12 However, a preventive effect of vitamin on the sperm exposed to of cimetidine-treated rats; thus, the remaining concentration these drugs has not yet been reported. in these animals was about 60 million of spermatozoa/mL In this study, the percentage of motile spermatozoa, the (12), whose 75% are motile and 60% are morphologically mitochondrial activity, the percentage of sperm with normal normal, according to the present study, indicating that these morphology, intact acrosomes, and normal DNA integrity cells were able to fertilize the oocytes. Even if the fertilizing was higher in the animals from CMT/B G than in CMTG. spermatozoon carries some DNA damage, it is known that 12 The improvement in the acrosome, sperm tail morphology the oocyte is able to repair, to some extent, the damage after and motility indicates a beneficial effect of this vitamin fertilization (58). on spermiogenesis (Figure 8). A protective effect of vitamin In spite of fertility potential was not affected by the treatment, B on the integrity of the sperm membrane, acrosome sperm quality was significantly impaired by cimetidine treatment. (62) and motility (64, 65) has also been demonstrated in Therefore, we cannot exclude a possible impact of the treatment, other studies. via paternal transgenerational inheritance, on the long term Frontiers in Endocrinology | www.frontiersin.org 12 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter TABLE 2 | Fertility parameters of the females (n = 12) naturally mated with male rats (n = 6) from CG, B G, CMTG and CMT/B G. 12 12 Parameters CG B G CMTG CMT/B G 12 12 (n = 12) (n = 12) (n = 12) (n = 12) Fertility potential (%) 96.65 (91.85–100.0) 96.45 (91.7–100.0) 87.5 (79.28–92.9) 93.8 (85.13–100.0) Pre-implantation loss (%) 3.35 (0.0–8.15) 3.55 (0.0–8.3) 12.5 (7.1–20.75) 6.3 (0.0–14.88) Post-implantation loss (%) 0.0 (0.0–0.0) 0.0 (0.0–8.5) 7.4 (0.0–13.0) 2.95 (0.0–7.1) Corpora lutea number 13.8 ± 1.4 13.4 ± 1.6 13.9 ± 1.7 14.8 ± 1.7 Implantation number 13.2 ± 1.2 12.8 ± 1.7 12.0 ± 2.0 13.3 ± 2.8 Resorption number 0.0 (0.0–0.0) 0.0 (0.0–1.0) 1.0 (0.0–1.75) 0.5 (0.0–1.0) Number of live fetuses 12.9 ± 1.0 12.3 ± 1.6 11.2 ± 1.6 12.2 ± 2.9 Fetus weight (g) 2.29 ± 0.16 2.53 ± 0.41 2.63 ± 0.41 2.44 ± 0.19 Placental weight (g) 0.50 ± 0.07 0.49 ± 0.09 0.56 ± 0.05 0.54 ± 0.06 Values expressed as median and interquartile intervals (Kruskal-Wallis test). Values expressed as mean ± SD (One-way ANOVA). FIGURE 8 | Representative scheme of the mechanism of action of cimetidine (A) and the preventive effect of vitamin B supplementation during the treatment (B) in the testes and sperm quality. In (A), cimetidine impairs LC steroidogenic activity, reducing 17β-HSD6 and the serum T levels. The low androgenic supply to the seminiferous epithelium impairs spermiogenesis and, subsequently, sperm quality, including: morphology, motility, mitochondrial activity as well as acrosome and DNA integrity. In (B), the supplementation of cimetidine-treated animals with vitamin B is able to prevent the harmful effect of cimetidine on the LC, maintaining the normal T levels, tubular androgenization and spermatogenic process (spermiogenesis), avoiding damage to sperm parameters. In CMT/B G, testosterone levels were similar to CG, activity as well as the serum testosterone at normal levels in and this result was consonant with the strong 17β-HSD6 rats following treatment with an androgen disrupter (Figure 8). immunofluorescence in the Leydig cells and the high levels Further studies are necessary to clarify the mechanism by which of this enzyme in the testicular extracts in the supplemented this vitamin maintains steroidogenesis. animals, confirming the preventive effect of vitamin B against the antiandrogenic effect of cimetidine. Adult Leydig cells express CONCLUSION high levels of a transmembrane protein amnionless (66, 67). This protein participates in the vitamin B transport and absorption Although the fertility potential was not impaired by cimetidine in testes, suggesting that the steroidogenic activity seems to in rodents, this drug induced significant sperm quality changes, depend on the transport of vitamin B in Leydig cells (66). including DNA fragmentation. Thus, considering a possible Except for a study in which rats fed with diet containing high paternal epigenetic and transgenerational inheritance, further levels of vitamin B showed high serum testosterone levels 12 studies focusing on the impact of cimetidine on the long term (68), studies showing preventive effect of vitamin B against offspring health are necessary. The concomitant beneficial effect androgenic disruption are inexistent in the literature. Our results of vitamin B either in the androgenic maintenance or in showed that vitamin B is able to maintain the steroidogenic the sperm parameters confirms that the cimetidine-induced Frontiers in Endocrinology | www.frontiersin.org 13 July 2019 | Volume 10 | Article 309 Beltrame et al. Vitamin B Effect Against Androgen Disrupter androgenic failure is the main cause of spermatic changes, and in fertility evaluation. FdS carried out the mitochondrial points to a potential preventive role of vitamin B on the Leydig activity analysis and western blot. FB and PC carried out the cell steroidogenesis. Therefore, vitamin B may be a potential immunohistochemistry reaction. VV, SM, and PC contributed therapeutic agent for the maintenance of sperm quality and the in the critical analysis of results, statistical analysis and in androgenic status of patients exposed to androgen disrupters. the revision of the manuscript. FB and ES-C examined and selected the images and participated in the manuscript design. All the authors read and approved the final version of ETHICS STATEMENT the manuscript. The protocol regarding the treatment of the animals used in this study was approved by the Ethical Committee for Animal FUNDING Research of São Paulo Federal University-UNIFESP/EPM, Brazil (CEUA n 7950060514) and by Ethics Committee of Dental This work was supported by São Paulo Research Foundation- School—São Paulo State University (UNESP), Araraquara/SP, FAPESP (grant numbers: 2012/23845-3; 2013/25322-0; Brazil (CEUA n 28/2014). 2019/11525-3) and CNPq. AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS ES-C coordinated the study. SM and VV contributed to the We thank Luis Antônio Potenza and Pedro Sérgio Simões for methodological design of the experiment. FB carried out the technical assistance; the Laboratory of Experimental Pathology treatment of animals. FB, FdS, and ES-C collected and carried and Biomaterials of Dental School-São Paulo State University out the histological processing. RC cooperated in the processing (FOAr/UNESP) for the assistance in spectrophotometric of sperm samples. FB carried out the sperm analysis (motility analysis; and Sérgio Valladão, from São Lucas Clinical and morphology, acrosome integrity, and comet assay), TBA and Microbiological Laboratory (Araraquara, SP), for the hormonal measurement and the assistance in the sperm test, fertility evaluation and immunofluorescence reaction. VV cooperated in comet assay. FdS, ES-C, and VV cooperated motility analysis. REFERENCES 11. Beltrame FL, Caneguim BH, Miraglia SM, Cerri PS, Sasso-Cerri E. Vitamin B12 supplement exerts a beneficial effect on the seminiferous 1. Drobnis EZ, Nangia AK. Male reproductive functions disrupted epithelium of cimetidine-treated rats. Cells Tissues Organs. (2011) 193:184–94. by pharmacological agents. Adv Exp Med Biol. 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