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. 2019 Jul 10;10:309.
doi: 10.3389/fendo.2019.00309. eCollection 2019.

Vitamin B 12 Prevents Cimetidine-Induced Androgenic Failure and Damage to Sperm Quality in Rats

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Free PMC article

Vitamin B 12 Prevents Cimetidine-Induced Androgenic Failure and Damage to Sperm Quality in Rats

Flávia Luciana Beltrame et al. Front Endocrinol (Lausanne). .
Free PMC article

Abstract

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 B12 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 B12 is able to prevent the harmful effect of this drug on steroidogenesis and sperm parameters. Adult male rats were treated for 52 consecutive days as follows: cimetidine group (100 mg/kg of cimetidine), cimetidine/vitamin B12 group (100 mg/kg of cimetidine + 3 μg vitamin B12), vitamin B12 group (3 μg vitamin B12) and control group (saline). Serum testosterone levels and immunofluorescence associated to western blot for detection of 17β-HSD6 were performed. Sperm morphology and motility, mitochondrial activity, acrosome integrity, DNA integrity by Comet assay, lipid peroxidation as well as fertility potential were analyzed in all groups. Apoptotic spermatids were also evaluated by caspase-3 immunohistochemistry. In the cimetidine-treated animals, reduced serum testosterone levels, weak 17β-HSD6 levels and impaired spermiogenesis were observed. Low sperm motility and mitochondrial activity were associated with high percentage of sperm tail abnormalities, and the percentage of spermatozoa with damaged acrosome and DNA fragmentation increased. MDA levels were normal in all groups, indicating that the cimetidine-induced changes are associated to androgenic failure. In conclusion, despite the fertility potential of rats was unaffected by the treatment, the sperm quality was significantly impaired. Therefore, considering a possible sperm-mediated transgenerational inheritance, the long term offspring health needs to be investigated. The administration of vitamin B12 to male rats prevents the androgenic failure and counteracts the damage inflicted by cimetidine upon sperm quality, indicating that this vitamin may be used as a therapeutic agent to maintain the androgenic status and the sperm quality in patients exposed to androgen disrupters.

Keywords: DNA damage; androgenic dysfunction; cimetidine; sperm quality; spermiogenesis; vitamin B12.

Figures

Figure 1
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 B12. 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).
Figure 2
Figure 2
(A–D) Photomicrographs of testicular sections submitted to immunofluorescence for detection of 17β-HSD6. In CG, B12G and CMT/B12G (A,B,D), the 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 ≠ b; p < 0.05; n = 8). In (F), the 17β-HSD6 immunofluorescent area per μm2 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 ≠ 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, B12G and CMT/B12G while a weak band is detected in CMTG. Actin signal is observed in all groups. 17β-HSD6 optical density (OD) shows high 17β-HSD6 levels in the CG, B12G and CMT/B12G and low 17β-HSD6 levels in the CMTG (Values expressed as mean ± SD. One-way ANOVA followed by Tukey's test; a ≠ b; p < 0.05; n = 3).
Figure 3
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 ≠ b; p < 0.05; n = 8).
Figure 4
Figure 4
Percentage of motile spermatozoa (A) and quality of sperm movement (B) in animals from CG, B12G, CMTG and CMT/B12G. Values expressed as mean ± SD. (A) One-way ANOVA followed by Tukey's test (a ≠ b; p < 0.05; n = 8). (B) Two-way ANOVA followed by Tukey's test (a ≠ b ≠ c; p < 0.05; n = 8).
Figure 5
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 ≠ b ≠ 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 ≠ b; p < 0.05; n = 6).
Figure 6
Figure 6
(A,B) Representative photomicrographs of spermatozoa classified according to the acrosome integrity. In (A), spermatozoa with intact acrosome; in (B), spermatozoa with damaged acrosome. Bars: 7 μm. (C) Percentage of spermatozoa with intact acrosome in the animals from CG, B12G, CMTG and CMT/B12G. (Values expressed as mean ± SD. One-way ANOVA followed by Tukey's test; a ≠ b; p < 0.05; n = 6).
Figure 7
Figure 7
Malondialdehyde (MDA) levels in testis (A) and epididymal sperm (B) of rats from CG, B12G, CMTG and CMT/B12G. (Values expressed as mean ± SD. One-way ANOVA. n = 6).
Figure 8
Figure 8
Representative scheme of the mechanism of action of cimetidine (A) and the preventive effect of vitamin B12 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 B12 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.

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