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Resveratrol ameliorates bisphenol A-induced testicular toxicity in adult male rats: a stereological and functional study

Resveratrol ameliorates bisphenol A-induced testicular toxicity in adult male rats: a... Background: Bisphenol A (BPA) is one of the most widely used synthetic chemicals worldwide. BPA as an endocrine disruptor affects the reproductive systems through estrogenic and antiandrogenic proprieties. Resveratrol (RES) as a natural polyphenol and potent antioxidant exhibits protective effects against reproductive toxicity by inhibiting of oxidative stress. 48 male rats were divided into eight groups (n=6), including CONTROL, OLIVE OIL (0.5 ml/ day), Car‑ boxy methylcellulose (CMC) (1 ml of 10 g/l), RES (100mg/kg/day), low dose of BPA (25 mg/kg/day), high dose of BPA (50 mg/kg/day), low dose of BPA + RES, and high dose of BPA + RES. All treatments were done orally per day for 56 days. At the end of the 8th week, blood samples were collected for hormone assays. Then, the sperm parameters were analyzed, and the left testis was removed for stereological study. Results: We showed a significant decrease in sperm parameters in the low and high doses of BPA groups compared to control groups (P<0.05). The volume of testicular components as well as the diameter and length of seminifer‑ ous tubules significantly reduced (11‑64 %), and the total number of the testicular cell types decreased (34‑67 %) on average in the low and high doses of BPA groups. Moreover, serum follicle‑stimulating hormone (FSH), luteinizing hormone (LH), and testosterone hormones concentration showed a significant reduction in both doses of BPA groups (P<0.01). Nonetheless, treatment with RES could ameliorate all the above‑mentioned changes in the low and high doses of BPA groups (P<0.05). Conclusions: RES could prevent BPA‑induced testicular structural changes and sperm quality via improving gonado ‑ tropin hormones and testosterone levels. Keywords: Bisphenol A, Resveratrol, Testicular toxicity, Sperm parameters, Stereology *Correspondence: naseh@sums.ac.ir Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Zand Ave., Shiraz 71348‑45794, Iran Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 2 of 12 Rèsumè Contexte: Le bisphénol A (BPA) est l’un des produits chimiques synthétiques les plus utilisés dans le monde. Le BPA en tant que perturbateur endocrinien affecte le système reproducteur par le biais de ses propriétés œstrogéniques et anti‑androgènes. Le resvératrol (RES), en tant que polyphénol naturel et puissant antioxydant, présente des effets protecteurs contre la toxicité sur la reproduction en inhibant le stress oxydatif. Quarante‑huit rats mâles ont été divisés en huit groupes (n = 6), comprenant les groupes TÉMOIN, HUILE D’OLIVE (0,5 ml/jour), méthylcellulose Carboxyle (MCC) (1 ml de 10 g/L), RES (100 mg/kg/ jour), faible dose de 25 de BPA (25 mg/kg/jour), dose élevée de BPA (50 mg/ kg/jour), faible dose de BPA + RES et dose élevée de BPA + RES. Tous les traitements ont été effectués quotidienne ‑ ment par voie orale pendant 56 jours. À la fin de la 8ème semaine, des échantillons de sang ont été prélevés pour dosages hormonaux. Ensuite, les paramètres du sperme ont été analysés et le testicule gauche a été retiré pour une étude stéréologique. Résultats: Nous avons montré une diminution significative des paramètres spermatiques dans les groupes traités par doses faibles et doses élevées de BPA par rapport aux groupe témoin (P<0,05). Le volume des composants testicu‑ laires ainsi que le diamètre et la longueur des tubules séminifères ont été considérablement réduits (11‑64 %) ; le nombre total des types de cellules testiculaires a diminué (34‑67 %) en moyenne dans les groupes traités par doses faibles et doses élevées de BPA. De plus, la concentration sérique d’hormone folliculostimulante (FSH), lutéinisante (LH) et de testostérone a montré une réduction significative dans les groupes traités quelle que soit la dose de BPA (P<0,01). Néanmoins, le traitement par RES pourrait améliorer tous les changements mentionnés ci‑ dessus dans les groupes traités par doses faibles et élevées de BPA (P<0,05). Conclusions: Le RES pourrait avoir un effet positif sur les changements structurels testiculaires induits par le BPA, ainsi que la qualité du sperme, en améliorant les taux sériques d’hormones gonadotrophines et de testostérone. Mots‑clés: Bisphénol AResvératrolToxicité testiculaireParamètres du SpermeStéréologie In this regard, several animal studies have also con- Background firmed the reproductive toxicity of BPA in rats and mice Bisphenol A (BPA) is one of the most widely used syn- [14–16]. It has been demonstrated that BPA decreases thetic chemicals worldwide. It is found in large amount of testis weight, reduces diameter and thickness of semi- consumer products such as polycarbonate plastics, epoxy niferous tubules and leads to compromised spermato- resins, linings of cans, medical devices, dental sealants, genesis. These morphological alterations and abnormal and many other products that are part of our daily lives spermatogenesis seem to be induced by the reduction [1–3]. Public health has raised concerns about the wide- of reproductive hormone production and promotion of spread applications and toxic effects of BPA [4 ]. Exposure germ cell apoptosis [17, 18]. On the other hand, exposure of BPA can occur directly or indirectly through inhalation, to BPA is related to the reduced activity of antioxidant dermal exposure and ingestion [5, 6]. It has been reported enzymes, which could contribute to oxidative stress and that the main rout of exposure in humans is oral, which sperm damage [19, 20]. accounts about 90% of BPA exposures. It has been shown Resveratrol (RES; trans-3,5,4’-trihidroxy-trans-stilbene), that BPA contributes to the cause of several endocrine as a natural polyphenol and potent antioxidant is found disorders including reproductive dysfunction, infertility, in a wide range of foods, especially grapes, berries, and precocious puberty and hormone dependent tumors [7, peanuts [21]. Several reports have demonstrated that RES 8]. Evidences suggest that BPA exerts the toxic effects on exhibits the protective effects against reproductive toxicity the reproductive system via different mechanisms. BPA by suppressing lipid peroxidation [22, 23]. Moreover, RES as an endocrine disruptor seems to mediate reproductive may improve sperm count and motility, as well as decrease failure through estrogenic and antiandrogenic proprieties germ cell apoptosis by stimulating the hypothalamic–pitu- [9]. BPA can interfere with estrogenic signaling pathways itary–gonad axis and enhancing blood testosterone levels by interacting with estrogen receptors (ERs), or by pro- [24]. Accordingly, for the first time this study was designed ducing a small but potent estrogenic metabolite [10]. BPA to evaluate the protective effects of RES against deleterious can also bind to the androgen receptor (AR) as an antago- effects of low (25 mg/kg/day) and high doses (50 mg/kg/ nist [11], which can disrupt the hypothalamic-pituitary- day) of BPA on the structure and function of testis using testicular axis, thereby affecting gene expression and the stereological assessment, hormonal measurements, and enzymatic activity of testicular steroidogenesis, leading to quantitative-qualitative study of sperm parameters. hypogonadotropic hypogonadism [12, 13]. B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 3 of 12 group received high dose of BPA (50 mg/kg/day) [28] for Materials and methods 56 days, BPA was diluted in olive oil and administered Animals daily orally at a dosing volume of 0.5 ml. BPA-LOW + Forty eight male Sprague-Dawley rats (age, 6–8 weeks RES group received orally with low dose of BPA plus old; weight, 180-210 g) were purchased from the Animal RES (100mg/kg/day) for 56 days, and BPA-HIGH + RES Laboratory Center of Shiraz University of Medical Sci- group received high dose of BPA plus RES (100mg/kg/ ences. The animals were kept under standard conditions day) orally for 56 days (Fig. 1). at room temperature (22 ± 2 °C), with normal humidity It should be noted that the dosages of BPA (CAS 80-05- and 12–12 h light-dark cycles. They also had free access 7, Sigma–Aldrich Co., St. Louis, USA) used in current to standard food and water. All animal experiments car- study were based on the previously reported as maxi- ried out in accordance with the National Institutes of mum permissible dose that have no observable side effect Health guide for the care and use of Laboratory animals on reproductive and developmental toxicity (50 mg/kg (NIH Publications No. 8023, revised 1978). Also, the BW/day) in rats [13, 29]. animal procedures were performed under the standard rules established by the Animal Care and Ethics Commit- Hormone measurements tee of Shiraz University of Medical Sciences (IR.SUMS. At the end of the 8th week (on day 56), fasted rats were REC.1398.392). killed by cervical dislocation and blood samples were collected from the heart through a cardiac puncture and Experimental design stored in heparin-free tubes. Then, the samples were cen - The rats were randomly divided into eight groups (n=6); trifuged at 3500 rpm for 15 min. The serum was obtained CONTROL group received distilled water orally per and stored at -70 °C for subsequent hormone evaluation. day for 56 days (spermatogenesis length), OLIVE OIL The serum levels of follicle-stimulating hormone (FSH; group received 0.5 ml/day Olive oil orally for 56 days, Category No. CK-30597), luteinizing hormone (LH; Cat- Carboxy methylcellulose (CMC) group received 1 ml of egory No. CK-E90904, and testosterone concentrations 10 g/l CMC orally [25] per day for 56 days, RES group (Category No. E90243) were determined by rat ELISA received 100 mg/kg/day RES that was diluted in CMC kits (From East. Bio Pharm Company) using a microplate and administered orally at a dosing volume of 1 ml [26, reader (Biotek, USA). Briefly, 100 μL of standard or sam- 27] for 56 days, BPA-LOW group received low dose ple was pipetted to each well and incubated for 2 hours at of BPA (25 mg/kg/day) orally for 56 days, BPA-HIGH Fig. 1 Flow chart of the experimental design Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 4 of 12 37 °C. After removing any unbound substances, 100 μL of degree of shrinkage “d (shr)” was calculated by the fol- anti-biotin antibodies was added to the wells. After wash- lowing formula: ing, 100 μL of avidin conjugated Horseradish Peroxidase (HRP) was added to each well and incubated for 1 hour 1.5 d(shr) = 1 −[Area(after)/Area(before)] at 37 °C. Then, 90 μL of 3,3’5,5’-Tetramethylbenzidine (TMB) substrate was added to each well and incubated Then, the total volume of the testis was evaluated with for 20 minutes at 37 °C. Finally, the color development regard to tissue shrinkage [V(shrunk)] using the follow- was stopped and the absorbance was determined at ing formula: 450 nm using a microplate reader. V(shrunken) = V(unshrunk) ×[1 − d(shr)] Spermatozoa counts, morphology and motility Immediately after blood collection, the proximal part of Estimation of the testicular components volume the vas deferens just distal to the cauda epididymis (10 The volume density of the testis sections was analyzed mm) was removed, and moved to a petri dish containing by a video microscopy system. In doing so, the point 3 mL normal saline solution. The suspension was gen - grid was superimposed on the microscopic images of tly shaken at 37°C for 5-10 min to diffuse the sperma - the H&E-stained sections (5μm thickness) on a moni- tozoa. The samples were counted in a hemocytometer. tor by the software designed at the Histomorphometry Ten fields were then randomly selected and evaluated and Stereology Research Center. The volume density “Vv for motility grading to distinguish the immotile sperms (structure/testis)” of the testicular components, includ- from those with progressive or non-progressive motility. ing seminiferous tubules, interstitial tissue, and germinal Also, the sperm smears were stained with 1% eosin Y for epithelium, was estimated by the point counting method assessing the morphology [30]. [33, 34]. Finally, the total volume of each component was There is two types of progressive motility: 1- rapid pro - obtained by the following formula: gressive motility, 2- slow progressive motility. The effi - cient passage of spermatozoa through cervical mucus is dependent on rapid progressive motility. V(structure) = Vv(structure/testis) × V(shrunk) We should add that it is necessary to distinguish between these two types of progressive motility. So Estimation of the length and diameter of seminiferous that neglecting the distinction between two progressive tubules sperm groups leads to ignoring the information in the The length density (Lv) of the seminiferous tubules was semen sample, and the removal of such useful informa- measured on the sampled tubules in an unbiased counting tion would impoverish the semen analysis [31]. frame applied on the 5 μm thick sections (H&E staining) [35], and calculated by the following formula: Stereological study The left testis was removed and weighed. Then, according Lv = 2�Q/[�P × (a/f)] to the immersion method, it was immersed in isotonic saline-filled jar for measuring the primary volume “V Where “ΣQ” is the total number of the selected tubules, (testicle)” [32]. Afterwards, the samples were fixed in 4% “ΣP” represents the total points superimposed on the buffered formaldehyde solution for stereological studies. testis, and “a/f” indicates the area of the counting frame. The orientator method was applied to obtain Isotropic The total length of the seminiferous tubules “L(tubules)” Uniform Random (IUR) sections [32]. About 8-12 slabs was calculated by multiplying the lengths density (Lv) by in each testis were collected through this procedure. To V(structure) [36]. estimate the shrinkage, a circle was punched out from a random testis slab by a trocar (diameter 5 mm), and the trocar radius was considered as the “area (before)” (πr2). L(tubules) = Lv × V(structure) After tissue processing, the area was calculated as the “area (after)”. After tissue processing and paraffin embed - The diameter of the seminiferous tubules was also ding, 5 and 25 μm sections were cut by the microtome measured on the sampled tubules in the counting frame. and were stained using Hematoxylin-Eosin (H&E). The The diameter was measured perpendicularly to the long areas of the circles were measured before processing axis of the tubules where the tubules were widest [35]. An (unshrunk) and after processing (shrunk) and finally, the average of 100 tubules were counted per testis. B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 5 of 12 Estimation of number of testicular cell types Statistical analysis A computer linked to a light microscope (Nikon E200, The data were expressed as mean ± standard error Japan) with 40× oil lens (NA=1.4) was used to assess the (SEM). The results were analyzed by one-way analysis total number of testicular cell types, including spermato- of variance (ANOVA) and Tukey’s post hoc test using gonia (A and B), spermatocytes, round spermatids (steps Graph Pad Prism 6 software (San Diego, CA, USA). 1–8 spermiogenesis), long spermatids (steps 9–16 sper- P<0.05 was considered to be statistically significant. miogenesis), Sertoli and Leydig cells. The total number of the testicular cell types was cal - Results culated using the optical disector method applied on the Spermatozoa count, normal morphology and motility H&E-stained sections (25μm thickness) [37]. In so doing, According to Table 1, a significant decrease was observed the microscopic fields were scanned by moving the micro - in the count, percentage of normal morphology, and scope stage at equal distances in X and Y directions based motility of spermatozoa in the rats exposed to low and on systematic uniform random sampling. The movement high doses of BPA groups compared to control group in Z direction was also performed using a microcator (P<0.05 and P<0.01, respectively). However, these param- (MT12, Heidenhain, Germany) fixed on the microscope eters in the BPA-LOW + RES and BPA-HIGH + RES stage. The Z-axis distribution from the sampled cells in groups improved compared to the BPA groups (P<0.01 different focal planes was plotted to determine the guard and P<0.05, respectively). zones and disector’s height [38]. The numerical density (Nv) was estimated using the following formula: Qualitative changes Qualitative evaluation of the testis has been presented in Fig.  2. The histological sections of the low and high Nv = �Q/(�A × h) × (t/BA) doses of BPA rats showed the structural changes, includ- ing atrophy and reduced number of seminiferous tubules. Where “ΣQ” was the number of each cell type nuclei Concomitant treatment of these groups with RES amelio- coming into focus, “ΣA” indicated the total area of the rated these destructive effects. unbiased counting frame, “h” represented the disector’s height, “t” was the mean section thickness, and “BA” was the microtome block advance. Finally, the total number Stereological assays of the testicular cell types was calculated by multiplying The volume of the testicle the numerical density (Nv) by V(structure): The results showed a significant reduction in the testicle volume by 11.7 % and 13.5 % in the rats exposed to low and high doses of BPA compared to the control groups, N(cells) = Nv × V(structure) respectively (P<0.01 and P<0.001). However, the tes- tis volume recovered considerably in the animals that Where, V(structure) was the total volume of the germi- received BPA-LOW + RES group compared to BPA- nal epithelium for the germinal layer cells and the total LOW group (P<0.01) (Fig. 3A). volume of the interstitial tissue for the Leydig cells. Table 1 Comparison of sperm parameters. Mean ± SEM of the Count (×106), Normal morphology (%), Motility (%), and Immotile (%) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. n = 6 in each group. The results were analyzed by one‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. * P<0.05, ** P<0.01 vs. CONTROL; ## P<0.01 vs. BPA‑LOW; $ P<0.05 vs. BPA‑HIGH Groups Count (×10 ) Normal Motility (%) morphology (%) Rapid Slow Non Immotile progressive progressive progressive CONTROL 6.08±1.8 89.6±5.08 34.6±4.5 20.8±3.2 21.1±2.4 21.3±6.2 OLIVE OIL 6.12±2.3 85.6±3.18 35.4±1.6 21.4±2.3 21.1±6.2 21.1±6.4 CMC 5.8±3.2 81.6±2.5 32.0±2.41 23.0±2.6 23.4±5.5 22.3±1.9 RES 6.52±2.2 91.5±3.05 36.7±6.4 22.0±3.8 21.6±7.7 19.7±4.2 BPA‑LOW 3.21±1.08* 54.9±10* 28.2±2.9* 24.4±3.1* 25.1±3.3* 26.3±2.7* BPA‑HIGH 2.63±1.4** 30.9±10** 23.2±2.5** 29.6±2.4** 30.2±2.3** 32.7±2.5** ## ## ## ## ## BPA‑LOW+RES 5.22±.99 67.7±15.2 33.5±1.6 26.4±2.6 24.4±5.5 26.7±1.9 $ $ $ $ $ BPA‑HIGH+RES 3.76± 1.9 44.5±8.2 29.1±2.7 25.3±2.1 26.4±3 27.2±1.2 Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 6 of 12 Fig. 2 Testicular histological evaluation. Representative photomicrographs of testis sections stained with hematoxylin & eosin (H&E) in the CONTROL (A), low dose of Bisphenol A (BPA‑LOW ) (B), high dose of BPA (BPA‑HIGH) (C), BPA‑LOW + resveratrol (RES) (D), and BPA‑HIGH + RES (E) groups. All plates are to the same scale (Scale bar = 200 μm). The images indicate the normal seminiferous tubules (asterisk), and atrophied seminiferous tubules (arrow) Fig. 3 The evaluation of volume. The box plots represents the volume of the testis (A), germinal epithelium (B), interstitial tissue (C), and seminiferous tubules (D) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. n = 6 in each group. The results were analyzed by one ‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. BPA‑LOW; $p < 0.05 and $$$p < 0.001 vs. BPA‑HIGH B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 7 of 12 The volume of germinal epithelium (P<0.01 and P<0.001, respectively). Nonetheless, the The total epithelial volume in rats treated with low and tubules length significantly was improved in the rats high doses of BPA decreased 43% and 64% in comparison treated with RES in the low and high doses of BPA groups to the control groups, respectively (P<0.001). Treatment (P<0.05 and P<0.01, respectively) (Fig. 4A). with RES ameliorated the epithelial volume changes in the low or high doses of BPA groups (P<0.001) (Fig. 3B). Number of spermatogonia A and B The total number of spermatogonia A reduced by 40.03% The volume of interstitial tissue and 55.2%, and spermatogonia B by 51.27% and 70.05% in The results indicated that the interstitial tissue volume the low and high doses of BPA compared to the control reduced 25.3% and 27.3% in the low and high doses of groups, respectively (P<0.001). However, treatment with compared to the control groups, respectively (P<0.01 RES increased these cells in the low and high doses of and P<0.05). However, this parameter significantly was BPA groups (P<0.001) (Fig. 5A and B). increased in the rats treated with RES in the low or high doses of BPA groups (P<0.05) (Fig. 3C). Number of spermatocytes Statistical analysis showed 34.85% and 53% reduction in The volume of seminiferous tubules the number of spermatocytes for both the low and high A significant reduction was seen in the total volume of doses of BPA compared to the control groups (P<0.001). seminiferous tubules by 26.2% and 34% in the low and high Treatment with RES ameliorated these changes in the doses of BPA compared to the control groups, respectively BPA-LOW + RES and BPA-HIGH + RES groups com- (P<0.01 and P<0.001). Nevertheless, seminiferous volume pared to the BPA groups (P<0.01 and P<0.001, respec- significantly was ameliorated in the BPA-LOW + RES and tively) (Fig. 5C). BPA-HIGH + RES groups compared to the BPA groups (P<0.01 and P<0.05, respectively) (Fig. 3D). Number of round and long spermatid The number of round spermatids decreased by 40.76% Diameter of the seminiferous tubules and 66.72%, and long spermatids by 28.7% and 60.35% The diameter of the seminiferous tubules decreased in the low and high doses of BPA, respectively compared 29.7% and 37.3% in rats treated with low and high doses to the control groups (P<0.001). Moreover, ameliorative of BPA compared to the control group (P<0.001). Treat- effects of RES on the number of these cells were seen ment with RES increased this parameter in the low and in rats treated with low and high doses of BPA groups high doses of BPA groups (P<0.01) (Fig. 4A). (P<0.001) (Figs. 5D and 6A). Length of the seminiferous tubules Number of Leyding and Sertoli cells The results showed that length of the seminiferous A significant reduction was seen in the number of Leydig tubules have reduced 20.6% and 29.8% in the low and cells by 45.78% and 62.85%, and Sertoli cells by 32.28% high doses of BPA compared to the control groups and 52.76% in the low and high doses of BPA than those Fig. 4 The evaluation of diameter and length of seminiferous tubules. The box plots shows the diameter (A), and the length (B) of seminiferous tubules in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. n = 6 in each group. The results were analyzed by one ‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. **p < 0.01, and ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01vs. BPA‑LOW; $$p < 0.01 vs. BPA‑HIGH Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 8 of 12 Fig. 5 Evaluation of the germinal cells number. The box plots represents the number of spermatogonia A (A), spermatogonia B (B), spermatocytes (C), and round spermatids (D) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. The results were analyzed by one ‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. BPA‑LOW; $$p < 0.01 and $$$p < 0.001 vs. BPA‑HIGH of the control groups, respectively (P<0.001). Treatment Discussion with RES recovered the number of Leydig cells and Ser- The current study revealed the ameliorative effects of toli cells in the BPA-LOW + RES (P<0.01 and P<0.05, RES on testicular damage induced by BPA in rats. The respectively), and BPA-HIGH + RES (P<0.001 and first part of our findings showed the deleterious effects P<0.01, respectively) groups compared to the BPA groups of two doses of BPA, 25 and 50 mg/kg/day for 8 weeks, (Fig. 6B and C) on sperm quality and structural changes of the testis. The earlier studies showed that 50 mg/kg/day is considered Hormone assays as maximum permissible dose that have no observable The gonadotropins assessment showed a significant side effect on reproductive and developmental toxicity reduction in serum LH and FSH levels in the BPA-LOW [39]. But we found that ingestion of BPA at these dosages (P<0.001 and P<0.01, respectively), and BPA-HIGH had adverse effects on count, morphology, and motil - (P<0.001) groups compared to the control group. Also, ity of spermatozoa. In line with our results, it has been the testosterone concentration of the rats given low or observed a reduction in epididymal sperm motility and high doses of BPA was lower than in the control group count in the rats exposed to BPA at the 10 and 50 mg/ (P<0.001). The RES exposure led to significant increase in kg in a dose dependent manner [40]. Also, it has been the serum LH and testosterone levels in the BPA-LOW demonstrated that BPA at 5 and 25 mg/kg/day reduced (P<0.01 and P<0.001, respectively), and BPA-HIGH sperm production, reserves and transit time through (P<0.05 and P<0.001, respectively) groups, while the the epididymis [13]. Moreover, long-term exposure to serum FSH levels significantly increased only in the BPA- 0.2 mg/kg BPA in rats led to decreased sperm count and HIGH + RES group (P<0.05) (Fig. 7). inhibited spermiation [41]. B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 9 of 12 Fig. 6 Evaluation of long spermatids, Leydig and Sertoli cells number. The box plots represents the number of long spermatids (A), Leydig (B), and Sertoli (C) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. The results were analyzed by one ‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. BPA‑LOW; $$p < 0.01 and $$$p < 0.001 vs. BPA‑HIGH Fig. 7 Serum concentrations of luteinizing hormone (LH), follicle‑stimulating hormone (FSH), and testosterone hormones. The column graphs represent the concentrations of LH (A), FSH (B), and testosterone (C) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. n = 6 in each group. The results were analyzed by one‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. **p < 0.01, and ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01vs. BPA‑LOW; $$p < 0.01 vs. BPA‑HIGH The reduction in sperm count and quality is in accord tubules and testicular abnormalities due to BPA. Loss with decreased stereological parameters. The changes of of the germinal epithelial cells was also seen after expo- structural indices including volume, diameter and length sure to both doses of BPA. A reduction in germinal epi- of seminiferous tubules suggest the atrophy of these thelial volume could be a consequence of decline in the Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 10 of 12 number of germinal cells. The reduction in sperm pro - LH, and testosterone increased in the cisplatin+RES- duction could be related to the disruption of spermato- treated rats compared to cisplatin group, thereby improv- genesis. Jin et  al., 2013 also reported that BPA exposure ing sperm parameters and testicular apoptosis [24]. could decrease sperm count via the reduction in type A Also, they showed that RES enhanced hormonal levels spermatogonial, spermatocytes and spermatids. BPA as well as sperm motility and count compared to control impaired spermatogenesis through suppressing repro- group. But in our study, there was no significant differ - ductive hormones and activating germ cells apoptosis ence between the RES and control groups. The difference mediated by Fas/FasL signaling pathway [42, 43]. Ser- between the results of our study and Shatti’s research toli cells are another type of cells in the seminiferous may be because of different route of administration and tubules, which have a supportive and nutrient function. dose of RES [24]. Since, Sertoli cells can affect the proliferation and differ - Also, another earlier study claimed that RES could entiation of germinal cells, and also help in the process ameliorate negative effects against BPA-induced repro - of spermatogenesis. So, it seems the loss of these sup- ductive toxicity in mice via reducing oxidative stress porting cells could be led to deficiency of supportive [51]. Our results support the contribution of repro- functions in BPA-treated rat, and cause the loss of sper- ductive hormones in the ameliorative effects of RES on matogenic cells. It has been indicated that Sertoli cells BPA-induced testicular toxicity in rats. Meanwhile, a are targets of pituitary-derived FSH and testosterone to reduction of oxidative stress, as shown by other stud- transduce signals into paracrine regulation of spermato- ies [52], may also be involved in RES protective effects, genesis [44–46]. Accordingly, Sertoli cell depletion fol- which required further studies to be confirmed. lowing BPA treatment in present study may be due to a One of the limitations of our study was that the sign- decrease in FSH and testosterone levels. On the other aling pathways that contribute to the amelioration of hand, testosterone secretion is produced in Leydig cells reproductive hormones by effect on the hypothalamic– of testicular interstitium in response to LH [47]. There - pituitary–gonadal axis in the RES treated rats following fore, the lack of LH stimulation in BPA-treated groups BPA, which led to spermatogenesis improvement were could justify the reduction of Leydig cells and interstitial not investigated. tissue atrophy and also the decrease of testosterone pro- duction. Testosterone is an essential hormone to main- tain normal spermatogenesis and prevention of germ cell Conclusion apoptosis in adult rats [48]. So, it is reasonable to assume In conclusion, the present study demonstrated the pro- that the inhibition of reproductive hormones production tective effects of RES against BPA-induced testicular may have contributed to spermatogenesis impairment structural changes and sperm quality via improving induced by BPA. Similarly, BPA could cause defective gonadotropin hormones and testosterone levels. spermatozoa by disruption of the hypothalamic–pitui- tary–gonadal axis, causing a state of hypogonadotropic Abbreviations hypogonadism [13, 42, 49]. Another possible hypothesis BPA: Bisphenol A; RES: Resveratrol; CMC: Carboxy methylcellulose; LH: Lutein‑ may be involved in spermatogenesis dysfunction is the izing hormone; FSH: Follicle‑stimulating hormone; ER: Estrogen receptor; AR: Androgen receptor. effects of BPA-induced oxidative damage. BPA exposure could induce ROS production by reducing the activity of Acknowledgements the antioxidant system [50]. The adverse effects of BPA This article was extracted from the thesis of Seyedeh‑Saeedeh Yahyavi’s, M.Sc. in Anatomy. This work was performed at the Histomorphometry and Stereol‑ on sperm count and quality due to oxidative stress have ogy Research Center and was financially supported by grant No. 97‑01‑21‑ been described by previous studies [40, 49]. 18338 from Shiraz University of Medical Sciences. Hereby, the authors would The second step of our study demonstrated the protec - like to thank Ms. A. Keivanshekouh at the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for improving the use of English in the tive effects of RES against BPA- induced testicular struc - manuscript. tural changes and sperm quality. Our results indicated that the concomitant treatment of the BPA groups with Authors’ contributions H.B: Designing the study, supervising laboratory works and revising the RES for 8 weeks could significantly restore the sperm manuscript. S.Y: Performing laboratory works and collecting the data. A.N. and parameters and prevent testicular atrophy and apoptosis E.A.A: Conceptualization, Methodology, Software. M.N: Analysis of the data, of the testicular cell types. Furthermore, RES enhanced writing and editing the manuscript. The authors read and approved the final manuscript. testosterone, FSH, and LH levels in BPA groups. The improvement of testicular structural and sperm quality Funding seems to be related to increased gonadotropin hormones This work was performed at the Histomorphometry and Stereology Research Center and was financially supported by grant No. 97‑01‑21‑18338 from Shiraz and testosterone levels. Consistent with these findings, University of Medical Sciences. previous reports have also shown that the levels of FSH, B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 11 of 12 Availability of data and materials hypothalamus–pituitary–testis axis and spermatogenesis. Front Endo‑ The datasets used and/or analyzed during the current study are available from crinol. 2014;5:1. https:// doi. org/ 10. 3389/ fendo. 2014. 00001. the corresponding author on reasonable request. 13. Wisniewski P, Romano RM, Kizys MM, Oliveira KC, Kasamatsu T, Giannocco G, et al. Adult exposure to bisphenol A (BPA) in Wistar rats reduces sperm quality with disruption of the hypothalamic–pituitary–testicular axis. Declarations Toxicology. 2015;329:1–9. https:// doi. org/ 10. 1016/j. tox. 2015. 01. 002 Epub 2015 Jan 6. Ethics approval and consent to participate 14. El Ghazzawy IF, Meleis AE, Farghaly EF, Solaiman A. Histological study All experimental procedures in the current study were done in accordance of the possible protective effect of pomegranate juice on bisphenol‑A with the National Institutes of Health guide for the care and use of laboratory induced changes of the caput epididymal epithelium and sperms of animals (NIH Publications No. 8023, revised 1978) and were approved by the adult albino rats. Alexandria J Med. 2011;47:125–37. Medical and Research Ethics Committee of Shiraz University of Medical Sci‑ 15. Tohei A, Suda S, Taya K, Hashimoto T, Kogo H. Bisphenol A inhibits testicu‑ ences, Shiraz, Iran (Approval No. IR.SUMS.REC.1398.392). All procedures were lar functions and increases luteinizing hormone secretion in adult male carried in accordance with the ARRIVE (Animal Research: Reporting in Vivo rats. Exp Biol Med. 2001;226:216–21. https:// doi. org/ 10. 1177/ 15353 70201 Experiments) guidelines. 22600 309. 16. Takahashi O, Oishi S. 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Resveratrol ameliorates bisphenol A-induced testicular toxicity in adult male rats: a stereological and functional study

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Abstract

Background: Bisphenol A (BPA) is one of the most widely used synthetic chemicals worldwide. BPA as an endocrine disruptor affects the reproductive systems through estrogenic and antiandrogenic proprieties. Resveratrol (RES) as a natural polyphenol and potent antioxidant exhibits protective effects against reproductive toxicity by inhibiting of oxidative stress. 48 male rats were divided into eight groups (n=6), including CONTROL, OLIVE OIL (0.5 ml/ day), Car‑ boxy methylcellulose (CMC) (1 ml of 10 g/l), RES (100mg/kg/day), low dose of BPA (25 mg/kg/day), high dose of BPA (50 mg/kg/day), low dose of BPA + RES, and high dose of BPA + RES. All treatments were done orally per day for 56 days. At the end of the 8th week, blood samples were collected for hormone assays. Then, the sperm parameters were analyzed, and the left testis was removed for stereological study. Results: We showed a significant decrease in sperm parameters in the low and high doses of BPA groups compared to control groups (P<0.05). The volume of testicular components as well as the diameter and length of seminifer‑ ous tubules significantly reduced (11‑64 %), and the total number of the testicular cell types decreased (34‑67 %) on average in the low and high doses of BPA groups. Moreover, serum follicle‑stimulating hormone (FSH), luteinizing hormone (LH), and testosterone hormones concentration showed a significant reduction in both doses of BPA groups (P<0.01). Nonetheless, treatment with RES could ameliorate all the above‑mentioned changes in the low and high doses of BPA groups (P<0.05). Conclusions: RES could prevent BPA‑induced testicular structural changes and sperm quality via improving gonado ‑ tropin hormones and testosterone levels. Keywords: Bisphenol A, Resveratrol, Testicular toxicity, Sperm parameters, Stereology *Correspondence: naseh@sums.ac.ir Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Zand Ave., Shiraz 71348‑45794, Iran Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 2 of 12 Rèsumè Contexte: Le bisphénol A (BPA) est l’un des produits chimiques synthétiques les plus utilisés dans le monde. Le BPA en tant que perturbateur endocrinien affecte le système reproducteur par le biais de ses propriétés œstrogéniques et anti‑androgènes. Le resvératrol (RES), en tant que polyphénol naturel et puissant antioxydant, présente des effets protecteurs contre la toxicité sur la reproduction en inhibant le stress oxydatif. Quarante‑huit rats mâles ont été divisés en huit groupes (n = 6), comprenant les groupes TÉMOIN, HUILE D’OLIVE (0,5 ml/jour), méthylcellulose Carboxyle (MCC) (1 ml de 10 g/L), RES (100 mg/kg/ jour), faible dose de 25 de BPA (25 mg/kg/jour), dose élevée de BPA (50 mg/ kg/jour), faible dose de BPA + RES et dose élevée de BPA + RES. Tous les traitements ont été effectués quotidienne ‑ ment par voie orale pendant 56 jours. À la fin de la 8ème semaine, des échantillons de sang ont été prélevés pour dosages hormonaux. Ensuite, les paramètres du sperme ont été analysés et le testicule gauche a été retiré pour une étude stéréologique. Résultats: Nous avons montré une diminution significative des paramètres spermatiques dans les groupes traités par doses faibles et doses élevées de BPA par rapport aux groupe témoin (P<0,05). Le volume des composants testicu‑ laires ainsi que le diamètre et la longueur des tubules séminifères ont été considérablement réduits (11‑64 %) ; le nombre total des types de cellules testiculaires a diminué (34‑67 %) en moyenne dans les groupes traités par doses faibles et doses élevées de BPA. De plus, la concentration sérique d’hormone folliculostimulante (FSH), lutéinisante (LH) et de testostérone a montré une réduction significative dans les groupes traités quelle que soit la dose de BPA (P<0,01). Néanmoins, le traitement par RES pourrait améliorer tous les changements mentionnés ci‑ dessus dans les groupes traités par doses faibles et élevées de BPA (P<0,05). Conclusions: Le RES pourrait avoir un effet positif sur les changements structurels testiculaires induits par le BPA, ainsi que la qualité du sperme, en améliorant les taux sériques d’hormones gonadotrophines et de testostérone. Mots‑clés: Bisphénol AResvératrolToxicité testiculaireParamètres du SpermeStéréologie In this regard, several animal studies have also con- Background firmed the reproductive toxicity of BPA in rats and mice Bisphenol A (BPA) is one of the most widely used syn- [14–16]. It has been demonstrated that BPA decreases thetic chemicals worldwide. It is found in large amount of testis weight, reduces diameter and thickness of semi- consumer products such as polycarbonate plastics, epoxy niferous tubules and leads to compromised spermato- resins, linings of cans, medical devices, dental sealants, genesis. These morphological alterations and abnormal and many other products that are part of our daily lives spermatogenesis seem to be induced by the reduction [1–3]. Public health has raised concerns about the wide- of reproductive hormone production and promotion of spread applications and toxic effects of BPA [4 ]. Exposure germ cell apoptosis [17, 18]. On the other hand, exposure of BPA can occur directly or indirectly through inhalation, to BPA is related to the reduced activity of antioxidant dermal exposure and ingestion [5, 6]. It has been reported enzymes, which could contribute to oxidative stress and that the main rout of exposure in humans is oral, which sperm damage [19, 20]. accounts about 90% of BPA exposures. It has been shown Resveratrol (RES; trans-3,5,4’-trihidroxy-trans-stilbene), that BPA contributes to the cause of several endocrine as a natural polyphenol and potent antioxidant is found disorders including reproductive dysfunction, infertility, in a wide range of foods, especially grapes, berries, and precocious puberty and hormone dependent tumors [7, peanuts [21]. Several reports have demonstrated that RES 8]. Evidences suggest that BPA exerts the toxic effects on exhibits the protective effects against reproductive toxicity the reproductive system via different mechanisms. BPA by suppressing lipid peroxidation [22, 23]. Moreover, RES as an endocrine disruptor seems to mediate reproductive may improve sperm count and motility, as well as decrease failure through estrogenic and antiandrogenic proprieties germ cell apoptosis by stimulating the hypothalamic–pitu- [9]. BPA can interfere with estrogenic signaling pathways itary–gonad axis and enhancing blood testosterone levels by interacting with estrogen receptors (ERs), or by pro- [24]. Accordingly, for the first time this study was designed ducing a small but potent estrogenic metabolite [10]. BPA to evaluate the protective effects of RES against deleterious can also bind to the androgen receptor (AR) as an antago- effects of low (25 mg/kg/day) and high doses (50 mg/kg/ nist [11], which can disrupt the hypothalamic-pituitary- day) of BPA on the structure and function of testis using testicular axis, thereby affecting gene expression and the stereological assessment, hormonal measurements, and enzymatic activity of testicular steroidogenesis, leading to quantitative-qualitative study of sperm parameters. hypogonadotropic hypogonadism [12, 13]. B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 3 of 12 group received high dose of BPA (50 mg/kg/day) [28] for Materials and methods 56 days, BPA was diluted in olive oil and administered Animals daily orally at a dosing volume of 0.5 ml. BPA-LOW + Forty eight male Sprague-Dawley rats (age, 6–8 weeks RES group received orally with low dose of BPA plus old; weight, 180-210 g) were purchased from the Animal RES (100mg/kg/day) for 56 days, and BPA-HIGH + RES Laboratory Center of Shiraz University of Medical Sci- group received high dose of BPA plus RES (100mg/kg/ ences. The animals were kept under standard conditions day) orally for 56 days (Fig. 1). at room temperature (22 ± 2 °C), with normal humidity It should be noted that the dosages of BPA (CAS 80-05- and 12–12 h light-dark cycles. They also had free access 7, Sigma–Aldrich Co., St. Louis, USA) used in current to standard food and water. All animal experiments car- study were based on the previously reported as maxi- ried out in accordance with the National Institutes of mum permissible dose that have no observable side effect Health guide for the care and use of Laboratory animals on reproductive and developmental toxicity (50 mg/kg (NIH Publications No. 8023, revised 1978). Also, the BW/day) in rats [13, 29]. animal procedures were performed under the standard rules established by the Animal Care and Ethics Commit- Hormone measurements tee of Shiraz University of Medical Sciences (IR.SUMS. At the end of the 8th week (on day 56), fasted rats were REC.1398.392). killed by cervical dislocation and blood samples were collected from the heart through a cardiac puncture and Experimental design stored in heparin-free tubes. Then, the samples were cen - The rats were randomly divided into eight groups (n=6); trifuged at 3500 rpm for 15 min. The serum was obtained CONTROL group received distilled water orally per and stored at -70 °C for subsequent hormone evaluation. day for 56 days (spermatogenesis length), OLIVE OIL The serum levels of follicle-stimulating hormone (FSH; group received 0.5 ml/day Olive oil orally for 56 days, Category No. CK-30597), luteinizing hormone (LH; Cat- Carboxy methylcellulose (CMC) group received 1 ml of egory No. CK-E90904, and testosterone concentrations 10 g/l CMC orally [25] per day for 56 days, RES group (Category No. E90243) were determined by rat ELISA received 100 mg/kg/day RES that was diluted in CMC kits (From East. Bio Pharm Company) using a microplate and administered orally at a dosing volume of 1 ml [26, reader (Biotek, USA). Briefly, 100 μL of standard or sam- 27] for 56 days, BPA-LOW group received low dose ple was pipetted to each well and incubated for 2 hours at of BPA (25 mg/kg/day) orally for 56 days, BPA-HIGH Fig. 1 Flow chart of the experimental design Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 4 of 12 37 °C. After removing any unbound substances, 100 μL of degree of shrinkage “d (shr)” was calculated by the fol- anti-biotin antibodies was added to the wells. After wash- lowing formula: ing, 100 μL of avidin conjugated Horseradish Peroxidase (HRP) was added to each well and incubated for 1 hour 1.5 d(shr) = 1 −[Area(after)/Area(before)] at 37 °C. Then, 90 μL of 3,3’5,5’-Tetramethylbenzidine (TMB) substrate was added to each well and incubated Then, the total volume of the testis was evaluated with for 20 minutes at 37 °C. Finally, the color development regard to tissue shrinkage [V(shrunk)] using the follow- was stopped and the absorbance was determined at ing formula: 450 nm using a microplate reader. V(shrunken) = V(unshrunk) ×[1 − d(shr)] Spermatozoa counts, morphology and motility Immediately after blood collection, the proximal part of Estimation of the testicular components volume the vas deferens just distal to the cauda epididymis (10 The volume density of the testis sections was analyzed mm) was removed, and moved to a petri dish containing by a video microscopy system. In doing so, the point 3 mL normal saline solution. The suspension was gen - grid was superimposed on the microscopic images of tly shaken at 37°C for 5-10 min to diffuse the sperma - the H&E-stained sections (5μm thickness) on a moni- tozoa. The samples were counted in a hemocytometer. tor by the software designed at the Histomorphometry Ten fields were then randomly selected and evaluated and Stereology Research Center. The volume density “Vv for motility grading to distinguish the immotile sperms (structure/testis)” of the testicular components, includ- from those with progressive or non-progressive motility. ing seminiferous tubules, interstitial tissue, and germinal Also, the sperm smears were stained with 1% eosin Y for epithelium, was estimated by the point counting method assessing the morphology [30]. [33, 34]. Finally, the total volume of each component was There is two types of progressive motility: 1- rapid pro - obtained by the following formula: gressive motility, 2- slow progressive motility. The effi - cient passage of spermatozoa through cervical mucus is dependent on rapid progressive motility. V(structure) = Vv(structure/testis) × V(shrunk) We should add that it is necessary to distinguish between these two types of progressive motility. So Estimation of the length and diameter of seminiferous that neglecting the distinction between two progressive tubules sperm groups leads to ignoring the information in the The length density (Lv) of the seminiferous tubules was semen sample, and the removal of such useful informa- measured on the sampled tubules in an unbiased counting tion would impoverish the semen analysis [31]. frame applied on the 5 μm thick sections (H&E staining) [35], and calculated by the following formula: Stereological study The left testis was removed and weighed. Then, according Lv = 2�Q/[�P × (a/f)] to the immersion method, it was immersed in isotonic saline-filled jar for measuring the primary volume “V Where “ΣQ” is the total number of the selected tubules, (testicle)” [32]. Afterwards, the samples were fixed in 4% “ΣP” represents the total points superimposed on the buffered formaldehyde solution for stereological studies. testis, and “a/f” indicates the area of the counting frame. The orientator method was applied to obtain Isotropic The total length of the seminiferous tubules “L(tubules)” Uniform Random (IUR) sections [32]. About 8-12 slabs was calculated by multiplying the lengths density (Lv) by in each testis were collected through this procedure. To V(structure) [36]. estimate the shrinkage, a circle was punched out from a random testis slab by a trocar (diameter 5 mm), and the trocar radius was considered as the “area (before)” (πr2). L(tubules) = Lv × V(structure) After tissue processing, the area was calculated as the “area (after)”. After tissue processing and paraffin embed - The diameter of the seminiferous tubules was also ding, 5 and 25 μm sections were cut by the microtome measured on the sampled tubules in the counting frame. and were stained using Hematoxylin-Eosin (H&E). The The diameter was measured perpendicularly to the long areas of the circles were measured before processing axis of the tubules where the tubules were widest [35]. An (unshrunk) and after processing (shrunk) and finally, the average of 100 tubules were counted per testis. B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 5 of 12 Estimation of number of testicular cell types Statistical analysis A computer linked to a light microscope (Nikon E200, The data were expressed as mean ± standard error Japan) with 40× oil lens (NA=1.4) was used to assess the (SEM). The results were analyzed by one-way analysis total number of testicular cell types, including spermato- of variance (ANOVA) and Tukey’s post hoc test using gonia (A and B), spermatocytes, round spermatids (steps Graph Pad Prism 6 software (San Diego, CA, USA). 1–8 spermiogenesis), long spermatids (steps 9–16 sper- P<0.05 was considered to be statistically significant. miogenesis), Sertoli and Leydig cells. The total number of the testicular cell types was cal - Results culated using the optical disector method applied on the Spermatozoa count, normal morphology and motility H&E-stained sections (25μm thickness) [37]. In so doing, According to Table 1, a significant decrease was observed the microscopic fields were scanned by moving the micro - in the count, percentage of normal morphology, and scope stage at equal distances in X and Y directions based motility of spermatozoa in the rats exposed to low and on systematic uniform random sampling. The movement high doses of BPA groups compared to control group in Z direction was also performed using a microcator (P<0.05 and P<0.01, respectively). However, these param- (MT12, Heidenhain, Germany) fixed on the microscope eters in the BPA-LOW + RES and BPA-HIGH + RES stage. The Z-axis distribution from the sampled cells in groups improved compared to the BPA groups (P<0.01 different focal planes was plotted to determine the guard and P<0.05, respectively). zones and disector’s height [38]. The numerical density (Nv) was estimated using the following formula: Qualitative changes Qualitative evaluation of the testis has been presented in Fig.  2. The histological sections of the low and high Nv = �Q/(�A × h) × (t/BA) doses of BPA rats showed the structural changes, includ- ing atrophy and reduced number of seminiferous tubules. Where “ΣQ” was the number of each cell type nuclei Concomitant treatment of these groups with RES amelio- coming into focus, “ΣA” indicated the total area of the rated these destructive effects. unbiased counting frame, “h” represented the disector’s height, “t” was the mean section thickness, and “BA” was the microtome block advance. Finally, the total number Stereological assays of the testicular cell types was calculated by multiplying The volume of the testicle the numerical density (Nv) by V(structure): The results showed a significant reduction in the testicle volume by 11.7 % and 13.5 % in the rats exposed to low and high doses of BPA compared to the control groups, N(cells) = Nv × V(structure) respectively (P<0.01 and P<0.001). However, the tes- tis volume recovered considerably in the animals that Where, V(structure) was the total volume of the germi- received BPA-LOW + RES group compared to BPA- nal epithelium for the germinal layer cells and the total LOW group (P<0.01) (Fig. 3A). volume of the interstitial tissue for the Leydig cells. Table 1 Comparison of sperm parameters. Mean ± SEM of the Count (×106), Normal morphology (%), Motility (%), and Immotile (%) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. n = 6 in each group. The results were analyzed by one‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. * P<0.05, ** P<0.01 vs. CONTROL; ## P<0.01 vs. BPA‑LOW; $ P<0.05 vs. BPA‑HIGH Groups Count (×10 ) Normal Motility (%) morphology (%) Rapid Slow Non Immotile progressive progressive progressive CONTROL 6.08±1.8 89.6±5.08 34.6±4.5 20.8±3.2 21.1±2.4 21.3±6.2 OLIVE OIL 6.12±2.3 85.6±3.18 35.4±1.6 21.4±2.3 21.1±6.2 21.1±6.4 CMC 5.8±3.2 81.6±2.5 32.0±2.41 23.0±2.6 23.4±5.5 22.3±1.9 RES 6.52±2.2 91.5±3.05 36.7±6.4 22.0±3.8 21.6±7.7 19.7±4.2 BPA‑LOW 3.21±1.08* 54.9±10* 28.2±2.9* 24.4±3.1* 25.1±3.3* 26.3±2.7* BPA‑HIGH 2.63±1.4** 30.9±10** 23.2±2.5** 29.6±2.4** 30.2±2.3** 32.7±2.5** ## ## ## ## ## BPA‑LOW+RES 5.22±.99 67.7±15.2 33.5±1.6 26.4±2.6 24.4±5.5 26.7±1.9 $ $ $ $ $ BPA‑HIGH+RES 3.76± 1.9 44.5±8.2 29.1±2.7 25.3±2.1 26.4±3 27.2±1.2 Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 6 of 12 Fig. 2 Testicular histological evaluation. Representative photomicrographs of testis sections stained with hematoxylin & eosin (H&E) in the CONTROL (A), low dose of Bisphenol A (BPA‑LOW ) (B), high dose of BPA (BPA‑HIGH) (C), BPA‑LOW + resveratrol (RES) (D), and BPA‑HIGH + RES (E) groups. All plates are to the same scale (Scale bar = 200 μm). The images indicate the normal seminiferous tubules (asterisk), and atrophied seminiferous tubules (arrow) Fig. 3 The evaluation of volume. The box plots represents the volume of the testis (A), germinal epithelium (B), interstitial tissue (C), and seminiferous tubules (D) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. n = 6 in each group. The results were analyzed by one ‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. BPA‑LOW; $p < 0.05 and $$$p < 0.001 vs. BPA‑HIGH B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 7 of 12 The volume of germinal epithelium (P<0.01 and P<0.001, respectively). Nonetheless, the The total epithelial volume in rats treated with low and tubules length significantly was improved in the rats high doses of BPA decreased 43% and 64% in comparison treated with RES in the low and high doses of BPA groups to the control groups, respectively (P<0.001). Treatment (P<0.05 and P<0.01, respectively) (Fig. 4A). with RES ameliorated the epithelial volume changes in the low or high doses of BPA groups (P<0.001) (Fig. 3B). Number of spermatogonia A and B The total number of spermatogonia A reduced by 40.03% The volume of interstitial tissue and 55.2%, and spermatogonia B by 51.27% and 70.05% in The results indicated that the interstitial tissue volume the low and high doses of BPA compared to the control reduced 25.3% and 27.3% in the low and high doses of groups, respectively (P<0.001). However, treatment with compared to the control groups, respectively (P<0.01 RES increased these cells in the low and high doses of and P<0.05). However, this parameter significantly was BPA groups (P<0.001) (Fig. 5A and B). increased in the rats treated with RES in the low or high doses of BPA groups (P<0.05) (Fig. 3C). Number of spermatocytes Statistical analysis showed 34.85% and 53% reduction in The volume of seminiferous tubules the number of spermatocytes for both the low and high A significant reduction was seen in the total volume of doses of BPA compared to the control groups (P<0.001). seminiferous tubules by 26.2% and 34% in the low and high Treatment with RES ameliorated these changes in the doses of BPA compared to the control groups, respectively BPA-LOW + RES and BPA-HIGH + RES groups com- (P<0.01 and P<0.001). Nevertheless, seminiferous volume pared to the BPA groups (P<0.01 and P<0.001, respec- significantly was ameliorated in the BPA-LOW + RES and tively) (Fig. 5C). BPA-HIGH + RES groups compared to the BPA groups (P<0.01 and P<0.05, respectively) (Fig. 3D). Number of round and long spermatid The number of round spermatids decreased by 40.76% Diameter of the seminiferous tubules and 66.72%, and long spermatids by 28.7% and 60.35% The diameter of the seminiferous tubules decreased in the low and high doses of BPA, respectively compared 29.7% and 37.3% in rats treated with low and high doses to the control groups (P<0.001). Moreover, ameliorative of BPA compared to the control group (P<0.001). Treat- effects of RES on the number of these cells were seen ment with RES increased this parameter in the low and in rats treated with low and high doses of BPA groups high doses of BPA groups (P<0.01) (Fig. 4A). (P<0.001) (Figs. 5D and 6A). Length of the seminiferous tubules Number of Leyding and Sertoli cells The results showed that length of the seminiferous A significant reduction was seen in the number of Leydig tubules have reduced 20.6% and 29.8% in the low and cells by 45.78% and 62.85%, and Sertoli cells by 32.28% high doses of BPA compared to the control groups and 52.76% in the low and high doses of BPA than those Fig. 4 The evaluation of diameter and length of seminiferous tubules. The box plots shows the diameter (A), and the length (B) of seminiferous tubules in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. n = 6 in each group. The results were analyzed by one ‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. **p < 0.01, and ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01vs. BPA‑LOW; $$p < 0.01 vs. BPA‑HIGH Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 8 of 12 Fig. 5 Evaluation of the germinal cells number. The box plots represents the number of spermatogonia A (A), spermatogonia B (B), spermatocytes (C), and round spermatids (D) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. The results were analyzed by one ‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. BPA‑LOW; $$p < 0.01 and $$$p < 0.001 vs. BPA‑HIGH of the control groups, respectively (P<0.001). Treatment Discussion with RES recovered the number of Leydig cells and Ser- The current study revealed the ameliorative effects of toli cells in the BPA-LOW + RES (P<0.01 and P<0.05, RES on testicular damage induced by BPA in rats. The respectively), and BPA-HIGH + RES (P<0.001 and first part of our findings showed the deleterious effects P<0.01, respectively) groups compared to the BPA groups of two doses of BPA, 25 and 50 mg/kg/day for 8 weeks, (Fig. 6B and C) on sperm quality and structural changes of the testis. The earlier studies showed that 50 mg/kg/day is considered Hormone assays as maximum permissible dose that have no observable The gonadotropins assessment showed a significant side effect on reproductive and developmental toxicity reduction in serum LH and FSH levels in the BPA-LOW [39]. But we found that ingestion of BPA at these dosages (P<0.001 and P<0.01, respectively), and BPA-HIGH had adverse effects on count, morphology, and motil - (P<0.001) groups compared to the control group. Also, ity of spermatozoa. In line with our results, it has been the testosterone concentration of the rats given low or observed a reduction in epididymal sperm motility and high doses of BPA was lower than in the control group count in the rats exposed to BPA at the 10 and 50 mg/ (P<0.001). The RES exposure led to significant increase in kg in a dose dependent manner [40]. Also, it has been the serum LH and testosterone levels in the BPA-LOW demonstrated that BPA at 5 and 25 mg/kg/day reduced (P<0.01 and P<0.001, respectively), and BPA-HIGH sperm production, reserves and transit time through (P<0.05 and P<0.001, respectively) groups, while the the epididymis [13]. Moreover, long-term exposure to serum FSH levels significantly increased only in the BPA- 0.2 mg/kg BPA in rats led to decreased sperm count and HIGH + RES group (P<0.05) (Fig. 7). inhibited spermiation [41]. B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 9 of 12 Fig. 6 Evaluation of long spermatids, Leydig and Sertoli cells number. The box plots represents the number of long spermatids (A), Leydig (B), and Sertoli (C) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. The results were analyzed by one ‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. BPA‑LOW; $$p < 0.01 and $$$p < 0.001 vs. BPA‑HIGH Fig. 7 Serum concentrations of luteinizing hormone (LH), follicle‑stimulating hormone (FSH), and testosterone hormones. The column graphs represent the concentrations of LH (A), FSH (B), and testosterone (C) in the CONTROL, OLIVE OIL, carboxy methylcellulose (CMC), resveratrol (RES), low dose of Bisphenol A (BPA‑LOW ), high dose of BPA (BPA‑HIGH), BPA‑LOW + RES, and BPA‑HIGH + RES groups. n = 6 in each group. The results were analyzed by one‑ way analysis of variance (ANOVA) and Tukey’s post hoc test. Data are presented as mean ± SEM. **p < 0.01, and ***p < 0.001 vs. CONTROL; #p < 0.05, ##p < 0.01vs. BPA‑LOW; $$p < 0.01 vs. BPA‑HIGH The reduction in sperm count and quality is in accord tubules and testicular abnormalities due to BPA. Loss with decreased stereological parameters. The changes of of the germinal epithelial cells was also seen after expo- structural indices including volume, diameter and length sure to both doses of BPA. A reduction in germinal epi- of seminiferous tubules suggest the atrophy of these thelial volume could be a consequence of decline in the Bordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 10 of 12 number of germinal cells. The reduction in sperm pro - LH, and testosterone increased in the cisplatin+RES- duction could be related to the disruption of spermato- treated rats compared to cisplatin group, thereby improv- genesis. Jin et  al., 2013 also reported that BPA exposure ing sperm parameters and testicular apoptosis [24]. could decrease sperm count via the reduction in type A Also, they showed that RES enhanced hormonal levels spermatogonial, spermatocytes and spermatids. BPA as well as sperm motility and count compared to control impaired spermatogenesis through suppressing repro- group. But in our study, there was no significant differ - ductive hormones and activating germ cells apoptosis ence between the RES and control groups. The difference mediated by Fas/FasL signaling pathway [42, 43]. Ser- between the results of our study and Shatti’s research toli cells are another type of cells in the seminiferous may be because of different route of administration and tubules, which have a supportive and nutrient function. dose of RES [24]. Since, Sertoli cells can affect the proliferation and differ - Also, another earlier study claimed that RES could entiation of germinal cells, and also help in the process ameliorate negative effects against BPA-induced repro - of spermatogenesis. So, it seems the loss of these sup- ductive toxicity in mice via reducing oxidative stress porting cells could be led to deficiency of supportive [51]. Our results support the contribution of repro- functions in BPA-treated rat, and cause the loss of sper- ductive hormones in the ameliorative effects of RES on matogenic cells. It has been indicated that Sertoli cells BPA-induced testicular toxicity in rats. Meanwhile, a are targets of pituitary-derived FSH and testosterone to reduction of oxidative stress, as shown by other stud- transduce signals into paracrine regulation of spermato- ies [52], may also be involved in RES protective effects, genesis [44–46]. Accordingly, Sertoli cell depletion fol- which required further studies to be confirmed. lowing BPA treatment in present study may be due to a One of the limitations of our study was that the sign- decrease in FSH and testosterone levels. On the other aling pathways that contribute to the amelioration of hand, testosterone secretion is produced in Leydig cells reproductive hormones by effect on the hypothalamic– of testicular interstitium in response to LH [47]. There - pituitary–gonadal axis in the RES treated rats following fore, the lack of LH stimulation in BPA-treated groups BPA, which led to spermatogenesis improvement were could justify the reduction of Leydig cells and interstitial not investigated. tissue atrophy and also the decrease of testosterone pro- duction. Testosterone is an essential hormone to main- tain normal spermatogenesis and prevention of germ cell Conclusion apoptosis in adult rats [48]. So, it is reasonable to assume In conclusion, the present study demonstrated the pro- that the inhibition of reproductive hormones production tective effects of RES against BPA-induced testicular may have contributed to spermatogenesis impairment structural changes and sperm quality via improving induced by BPA. Similarly, BPA could cause defective gonadotropin hormones and testosterone levels. spermatozoa by disruption of the hypothalamic–pitui- tary–gonadal axis, causing a state of hypogonadotropic Abbreviations hypogonadism [13, 42, 49]. Another possible hypothesis BPA: Bisphenol A; RES: Resveratrol; CMC: Carboxy methylcellulose; LH: Lutein‑ may be involved in spermatogenesis dysfunction is the izing hormone; FSH: Follicle‑stimulating hormone; ER: Estrogen receptor; AR: Androgen receptor. effects of BPA-induced oxidative damage. BPA exposure could induce ROS production by reducing the activity of Acknowledgements the antioxidant system [50]. The adverse effects of BPA This article was extracted from the thesis of Seyedeh‑Saeedeh Yahyavi’s, M.Sc. in Anatomy. This work was performed at the Histomorphometry and Stereol‑ on sperm count and quality due to oxidative stress have ogy Research Center and was financially supported by grant No. 97‑01‑21‑ been described by previous studies [40, 49]. 18338 from Shiraz University of Medical Sciences. Hereby, the authors would The second step of our study demonstrated the protec - like to thank Ms. A. Keivanshekouh at the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for improving the use of English in the tive effects of RES against BPA- induced testicular struc - manuscript. tural changes and sperm quality. Our results indicated that the concomitant treatment of the BPA groups with Authors’ contributions H.B: Designing the study, supervising laboratory works and revising the RES for 8 weeks could significantly restore the sperm manuscript. S.Y: Performing laboratory works and collecting the data. A.N. and parameters and prevent testicular atrophy and apoptosis E.A.A: Conceptualization, Methodology, Software. M.N: Analysis of the data, of the testicular cell types. Furthermore, RES enhanced writing and editing the manuscript. The authors read and approved the final manuscript. testosterone, FSH, and LH levels in BPA groups. The improvement of testicular structural and sperm quality Funding seems to be related to increased gonadotropin hormones This work was performed at the Histomorphometry and Stereology Research Center and was financially supported by grant No. 97‑01‑21‑18338 from Shiraz and testosterone levels. Consistent with these findings, University of Medical Sciences. previous reports have also shown that the levels of FSH, B ordbar et al. Basic and Clinical Andrology (2023) 33:1 Page 11 of 12 Availability of data and materials hypothalamus–pituitary–testis axis and spermatogenesis. Front Endo‑ The datasets used and/or analyzed during the current study are available from crinol. 2014;5:1. https:// doi. org/ 10. 3389/ fendo. 2014. 00001. the corresponding author on reasonable request. 13. Wisniewski P, Romano RM, Kizys MM, Oliveira KC, Kasamatsu T, Giannocco G, et al. Adult exposure to bisphenol A (BPA) in Wistar rats reduces sperm quality with disruption of the hypothalamic–pituitary–testicular axis. Declarations Toxicology. 2015;329:1–9. https:// doi. org/ 10. 1016/j. tox. 2015. 01. 002 Epub 2015 Jan 6. Ethics approval and consent to participate 14. El Ghazzawy IF, Meleis AE, Farghaly EF, Solaiman A. Histological study All experimental procedures in the current study were done in accordance of the possible protective effect of pomegranate juice on bisphenol‑A with the National Institutes of Health guide for the care and use of laboratory induced changes of the caput epididymal epithelium and sperms of animals (NIH Publications No. 8023, revised 1978) and were approved by the adult albino rats. Alexandria J Med. 2011;47:125–37. Medical and Research Ethics Committee of Shiraz University of Medical Sci‑ 15. Tohei A, Suda S, Taya K, Hashimoto T, Kogo H. Bisphenol A inhibits testicu‑ ences, Shiraz, Iran (Approval No. IR.SUMS.REC.1398.392). All procedures were lar functions and increases luteinizing hormone secretion in adult male carried in accordance with the ARRIVE (Animal Research: Reporting in Vivo rats. Exp Biol Med. 2001;226:216–21. https:// doi. org/ 10. 1177/ 15353 70201 Experiments) guidelines. 22600 309. 16. Takahashi O, Oishi S. 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Testicular toxic changes induced by At BMC, research is always in progress. bisphenol A in adult albino rats: a histological, biochemical, and Learn more biomedcentral.com/submissions

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Basic and Clinical AndrologySpringer Journals

Published: Jan 6, 2023

Keywords: Bisphenol A; Resveratrol; Testicular toxicity; Sperm parameters; Stereology

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