Resveratrol reestablishes spermatogenesis after testicular injury in rats caused by 2,5-hexanedione

Several investigations have confirmed that environmental toxins and their metabolic products, which can include insecticides, fumigants, herbicides, paints, diluents and detergents, could decrease the mobility and density of sperm, increase the incidence of teratospermia, induce structural abnormalities of spermatic membrane protein and nucleoprotein and even lead to male infertility and embryonic developmental anomalies.^1,2 Because of the multiplicity of testicular targets and the complexity of the exposures little data is available concerning the impaired mechanism of procreation and the protective effects of some antagonisms to testicular toxicants.

Chronic exposure to 2,5-hexanedione (2,5-HD), a toxic metabolite of the common industrial solvents n-hexane and methyl n-butyl ketone, can induce peripheral polyneuropathy³ and testicular injury.⁴ Researches have now revealed that 2,5-HD can damage testis by impairing Sertoli cell function, which is the most important cell for producing the nurturing environment for germ cells and forming the blood-testis barrier.⁵ For its established damaging effect to testis, 2,5-HD has been utilized as an agent in several studies of blocking the toxic reaction.

Resveratrol (3,5,4′-trihydroxystilbene, RES), a natural phytoalexin, has exhibited a wide range of biological activities in previous studies, including anti- inflammatory,⁶ antioxidant,⁷ antivirus and antitumor⁸ properties. However, the mechanism of RES action has not been totally elucidated. Some recent researches demonstrated that RES could increase sperm output⁹ and protect sperm from apoptosis caused by physical damage, such as testicular torsion.^10-12 Whether RES could inhibit adverse effects of chemical toxicants, such as 2, 5-HD, to testis was unclear. This experimental study was designed to evaluate the protective effect of RES on germ cells after exposure to 2, 5-HD.

Animals and treatments
A total of 40 male Sprague-Dawley rats, approximately 70–80 days of age, purchased from Beijing Institute of Heart Lung and Blood Vessel Diseases, were maintained under controlled conditions at 22–23°C ambient temperature, 50%–55% relative humidity and a 12L:12D cycle with free access to standardized rat chow and tap water.

The rats were divided into 5 groups (8 rats per group), namely, normal group A, induced abnormality group B and RES treatment groups C, D and E. Initially, groups B, C, D and E were treated with 1% 2, 5-HD in drinking water for 5 weeks at an average dose/rat of 3.1 mmol∙kg^-1∙d^-1. Because of its low solubility in water RES was suspended in 5 g/L carboxymethylcellulose. The dose was adjusted according to the rat's weight to ensure a constant dose level and was freshly prepared immediately before each administration. Groups C, D and E were respectively treated with different doses of RES, 20 mg∙kg^-1∙d^-1, 40 mg∙kg^-1∙d^-1 and 80 mg∙kg^-1∙d^-1, for another 9 weeks, while groups A and B were administrated carboxymethylcellulose at the same time. All rats were sacrificed after the terminal day of the experiment.

Body and organ weights
The body weights were collected on the first day and the terminal day (prior to necropsy) of the last 9-week period and all the data were used to calculate individual body weigh gain/loss over the period of treatment. The weights of the testis were also recorded for each animal. Testes were trimmed of fat prior to recording their weights. The right testes were processed for histopathology, and the left testes were frozen at –80°C until protein analysis.

Histological examination of testes
The right testis were fixed in 10% neutral formalin, embedded in paraffin and sectioned at 5 µm. The slides were dewaxed in dimethylbenzene for 5–10 minutes, and then transferred in alcohol/dimethylbenzene (1:1) for 5 minutes, afterwards, transferred by turn in 100%, 95%, 85%, 70% of alcohol for 2–5 minutes and finally transferred in distilled water. The slides were immerged into hematoxylin for 5–15 minutes then washed. The slides were fixed in 0.5% of muriatic alcohol for 10 seconds and then washed with water for 15 minutes. The slides were immerged into 0.1% to 0.5% eosin for 1–5 minutes, and then dehydrated by turn in 70%, 85%, 95% and 100% alcohol for 2–3 minutes. At last, mean number (5 fields per tubule) and diameter (20 tubules per animal) of the seminiferous tubules were measured.

Immunohistochemical staining for c-kit in testes
The right testis were fixed in 10% neutral formalin, embedded in paraffin and sectioned at 5 µm. Slides were dewaxed and endogenous peroxidase was quenched in 1% H₂O₂ in methanol for 30 minutes. Sections were sequentially permeabilized three times, followed by DNA denaturation and nonspecific binding. After that, rabbit polyclonal antibody made against the amino acid sequence found at the carboxy terminus of c-kit (pc34, Sigma) was incubated with sections overnight at 4°C. Tubules were then washed with PBS and incubated with peroxidase-conjugated goat anti-rabbit IgG (Dingguo Biotechnology, China, 1:250 dilutions) for 5 hours. At last, diaminobenzidine was used as the color substrate and haematoxylin was used as the additional nuclear counterstain. When the above process had been completed, pathological images were captured and analyzed with VNT Quant Lab-MD system.

Reverse transcription PCR
Total testes RNAs were isolated from the five different groups using TRI Reagent (Invitrogen Inc, USA). The RNA samples were first treated with Deoxyribonuclease I (Fermentas Inc, USA) and converted into cDNA using oligo (dT)18 primers and reverse transcriptase (Fermentas Inc, USA). To amplify c-kit and GAPDH, PCR was carried out as the following conditions: c-kit, initial denaturation for 3 minutes at 95°C, followed by 29 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds; GAPDH, initial denaturation for 3 minutes at 95°C, followed by 29 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 30 seconds. Amplified products were analyzed on 1.5% agarose gel. The intensities of the autoradiographic bands were estimated through Image Master Total Lab v1.11 software. The results corresponded to the optical densities (ODs) of autoradiogram relative to two different PCR products. Because of the constancy of the GAPDH expression in tissues, it was chosen as an internal standard. The ODs corresponding to c-kit and GAPDH in each individual sample were expressed in arbitrary units as a ratio of c-kit over GAPDH densitometric intensities. The following are PCR primers for c-kit (sense primer, 5′-ACAGGCAGAC- GCCGCACTTTAT-3′; antisense primer, 5′-CATCGGGC- TCCGTTTCTACTCA-3′), and GAPDH (sense primer, 5′-ATTAAAGAACAGGCTCTTAGCACA-3′; antisense primer, 5′-AGACGCCAGTAGACTCCACGAC-3′). All of the primers were obtained from Sangog Ltd (Shanghai, China).

Western blotting
Total protein was isolated from another portion of the left testis, by using 1 ml of ice cold TNE lysis buffer containing 0.01 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 0.01 mmol/L EDTA (pH 7.4), 0.5% deoxycholate, 200 μg/ml PMSF and 50 μg/ml protease inhibitor cocktail (p2714, Sigma, USA). Insoluble materials were removed by centrifugation at 15 000 ×g for 15 minutes at 4°C. The concentration of the extracted protein was measured spectrophotometrically with Coomassie G-250. Aliquots of 50 μg protein were separated by 8% SDS-PAGE and electro-transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were initially blocked with 5 % nonfat dry milk in TBS for 2 hours and then incubated with the primary antibodies (anti-c-kit and anti-tubulin were bought from Sigma Inc, USA) all at a 1:500 dilution in 5% nonfat dry milk/TBS at 4°C for overnight. The secondary horse radish peroxidase-conjugated antibody goat anti-rabbit antibody for c-kit or goat anti-mouse antibodies for tubulin (Boshide Inc, China) were diluted to 1:500 and 1:200 in 5% nonfat dry milk/TBS (0.1% Tween-20), respectively. After washing three times with TBS, the membranes were incubated with the secondary antibodies for 2 hours at room temperature. After washing three times with TBS again, super ECL plus Detection Reagent (Applygen Technologies Inc, China) was used to detect immunopositive protein bands.

Statistical analysis
All data were expressed mean ± standard deviation (SD). Statistical significance of the differences observed between experimental groups was determined by one-way analysis of variable (ANOVA) using SPSS11.0. A P value less than 0.05 was considered statistically significant.

Effect of RES on physical signs, body weight and testis weight
Toxicity of 2,5-HD led to the abnormal changes of physical signs, loss of body weight and testis atrophy (Table 1). 2,5-HD-treated rats were physically weak, had grey-yellow color patterns, Cutis laxa, feeble and cyanotic limbs. After 9 weeks of RES treatment, aforesaid physical signs in RES treatment groups were obviously improved being close to the normal group A. Body weight gain and testis weight of the RES treatment groups were higher than in group B (P <0.01), and the amelioration degree was greater with the increasing dose of RES (P <0.01); however, body weight and testis weight of the RES groups were still different from the normal group A (P <0.01) (Table 1).

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Table 1. Body weight gains, testis weights and morphological normal seminiferous tubules after RES treatment for 9 weeks

Histomorphological analysis of seminiferous tubules
Seminiferous tubules were observed in all groups. HE staining showed the seminiferous tubules of group A with big lumens and clear cell layers from outside to inside which were divided into Sertoli cells, spermatogonia, primary spermatocytes, second spermatocytes, spermatids and sperms. Seminiferous tubules of group B appeared deformed with emarcid lumens, physiological progression of spermatogenesis in group B was stagnant in undifferentiated type A spermatogonia. However, the testes of groups C, D and E could generate nascent seminiferous tubules, whose shape was approximate to that of normal group A; meanwhile, there were more regular lumens and clearer cell layers in groups D and E than in group C, macroscopicly (Figure 1).

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Figure 1. Histomorphological analysis of seminiferous tubules (HE staining, original magnification ×400). A: normal group A; B: abnormal group B; C: RES-treated group C; D: RES-treated group D; E: RES-treated group E. RES: resveratrol.

After the number and diameter of seminiferous tubules in every group was counted, we found that values for the three RES treated groups were significantly more than those of Group B, whereas, still less than those of group A. And among RES groups, the number and diameter of seminiferous tubules were closer to normal with an increasing RES drug dose (Table 1).

Immunohistochemical staining for c-kit in testes
c-kit protein is one of critical proteins correlated with proliferation and differentiation of spermatogenic cells. It may play a role in preparing the germinal cells to enter meiosis. The c-kit gene mutation could result in the abnormal generation and migration of spermatogenic cells and ultimately in infertility.¹³

The data of immunohistochemical staining showed that the c-kit protein expression in the normal group A in undifferentiated type A spermatogonia was negative; and differentiating type A spermatogonia, type B spermatogonia and preleptotene spermatocytes, which are differentiated from undifferentiated type A spermatogonia, all exhibited c-kit positive. At the phase from leptotene spermatocytes to spermatids, c-kit expression disappeared. However, as shown in group B, c-kit positive staining can not be detected in seminiferrous epithelium after 2, 5-HD exposure. That means 2, 5-HD induced dyszoospermia may be related to impaired c-kit protein or gene expression. We observed the same expression profile of c-kit in the RES treatment groups as group A. Furthermore, c-kit expression in spermatogenic cells of the RES treated group C were stained lightly, not like groups D and E; that means the seminiferous tubules in group C had not recovered fully (Figure 2).

c-kit expression of RT-PCR and Western blot evaluation
To determine the therapeutic effect of RES on infertility rats induced by 2.5-HD, we performed RT-PCR and Western blot to detect c-kit expression. After collecting the values of mRNA and protein band density we compared the difference among these data. RT-PCR showed that the c-kit mRNA expression was weakest in group B, when the GAPDH mRNA was used as an internal standard (Figure 3). And as observed in Western blot, the expression of c-kit protein was markedly absent in group B; whereas, its expression in other groups was positive (Figure 4). Through analyzing the band density

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Figure 3. mRNA expression of c-kit in five groups of testis tissues. Lane A: normal group A; Lane B: abnormal group B; Lane C: RES-treated group C; Lane D: RES-treated group D; Lane E: RES-treated group E. RES: resveratrol; M: marker.

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Figure 4. Protein expression of c-kit in five groups of testis tissues by PAGE electrophoresis. Lane A: normal group A; Lane B: abnormal group B; Lane C: RES-treated group C; Lane D: RES-treated group D; Lane E: RES-treated group E. RES: resveratrol.

of c-kit mRNA and protein, we found that c-kit expression increased gradually along with the increasing RES dose used during the therapeutic period (Table 2). Combining these results, we presumed that RES could ameliorate dyszoospermia induced by 2, 5-HD through activating the transcription of c-kit mRNA and upregulating expression of c-kit protein.

The findings of the previous studies showed that increasing environmental toxins could interrupt the physiological spermatogenesis and lead to male infertility. Among these toxins, 2, 5-HD has demonstrated that it can damage testis through impairing Sertoli cell function, which is the most important cell for producing a nurturing environment for germ cells, forming the blood-testis barrier⁵ and the shedding of germ cells from the seminiferous epithelium.⁶ Boekelheide found that the malignant alterations in testis could continue for more than 70 weeks after a five week exposure to 2,5-HD.⁵ Because its injuring effect to testis is stable and definite, 2,5-HD has been utilized as an injury agent in several studies. Our study also confirmed this testicular injury effect could continue for at least nine weeks.

Previous researches discovered that RES, which is a natural phytoalexin found in a wide variety of plant species including grapes, had exhibited a wide range of biological activities, including anti-inflammatory,⁶ antioxidant,⁷ antivirus and antitumor⁸ properties. RES has further demonstrated that it can not only increase sperm output⁹ and improve sperm development,⁹ but also protect sperm from DNA damage by benzo(a)pyrene¹⁰ and apoptosis caused by ischemia-reperfusion (I/R) injury in testicular torsion cases.^11,12 Currently RES is gradually becoming a really important bioactive agent to promote germ cell growth and avoid damage. However, whether RES is still effective for patients that have dyszoospermia is not clear. Our study was just intended to determine if RES could make sperm, damaged by toxins, re-differentiate.

The major result in our study was the demonstration that, after 2,5-HD-induced dyszoospermia, RES could promote the regrowth of emarcid seminiferous tubules and mediate the resumption of c-kit gene expression and the reappearance of c-kit protein. This reversal effect by RES correlated with the use of increasing dosage of RES. Accordingly, we demonstrated RES could have therapeutic effects not only at the cellular level but at the gene and protein levels as well.

The mechaniasm of RES on dyszoospermia might correlate with the following two factors. First, through the feedback regulation of hypothalamic-pituitary-gonadal axis, RES, which has a similar polyphenol structure to estrogen,^14-16 could elevate the concentration of follicle-stimulating hormone (FSH) and testosterone (T).⁹ Other in vitro studies proved that the expression of c-kit protein in the germ/Sertoli cell co-culture system could be enhanced with FSH or T alone; and FSH manifested a synergistic effect with T in stimulating germ cell proliferation.¹⁷ Therefore, RES could promote spermatogenesis by binding to ER as an agonist. On the other hand, the effects of RES could also be mediated by counteraction of constitutive oxidative stress within the seminiferous tubules. Male germ cells are capable of producing reactive oxygen species (ROS), and a certain amount of free radicals generated in the respiratory chain are necessary for the normal function of sperm cells. In cases of overproduction of ROS, the antioxidant potential of sperm cells could be exhausted and oxidative stress might develop.¹⁸ 2,5-HD could result in dysfunction of the cellular antioxidant system and accumulation of ROS,¹⁹ which could be another reason for 2, 5-HD-induced dyszoospermia. Recently, RES was found to be an effective scavenger of ROS as well as showing antioxidant ability in cells producing ROS.⁷ RES could effectively counteract lipid peroxidation in cell membranes and DNA damage caused by ROS.²⁰ Therefore, RES could also be acting by counteraction of constitutive oxidative stress to decrease the steady-state levels of ROS within the seminiferous tubules, thus ameliorating 2,5-HD-induced dyszoospermia and renewing spermatogenesis.

However, compared with normal group, the RES treatment groups had fewer seminiferous tubules with narrower diameters and decreased expression of c-kit gene and protein. This result might be on account of the short-term therapy and the insufficient dosage. Conventionally, RES was thought to be an atoxic substance. Juan and Crowell demonstrated that oral administration of RES in rats at a dose of 20 mg∙kg^-1∙d^-1 for 90 days and at a dose of 300 mg∙kg^-1∙d^-1 for 28 days were not harmful to male rats.^9,21 Therefore, additional studies are required to identify the more suitable dose and course of treatment to resume spermatogenesis.

In conclusion, the data presented in this article displayed a novel activity of RES and provided a basis for clinical therapy to male infertility. Moreover, these findings would guide the clinical application of RES to resume dyszoospermia induced by environmental toxins or chemotherapeutics. Nevertheless, the mechanism of RES that creates an anastatic effect on toxicity-damaged seminiferous tubules is intricate and needs further researche.

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