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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2017 Dec 29;98(6):312–328. doi: 10.1111/iep.12251

Resveratrol attenuates reproductive alterations in type 1 diabetes‐induced rats

Joana Noguères Simas 1, Talita Biude Mendes 1, Camila Cicconi Paccola 1, Vanessa Vendramini 1, Sandra Maria Miraglia 1,✉
PMCID: PMC5826946  PMID: 29285813

Summary

The progression of diabetes mellitus leads to several complications including overproduction of reactive oxygen species and reproductive alterations. As resveratrol (RES) is a powerful anti‐oxidant and an anti‐apoptotic compound, we hypothesized that side effects of type‐1 diabetes (DM1) on male reproduction could be reduced by the RES treatment. Eighty‐four prepubertal male rats were distributed into seven groups: sham‐control (SC), RES‐treated (R), resveratrol‐vehicle‐treated (RV), diabetic (D), diabetic‐insulin‐treated (DI), diabetic‐RES‐treated (DR), diabetic‐insulin and RES‐treated (DIR). DM1 was induced by a single intraperitoneal streptozotocin (STZ) injection (65 mg/kg) on the 30th day postpartum (dpp). Animals of DR, DIR and R groups received 150 mg/day of RES by gavage for 43 consecutive days (from the 33 to 75 dpp). DI and DIR rats received subcutaneous injections of insulin (1 U/100 g b.w./day) from 5th day after the DM1 induction. The blood glucose level was monitored. At 75 dpp, the euthanasia was performed for morphometric and biometric testicular analyses, spermatic evaluation and hormonal doses. In the D group, the blood glucose level was higher than in the DR, DI and DIR groups. Besides morphometric testicular measurements, testosterone and estradiol doses were lower in D group than in DR and DIR groups; LH dose was also lower than in DR. The preputial separation age was delayed in diabetes‐induced groups. The DR and DIR groups showed an improvement in sperm mitochondrial activity, epididymal sperm counts and the frequency of morphologically normal sperms. RES treatment improved glycaemic level, sperm quantitative and qualitative parameters and the hormonal profile in DM1‐induced rats and seems to be a good reproductive protector.

Keywords: diabetes, oxidative stress, resveratrol, sperm, testis

Diabetes mellitus (DM) is a chronic metabolic illness, characterized by hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism, which is mainly divided into two major forms (Alberti & Zimmet 1998): type 1 diabetes (DM1) and type 2 diabetes. DM1 is a complex immune‐mediated process resulting from massive destruction of insulin‐secreting pancreatic β‐cells that combined with predisposing genes and environmental factors lead to the stimulation of autoreactive lymphocytes and the cytokine‐inducing apoptosis of pancreatic β‐cells (Atkinson et al. 2014; Rasoul et al. 2016). As a consequence the insulin required is no longer produced (I. D. F. Diabetes‐Atlas, [Link]). Type 2 diabetes (DM2) is widely known as non‐insulin‐dependent diabetes mellitus, where the pancreatic β‐cells may produce altered or impaired insulin, leading to hyperinsulinaemia and/or insulin resistance (American Diabetes 2016).

Nowadays, there are more than 415 million diabetic people worldwide, and over the next few years, about 642 million individuals will develop the disease (I. D. F. Diabetes‐Atlas [Link]). Furthermore, according to the International Diabetes Federation, more than 86,000 children are diagnosed with DM1 every year and the occurrence of this endocrine and metabolic illness in childhood (De Beaufort 2006; Patterson et al. 2009) is increasing from 3% to 5% per year.

It is already known that the series of complications are related to diabetes' pathogenesis include atherosclerosis, coronary artery disease, peripheral vascular disease, hypertension, nephropathy, neuropathy, retinopathy, among others (Amos et al. 1997), and may include reproductive abnormalities (Laaksonen et al. 2004; De Berardis et al. 2007). Some of the damages on male reproductive tract are erectile dysfunction, retrograde ejaculation and loss of libido, accompanied by abnormal spermatogenesis, followed by alterations in histological characteristics of seminiferous epithelium and changes in sperm count and motility (Alves et al. 2013).

Oxidative stress is likely involved in the cell damage triggered by the hyperglycaemia, and it is one of the major causes of male infertility (Shrilatha & Muralidhara 2007; Ricci et al. 2009; Mohasseb et al. 2011; Kyathanahalli et al. 2014). Patients with DM1 had lower sperm DNA quality as a result of damage caused by increased reactive oxygen species (ROS) concentrations (Agbaje et al. 2008), which might be related to alterations in the hypothalamic–pituitary–gonadal axis (Dandona et al. 1996; Sexton & Jarow 1997). Another major complication of DM1 – the neuropathy – also seems to be triggered by oxidative stress (Vincent et al. 2004).

The imbalance between ROS levels and anti‐oxidant system is an important part of the aetiology of several complications related to DM, as reported in both clinical and experimental studies (Van Dam et al. 1995; Shrilatha & Muralidhara 2007). Moreover, increased levels of ROS may have a negative impact on the quality of sperm along with a reduction in the mechanisms of anti‐ROS defence, which can damage not only proteins in the membrane, but also nucleic acids (Sies 1997; Vincent et al. 2004; Xu et al. 2004). It may be due to the fact that spermatozoa display particular vulnerability to overproduction of free radicals, although only small amounts of ROS are needed to restore fertility capacity (De Lamirande et al. 1997). Furthermore, spermatozoa are unable to self‐repair from the injury promoted by ROS due to the absence of cytoplasmic enzyme repair systems (Agarwal et al. 2004). Besides, the membranes of germ cell lineage, as well as of the spermatozoa, are rich in polyunsaturated fatty acids (PUFAs), which are very prone to lipid peroxidation and highly vulnerable to oxygen‐induced stress (Aitken 1995; Griveau & Lannou 1997; Agarwal et al. 2014).

A series of reports have demonstrated that the administration of anti‐oxidant substances to diabetic rats reduces lipid peroxidation, promoting protective action against testicular oxidative injuries and germ cell apoptosis (Mohasseb et al. 2011). Regarding the scavenger properties of anti‐oxidants, the phenolic compounds could be a particularly important tool to control ROS production in diabetic profiles (Jang & Surh 2001).

Resveratrol (RES; 3,5,4′‐trihydroxystilbene), a phytoalexin present in more than 70 varieties of plants (Burns et al. 2002), has presented beneficial effects in the organism due to its anti‐oxidant, anti‐inflammatory, anti‐tumoral and anti‐apoptotic properties (Saiko et al. 2008). Yuluğ et al. (2014) reported the potential protective effect of RES against the damage caused by high ROS production in testicular ischaemia: they showed that the acute administration of RES reduced apoptosis in the rat seminiferous epithelium. Mendes et al. (2016) observed a reduction in apoptosis in the testes and an improvement of qualitative spermatic parameters in resveratrol‐treated varicocelized rats.

Furthermore, it has already been demonstrated that streptozotocin (STZ)‐treated rats display similar harmful effects on the male reproductive tract comparative to those observed in patients with DM1 (Hassan et al. 1993; Soudamani et al. 2005; Agbaje et al. 2008; Navarro‐Casado et al. 2010). Thus, taking into account that the oxidative stress may represent an important harmful factor involved in the reproductive complications observed in patients with diabetes, we aimed to investigate whether RES, an anti‐apoptotic and anti‐oxidant substance, could be an effective adjuvant against the late reproductive damage caused by ROS in rats with STZ‐induced DM1 from prepuberty. In addition, we also aimed to investigate whether the treatment only with RES would be more efficient than the insulin treatment against the reproductive damage observed in DM1.

Materials and methods

Animals

Eighty‐four male Wistar rats (Rattus norvegicus albinus) were obtained from the mating of females and males acquired from the Center of the Development of Experimental Models for Medicine and Biology (CEDEME‐UNIFESP, Sao Paulo, Brazil). Rats at day 30 postpartum (30 dpp) were distributed into seven groups according to the treatment applied. Up to four rats pertaining to the same group were maintained in polypropylene cages (40 cm × 30 cm × 15 cm) under controlled conditions: hygiene, photoperiod (12‐h light/dark cycle), humidity (60%) and temperature (22–23°C). Free access to water and commercial laboratory chow (Nuvilab CR1; Nuvital Nutrientes, Colombo, Paraná, Brazil) was provided.

Ethical approval statement

This study followed the ethical principles adopted by the Brazilian College of Animal Experimentation. It was approved by the Ethical Committee for Animal Research of the Federal University of Sao Paulo, Brazil (Protocol number (0315/12)).

Experimental design

The prepubertal male rats were distributed into seven groups (n = 12 each); each group contained rats from different dams. The groups were named according to the treatments: 1) sham‐control (SC group), which received a single dose of the STZ's diluent (citrate buffer, pH 4.5) at 30 dpp, by intraperitoneal route; 2) resveratrol‐vehicle (RV group), which was only treated with carboxymethyl cellulose (the RES's diluent) by gavage from the 33 dpp up to the 75 dpp; 3) resveratrol‐treated (R group), which received a dose of RES 150 mg/kg of body weight/ daily/once a day from the 33 dpp up to the 75 dpp; 4) STZ‐induced‐diabetic (D group), treated by a single intraperitoneal injection of streptozotocin (65 mg/kg b.w. in 0.1 M of citrate buffer, pH 4.5) at 30 dpp; 5) STZ‐induced‐diabetic at 30 dpp and treated with RES (DR group) as previously mentioned; 6) STZ‐induced‐diabetic and treated with insulin (DI group) as mentioned in the following item; and 7) STZ‐induced‐diabetic and treated with insulin and RES (DIR group). The protocol implemented in our study (using the single dose of STZ 65 mg/kg b.w.) to induce DM1 in prepubertal rats was based on the studies of Masiello et al. (1975), Lambertucci et al. (2013) and Furman (2015). According to Masiello et al. (1975), higher doses of STZ are required to cause diabetes in younger rats, as their pancreatic β‐cells are more resistant to the damage triggered by this drug.

Experimental diabetes induction and blood glucose measurement (BGM)

DM1 was induced at 30 dpp. Following the STZ treatment, an oral glucose solution was offered to the animals for two subsequent days in a row (2.5% and 5.0%, respectively) to avoid sudden hypoglycaemia resulting from the massive destruction of pancreatic beta cells (Krishnan et al. 2011; Suresh et al. 2013). After the DM1 induction, all animals were daily weighed and scrutinized until euthanasia at 75 dpp.

BGM was obtained at 30 dpp (before STZ injection), 33 dpp (after STZ injection), 45 dpp (peripuberty), 64 dpp (late puberty) and at 75 dpp (young adult – day of the euthanasia). After 10 h of fasting, the rats of SC, RV, R, D, DR, DI and DIR groups had the distal region of their tail pierced with lancets (Accu‐check softclix Roche®, Mannheim, Germany) for the collection of one drop of blood, which was carefully placed in reactive stripes (Accu‐Chek Performa Roche®) for blood glucose measurement in proper glucometer (Accu‐Chek Performa Roche®). The rats with BGM higher than 200 mg/dl 3 days after STZ induction were considered diabetic (Parthasarathy et al. 2009; Lambertucci et al. 2013; Ozcan et al. 2012; Cornejo‐Garrido et al. 2014; Emordi et al. 2016).

Resveratrol and insulin therapies

There are two isoforms of RES: trans‐resveratrol and cis‐resveratrol. In the current research, the trans‐resveratrol was used as the trans‐isomer stability and bioavailability are greater than those from the cis‐isomer (Domínguez et al. 2001; Chen et al. 2007; Das et al. 2008), remaining stable at least for 42 h (Trela & Waterhouse 1996). The trans‐resveratrol used here was extracted from the roots of Polygonum cuspidatum (Sieb‐Xi'an Pharmpro Union Co., Ltd., Xi'an, China).

Resveratrol at higher doses can decrease lipid peroxidation more efficiently and might act as a free radical scavenger according to Bishayee et al. (2010). The oxidative stress is an important and primary cause of male reproductive damage in different adverse conditions causing infertility. Moreover, in a previous study, we found that a higher dose of RES improved significantly the reproductive damage in varicocelized rats (Mendes et al. 2016). Taking into account these subjects, we decided to administer a high RES dose.

Thus, animals of the DR, DIR and R groups received a dose of 150 mg/kg bw/day of RES (by gavage route using an 18‐G stainless steel needle – Thomas Scientific) for 43 consecutive days (from 33 to 75 dpp) as previously described. The procedure was always performed in the morning (before 11 AM), as bioavailability of plasma concentrations of trans‐resveratrol was higher after morning administration (Almeida et al. 2009). Considering that RES has low solubility in water, the phytoalexin was suspended in 10 g/L carboxymethyl cellulose (Juan et al. 2005; Jiang et al. 2008).

For insulin treatment, rats from DI and DIR groups received daily subcutaneous injections of neutral protamine Hagedorn (Humulin®, Eli Lilly and Company, Indianapolis, IN, USA) (Haughton et al. 1999; Rastelli et al. 2005) from 35 to 75 dpp.

Preputial separation (PS)

Attempting to evaluate sexual development of the animals, preputial separation (PS) was inspected on a daily basis from 33 dpp until the completion of the process, considering three anatomical features: (i) start of separation; (ii) incomplete preputial separation, when the glans penis is still covered by prepuce or foreskin; and (iii) complete preputial separation. The degree of separation was measured using manual retraction of the prepuce and a magnifying glass to improve the visualization during the observation (Lewis et al. 2002).

Plasma collection and hormone measurements

At 75 dpp, the animals were weighed and the euthanasia performed through CO2 inhalation (Cartner et al. 2007). Heparin (130 UI/kg, Clexane; Sanofi Winthrop Industrie, Paris, France) was administered by a single intraperitoneal injection 10 min before euthanasia for further blood collection. The inferior vena cava was dissected for the procedure; then, the plasma was separated and stored at −20°C for the hormonal analyses (Oliva & Miraglia 2009). The dose of plasmatic luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) was assessed using a Multiplex® Map Rat Pituitary Magnetic Bead Panel with sensibility of 4.9 pg/ml for LH and 47.7 pg/ml for FSH. Estradiol (E2) and testosterone (T) plasmatic levels were measured by enzyme‐linked immunosorbent assay (ELISA), using commercial kits CEA461Ge and CEA458Ge, respectively (USCN®, Life Science Inc., Texas, TX, USA), and following the manufacturer's instruction. The detection limits were 4.38 pg/mL for E2 and 43.7 pg/mL for T. The intra‐assay precision (% CV) was <10% and the interassay precision was <12% for E2 and T.

Histological procedures

Straightaway after the euthanasia and blood collection, testes, epididymides, seminal vesicles (full and empty) and ventral prostate of the rats were removed and weighed through a semi‐analytical electronic scale (Marte‐AS1000; Marte Científica, Santa Rita do Sapucaí, MG, Brazil).

The relative weight of the testes (mg of testicular weight⁄100 g of body weight) was also calculated; in sequence, the left testes were fixed by immersion in Bouin's fixative for 48 h (Russell 1990); consecutively, specific fragments from these testes were processed for paraffin embedding (P‐3683, Sigma 158 Chemical Co., Bellefonte, PA, USA). Meanwhile, the right epididymides and testes were collected and frozen for spermatic analysis. For histopathological and morphometric analyses, two 4‐μm‐thick non‐consecutive testicular sections (10 cross‐sections of same thickness from each side) were obtained per rat and submitted to the periodic acid–Schiff histochemical method with Harris's haematoxylin counterstaining (PAS + H) (Leblond & Clermont 1952).

Morphometric analysis

To obtain the seminiferous tubule diameter and seminiferous epithelium height, a Leica Application Suite LAS 4.5 (Leica – Wetzlar, Hessen, Germany) image analysis system was used. Fifty seminiferous tubule cross‐sections per testis were randomly sampled and measured using a ×20 objective lens. When sections were slightly oblique, only the minor axis was computed (Miraglia et al. 1990; Stumpp et al. 2004; Miranda‐Spooner et al. 2016).

Histopathological analysis

The histopathological analysis of the testicular sections was performed under a light microscope (n = 7/group). Two non‐consecutive testicular sections were analysed per animal using a Leica Application Suite LAS 4.5 (Leica – Wetzlar) image analysis system under ×40 objective lens and a digital camera attached to the light microscope. A histopathological score was given based on previous studies performed by our group (Oliva & Miraglia 2009; Cabral et al. 2014). Two hundred tubular sections per animal were systematically randomly sampled and analysed. Tubular sections presenting histopathological alterations were categorized in a crescent score (1–3) according to the level of seminiferous epithelium damage: 1 = 0–33.4%; 2 = 34.5–66.8%; and 3 = 67.8–100%.

Sperm quantitative analysis: sperm count, daily production and transit time

The right testes and epididymides were collected after the euthanasia (75 dpp) and frozen at −20°C; subsequently, they were thawed, decapsulated and homogenized on the day of analysis.

Homogenization‐resistant testicular spermatids (step 19) and sperms were collected from the right testes and from the caput/corpus and cauda of the right epididymis respectively for sperm count and for the determination of daily sperm production and sperm transit time (Robb et al. 1978; Blazak et al. 1993; Kempinas et al. 1998). For this purpose testicular spermatids and epididymal sperm number were expressed by organ (sperm/organ) and gram of organ (sperm/g/organ). The daily sperm production (DSP) was determined by dividing the total number of homogenization‐resistant spermatids per testis by 6.1 as this is the length of time which spermatids are kept in the epithelium during the seminiferous cycle (Robb et al. 1978; Blazak et al. 1993; Kempinas et al. 1998). The sperm transit time through the portions of the epididymis (caput+corpus and cauda) was calculated by dividing the number of sperm in each portion by the DSP (Robb et al. 1978; Blazak et al. 1993; Kempinas et al. 1998).

Sperm qualitative analysis: sperm morphology and mitochondrial activity

(a) Sperm morphology

Samples of epididymal fluid (3 μL) were obtained from the cauda of the left epididymis (n = 7/group) and were homogenized in 2 mL of bidistilled water. One drop of this mixture was smeared on to a glass slide and air‐dried. The smears were stained according to Shorr/haematoxylin methodology (Shorr 1941). Two hundred sperms were randomly counted and morphologically evaluated according to the descriptions established by Filler (1993) and adapted for the experimental model (Miranda‐Spooner et al. 2016). The sperms were classified based on the criteria, which are as follows: shape of spermatozoa head; defects in the intermediate piece; defects in the tail; or with multiple abnormalities. The results were expressed as percentage of normal sperm forms.

(b) Mitochondrial activity

The classification of spermatozoa with regard to mitochondrial activity followed the scale proposed by Hrudka (1987): Class I (100% of the intermediate piece stained, indicating 100% of mitochondrial activity), Class II (over 50% of the intermediate piece stained, indicating low reduction in mitochondrial activity), Class III (<50% of stained intermediate piece, indicating high reduction in mitochondrial activity) and Class IV (no staining in the intermediate piece, indicating absence of mitochondria).

This methodology is based on the oxidation of 3,3′‐diaminobenzidine (DAB), by the enzyme cytochrome c‐oxidase, through a chain reaction in which the reagent is polymerized and deposited in the midpiece of the spermatozoa (Antoniassi et al. 2016; Miranda‐Spooner et al. 2016). For this, 50 μL of the sample collected from the left epididymis of each animal (n = 7/group) was added to solution containing 1 mg/mL of DAB in phosphate buffer solution (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH = 7.4) in a ratio of 1:2, and incubated for 1 h in the dark at 37°C. In order to avoid non‐specific staining of the intermediate piece, negative controls were made adding an aliquot of epididymal fluid previously inactivated at 70°C, for 5 min. After the incubation period, 10 μL of each aliquot was smeared on to a microscope slide (Precision Glass Line, China) and air‐dried. The smears were fixed in 10% formaldehyde for 10 min, washed with distilled water, dried again at room temperature, and finally covered by microscope cover slips with synthetic resin (Novo Entellan ‐ Merck KGaA, Darmstadt, Germany). Two hundred cells were counted using ×100 objective lens, in a differential interference contrast Olympus BX51 microscope (Olympus BX‐51TF, Olympus Co. Ltd., Tokyo, Japan).

Testicular oxidation level

After euthanasia, the left testis of each animal (n = 6/group) was collected and decapsulated, and 0.1 g samples were frozen at −20°C. The testicular tissues were then thawed and homogenized in 1.15% KCl solution using a tissue homogenizer (Ultra Stirrer – model ultra 80 II; CB Biotech, São Carlos, SP, Brazil) for 20 min; then, two aliquots were separated for malondialdehyde (MDA) and nitric oxide (NO) doses.

(a) Lipid peroxidation at testicular level: malondialdehyde (MDA) dosage

The lipid peroxidation product in the testis was evaluated through the MDA dose. MDA was quantified through a controlled reaction with thiobarbituric acid, generating thiobarbituric acid reactive substances (TBARS) (Abd‐Allah et al. 2009). A sample of 0.1 mL of the testicular homogenate was placed into a tube containing 1.5 mL of acetic acid (20%, pH 3.5), 0.2 mL of sodium dodecyl sulphate (SDS 8.1%), 1.5 mL of 2‐thiobarbituric acid (TBA, 0.8%) and 0.7 mL of water. Secondly, the tubes were shaken and incubated for 60 min in a water bath at 95°C. Subsequently, the tubes were cooled and centrifuged at 1878 g for 10 min. Finally, the samples were analysed photometrically with microcuvettes, using the enzyme‐linked immunosorbent assay (ELISA); the absorbance was measured at 532 nm using a spectrophotometer (Spectra Max Plus 384®; Molecular Devices, Sunnyvale, CA, USA). Concentration levels were expressed as malondialdehyde nMolar/g tissue according to the methodology described by Abd‐Allah et al. (2009).

(b) Nitric oxide measurement (NO)

The NO measurement was evaluated indirectly using a colorimetric assay, which measured the nitrite concentration from a standard curve with different concentrations of sodium nitrite. A sample corresponding to 500 μL of each testicular homogenate was placed inside a tube with 500 μL of absolute ethanol; in sequence, this homogenate was centrifuged at 1574 g (Centrifuge 5430 R; Eppendorf, Hamburg, Germany), for 10 min. Then, 300 μL of the sample's supernatant was added to 300 μl of vanadium chloride (VCl3, 0.8% in 1 M HCl). Simultaneously, another sample of reagents containing 750 μL Griess 1 plus and 750 μL of Griess 2 was prepared. Then, 500 μL of the previous supernatant sample diluted in vanadium was added (Miranda et al. 2001; Abd‐Allah et al. 2009). This final sample was incubated at room temperature for 30–35 min photometrically with microcuvettes, using the enzyme‐linked immunosorbent assay (ELISA); the absorbance was measured at 540 nm using a spectrophotometer (Spectra Max Plus 384®; Molecular Devices Sunnyvale). The concentrations of NO (nmol/g tissue) were determined from a standard curve of different concentrations of sodium nitrite (Miranda et al. 2001; Abd‐Allah et al. 2009).

Statistical analysis

Statistical analysis was performed using the statistical software sigma plot 12.0 (Systat Software Inc., San Jose, CA, USA). The data that passed in the normality and variability tests were analysed by the one‐way analysis of variance parametric test (anova). When there was statistical significance (p ≤ 0.05), the multiple comparison Student–Newman–Keuls or Bonferroni post‐tests were applied. On the other hand, when the data did not pass the tests of normality and variability, the variance analysis of Kruskal–Wallis, a nonparametric test, was applied followed by the Dunn's multicomparison post‐test, when significant (p ≤ 0.05).

Results

Two rats of the D group and three of the DI group died before the euthanasia (at 75 dpp). Thus, in order to maintain consistency of statistical data, they were replaced.

Blood glucose measurement (BGM)

Rats of the D group did not show reversal of hyperglycaemia during the study. On the day of the STZ induction, no significant differences were observed among the groups relating to BGM. Three days later (after STZ induction), all diabetic animals from D, DR, DI and DIR groups presented a significant BGM increase in comparison with the SC, RV and R groups, although they did not differ among them. In addition, 45 dpp diabetic rats that received either insulin, RES or both (DI, DR and DIR groups) presented at 45 dpp lower glycaemic level in comparison with those from the D group; however, these levels were still higher than in SC, RV and R groups. At 64 dpp and 75 dpp, D group showed a higher BGM than other groups (SC, RV, R, DR, DI and DIR), while DI, DR and DIR rats did not show significant differences in comparison with the SC, RV and R groups (Figure 1).

Figure 1.

Figure 1

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Blood glucose measurement (BGM) at different ages in non‐diabetic and diabetic groups. Values submitted to anova of Kruskal–Wallis followed by Dunn's test (P ≤ 0.05; n = 7). a: D, DR, DI and DIR > SC, RV and R; b: D > DR, DI, DIR > SC, RV and R; c: D > SC, RV, R, DR, DI and DIR.

Preputial separation (PS)

The D, DR, DI and DIR groups showed a significant delay in the time of preputial separation when compared with the SC, RV and R (Figures 2 and 3). Despite this delay, the PS was complete in all groups. However, no significant differences concerning this parameter were observed when all diabetic groups were compared among them.

Figure 2.

Figure 2

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Ages of preputial separation in non‐diabetic and diabetic groups. Values expressed in median and interquartile ranges. Values submitted to anova of Kruskal–Wallis followed by Dunn's test (P ≤ 0.05; n = 7). D, DR, DI and DIR > SC, RV and R.

Figure 3.

Figure 3

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(a) Evolution of preputial separation in rats from 30 to 40 dpp. At 30 dpp, the glans penis is covered by prepuce or foreskin; (b) incomplete preputial separation; (c) complete preputial separation. [Colour figure can be viewed at wileyonlinelibrary.com].

Biometric parameters

The rats of the D group showed a lower body weight at 75 dpp, when compared to those from SC, RV, R, DR, DI and DIR groups, while rats from the DR group showed increased body weight when compared to the D group. Similarly, rats from the DI group showed an improvement in this parameter compared to the D and DR groups. Furthermore, rats from the DIR group showed a higher body weight when compared to those from the D, DR and DI groups and showed similar values compared to the non‐diabetic groups (Table 1).

Table 1.

Body weight and reproductive organ weights of non‐diabetic and diabetic rats at 75 dpp. Values submitted to anova and Bonferroni test (*), expressed as mean ± SD, or submitted to anova of Kruskal–Wallis followed by Dunn's post‐test (†) and expressed as median and interquartile ranges (p ≤ 0.001; n = 7)

Parameters Groups
SC RV R D DR DI DIR
Body weight (g)(*) 334.0 (310.5–350.0) 327.0 (308.0–340.5) 327.0 (313.5–348.0) 219a (194.0–281.0) 270.0a (189.0–335.5) 314.0a (264.5–318.0) 314.0a (283.0–341.5)
Testicular absolute weight (g)(*) 1.68 (0.10–0.02) 1.62 (0.08–0.02) 1.66 (0.10–0.02) 1.09b (0.10–0.02) 1.52 (0.23–0.06) 1.63 (0.09–0.03) 1.57 (0.10–0.03)
Epididymis absolute weight (mg)(*) 48.5 (48.0–54.7) 51.5 (45.0–53.5) 54.0c (51.5–56.0) 37.5c (31.2–42.2) 50.0 (38.7–53.7) 46.0 (37.7–51.0) 49.5 (41.0–52.5)
Ventral prostate absolute weight (mg)(†) 75.5 (69.0–80.2) 73.5 (65.0–76.2) 75.0 (70.2–83.7) 17.0d (11.0–40.0) 39.5e (22.0–46.5) 42.5 (29.0–45.5) 32.0f (26.7–36.0)
Full seminal vesicle absolute weight (mg)(†) 96.0 (85.2–107.5) 89.5 (76.2–96.5) 91.5 (68.0–115.7) 55.5g (18.0–57.0) 77.0 (67.7–88.5) 79.0 (63.5–109.0) 66.5 (64.0–70.0)
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aD < DR < DI < DIR, SC, RV and R; bD < SC, RV, R, DR, DI, and DIR; cD < SC, RV, DR, DI, DIR < R; dD < SC, RV and R; eDR < SC; fDIR < SC and R; gD < SC, RV and R.

The testicular absolute weight was lower in the D group in comparison with the other groups (Table 1). Interestingly, the animals of the R group had a higher absolute weight of the epididymis than all other groups, while in rats from D group the same parameter was lower than in other groups (Table 1).

The absolute weight of the ventral prostate was significantly lower in almost all diabetic groups when compared to the SC control group, except for the DI group. In addition, a significant decrease in absolute weight of the seminal vesicle was observed only in the D group when compared to the non‐diabetic groups (Table 1).

On the other hand, the relative weights of the testes, epididymides and full seminal vesicles in the diabetic groups did not significantly differ when compared to those from non‐diabetic groups (Table S1). On the contrary, the relative weight of the ventral prostate was lower in the diabetic group in comparison with all the other groups (Table 1).

Spermatic parameters: sperm number, sperm morphology and mitochondrial activity

In the D, DI, DR and DIR groups, there was a significant reduction in both counts of mature spermatids and of daily sperm production comparatively to SC, RV and R groups. However, animals of the DR and DIR groups showed an improvement in absolute spermatid number and daily sperm production when compared to the D and DI groups (Table 2).

Table 2.

Testicular and epididymal quantitative spermatic parameters observed in the non‐diabetic and diabetic groups at 75 days of age. Values submitted to anova and Bonferroni test (*) and expressed as mean ± SD, or submitted to anova of Kruskal–Wallis followed by Dunn's post‐test (†) and expressed as median and interquartile ranges (p ≤ 0.001; n = 7)

Parameter Groups
SC RV R D DR DI DIR
Testis
Absolute spermatid number (×106/organ) 126.0 ± 10.7a 107.3 ± 13.3a 132.4 ± 5.2b 58.2 ± 11.7 83.7 ± 16.0c 62.9 ± 7.3 80.9 ± 11.0c
Relative spermatid number (×106/g of testis) 85.3 ± 6.9 76.3 ± 11.6 98.3 ± 17.7 50.6 ± 69.1d 83.8 ± 56.1 83.8 ± 56.1 72.0 ± 24.5
Daily sperm production (×106/day) 20.6 ± 1.7e 17.5 ± 2.1e 21.7 ± 2.4 9.5 ± 1.9e 13.7 ± 2.6e 10.3 ± 1.2e 13.2 ± 1.8e
Epididymal caput/corpus
Absolute sperm number (×106/organ)† 73.5 ± 9.4f 66.6 ± 4.9f 84.9 ± 15.7g 29.1 ± 6.6 43.7 ± 3.7h 34.6 ± 4.5 43.5 ± 5.7h
Relative sperm number (×106/g of organ)† 268.7i (256.2–273.7) 242.8i (240.2–231.3) 306.47i (283.7–326.9) 137.5j (111.9–160.0) 200.3 (187.5–217.5) 156.7j (149.3–165.0) 186.6 (178.7–212.8)
Sperm transit time (days)* 3.5 ± 0.5 3.8 ± 0.4 3.9 ± 0.4 3.0 ± 0.4k 3.2 ± 0.4 3.4 ± 0.6 3.2 ± 0.4
Epididymal cauda
Absolute sperm number (×106/organ)* 125.7 ± 19.5l 109.9 ± 15.5l 153.3 ± 13.0l 43.5 ± 11.3l 85.5 ± 14.9l 60.3 ± 13.2l 75.7 ± 15.3l
Relative sperm number (×106/g of organ)* 680.4 ± 89.9m 544.2 ± 89.7n 781.5 ± 49.3m 307.8 ± 58.9 522.5 ± 65.9 389.4 ± 54.7n 452.2 ± 52.7o
Sperm transit time (days)* 6.0 ± 0.5 5.8 ± 0.8 7.2 ± 0.6 4.5 ± 0.8p 6.2 ± 0.7 5.9 ± 1.7 5.7 ± 1.3
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aSC and RV > D, DR, DI and DIR; bR > SC, RV, D, DR, DI and DIR; cDR and DIR > D and DI; dD < SC, RV, R, DR, DI and DIR; eD and DI < DR and DIR < RV and SC < R; fSC and RV > D, DR, DI and DIR; gR > RV, D, DR, DI and DIR; hDR and DIR > D and DI; iSC, RV and R > D, DR, DI and DIR; jD and DI < DR and DIR; kD < R; lD, DR, DI and DIR < SC and RV < R; mSC and R > RV, D, DR, DI and DIR; nRV and DR > D and DI; oDIR > D; pD < R.

Sperm counts in the epididymal portions are also demonstrated in Table 2. In the epididymal caput/corpus portion, there was a reduction in absolute sperm count in all diabetic groups in comparison with SC, RV and R groups. In addition, in this region, there was a reduction in absolute and relative epididymal sperm count in D and DI rats in comparison with the DR and DIR rats. In the epididymal cauda, a lower absolute and relative sperm count was observed in the D group comparatively to the DR and DIR and to the SC, RV and R groups. A reduction in sperm transit time in both epididymal caput/corpus and epididymal cauda was also noted in the D group in comparison with the R group (Table 2).

The diabetic animals showed a decrease in the frequency of morphologically normal sperm in comparison with SC, RV and R groups. They presented several types of abnormalities, including alterations of the head (banana‐shaped, detached, backward bent) and tail (coiled, bent, broken). The most frequent alterations occurred in diabetic groups were bent tail, sperm head detachment from its tail and banana‐shaped head sperm. Furthermore, spermatozoa with multiple defects were also frequently noted in diabetic groups, but very rarely observed in non‐diabetic ones. However, the frequency of morphologically normal sperm was still significantly lower in D and DI than in DR and DIR groups (Figure 4).

Figure 4.

Figure 4

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Morphological sperm analysis of non‐diabetic and diabetic groups at 75 dpp. Values expressed in median and interquartile ranges. Values submitted to anova of Kruskal–Wallis followed by Dunn's test (p ≤ 0.05; n = 7). a: SC > D, DI and DIR; b: RV and R > D, DR, DI and DIR; c: DR and DIR > D and DI (p ≤ 0.05).

Sperm mitochondrial activity also showed a significant drop in the diabetic groups. Thus, while the D group had a lower number of Class I sperms in comparison with SC, RV, R, DR and DIR, all diabetic groups presented a higher number of Class II in relation to non‐diabetic ones. In addition, the DR, DI and DIR groups presented a decrease in Class III sperm and an increase in Class I in comparison with the D group. Interestingly, regarding the Class IV of mitochondrial activity, the frequency from the D group was higher than that from all other groups, while in the DI group it was higher than in the DIR group; in this last group, the frequency of Class IV sperm was higher than that observed in the DR group and in the non‐diabetic rats (Table 3 and Figure 5).

Table 3.

Analysis of mitochondrial activity in non‐diabetic and diabetic groups at 75 days of age. Values submitted to anova and Bonferroni test (*), expressed as mean ± SD, or submitted to anova of Kruskal–Wallis followed by Dunn's post‐test (†), expressed as median and interquartile ranges (p ≤ 0.001; n = 7)

Mitochondrial activity Groups
SC RV R D DR DI DIR
Class I(†) 93.5 (92.0–95.0) 94.5 (91.0–96.0) 97.0 (96.0–98.5) 57.5a (53.5–62.0) 82.5 (81.0–85.0) 76.5 (69.5–78.0) 78.0 (75.0–85.0)
Class II(*) 4.2 ± 1.4 4.2 ± 2.3 2.6 ± 2.3 22.7 ± 4.9b 13.4 ± 4.9b 15.1 ± 3.4b 11.7 ± 3.6b
Class III(†) 1.5 (1.0–2.5) 1.5 (1.0–2.0) 0.5 (0.0–1.0) 11.0c (9.5–16.0) 5.0d (3.0–7.5) 5.5d (4.5–7.0) 5.5d (4.5–7.0)
Class IV(†) 0.0 (0.0–0.5) 0.5 (0.0–1.0) 0.0 (0.0–0.0) 6.5e (5.5–8.5) 0.0 (0.0–2.0) 3.5e (2.5–5.0) 2.0e (1.0–4.5)
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aD < SC, RV, R, DR and DIR; bD, DR, DI and DIR > SC, RV and R; cD > SC, RV, R, DR, DI and DIR; dDR, DI and DIR > SC, RV and R; eD > DI > DIR > SC, RV, R and DR.

Figure 5.

Figure 5

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Examples of mitochondrial activity classification using DAB staining in sperm (a–c) at 75 dpp. (a) Class I sperm showing 100% active mitochondria; (b) Class II sperm (long arrow) showing >50% of active mitochondria and Class III sperm (short arrow) with <50% of active mitochondria; (c) Class IV sperm with 100% of inactive mitochondria. [Colour figure can be viewed at wileyonlinelibrary.com].

Hormone levels

The LH plasma level was lower in the D group when compared to the SC, RV, R, DR and DI groups (Figure 6a). The FSH plasma level in animals from the D group was lower when compared to other groups, but no statistical significance was observed (Figure 6b). Estradiol level was also reduced in D, DI and DIR groups when compared to the SC, RV, R and DR groups and this last one did not significantly differ from non‐diabetic groups (Figure 6c).

Figure 6.

Figure 6

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Plasmatic hormone levels of LH (a), FSH (b), estradiol (c) and testosterone (d) of non‐diabetic and diabetic groups at 75 dpp. Values submitted to anova of Kruskal–Wallis and Dunn's post‐test and expressed as median and interquartile ranges (p ≤ 0.05; n = 10). a: D < SC, RV, R, DR and DI; b: D, DI and DIR < SC, RV, R and DR; c: D < SC, RV, R, DR, DI and DIR.

The testosterone plasma level was lower in the D group than in all other groups (Figure 6D). In diabetic rats that were treated with resveratrol and/or insulin, the testosterone levels approached the normal.

Oxidative stress

There was an increase in MDA concentration, a marker of lipid peroxidation, in the D group animals in comparison with all other groups (Figure 7a). The concentration of NO (indirectly calculated by the nitrite dose) was also significantly increased in the D group when compared to the SC, DR and DI groups (Figure 7b).

Figure 7.

Figure 7

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Testicular levels of malondialdehyde (MDA) concentration (a) and nitrite concentration (b) at 75 dpp. Values submitted to anova of Kruskal–Wallis followed by Dunn's test and expressed as median and interquartile ranges (p ≤ 0.05; n = 6). a: D > SC, RV, R, DR, DI and DIR; b: D > SC, DR and DI.

Morphometric analysis

The tubular diameter of the androgen‐dependent seminiferous tubules (stages VII and VIII of the seminiferous epithelium cycle) was significantly lower in the D and DI groups than in SC, RV, R, DR and DIR groups. There was also a reduction in this parameter in the DR and DIR groups in comparison with the R and RV groups. Furthermore, this parameter was higher in the R group than in the SC group (Figure 8a). The seminiferous epithelium height was also significantly lower in the D and DI groups, than in the other groups; in addition, this measurement was lower in the D group in relation to the DI group (Figure 8b).

Figure 8.

Figure 8

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Measurements of the tubular diameter (a) and of the seminiferous epithelium height (b) in sections of androgen‐dependent tubules (stages VII and VIII) of non‐diabetic and diabetic groups at 75 dpp. Values were submitted to anova and SNK post‐test and expressed as mean ± SD (a) or anova of Kruskal–Wallis and to the Dunn's post‐test and expressed as median and interquartile ranges (b) (p ≤ 0.05; n = 7). a: R > SC; b: D and DI < SC, RV, R, DR and DIR; c: DR and DIR < RV and R; d: D and DI < SC, RV, R, DR, DI and DIR; e: D < DI.

Histopathological analysis

At 75 dpp, the animals of the SC, RV and R groups displayed normal histological characteristics of seminiferous epithelium and presented all typical cellular associations of the stages of the seminiferous epithelium cycle of an adult normal rat (Figure 9a–c). In all diabetic groups, there was a large germ cell desquamation leading to a cellular loss of the intermediate and adluminal layers of the seminiferous epithelium (Figure 9d–h). Vacuolization and folding of the seminiferous epithelium were also commonly observed in testicular sections of the D rats (Figure 9d1,d2). Rats of the D and DI groups usually presented a large amount of germinal lineage cells detached into the tubular lumen and cellular debris (Figure 9d,f).

Figure 9.

Figure 9

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Photomicrographs of testicular cross‐sections of non‐diabetic (SC, RV, R), diabetic (D) and diabetic‐treated rats (DI, DR, DIR) at 75 dpp. PAS+H. Non‐diabetic groups (a–c) show normal histological characteristics of the seminiferous epithelium (Ep); interstitial space (IT); tubular lumen (L). Note in the diabetic groups (d–h): detached cells into the tubular lumen (arrows), disorganized and discontinuous epithelium (triangles), intraepithelial vacuolization (asterisks). In the diabetic groups treated with resveratrol (DR and DIR groups), sections with normal cell associations are also present (e, h). Note also the epithelial invagination (e, f) and multinucleate cell (g – inset) in diabetic rats. [Colour figure can be viewed at wileyonlinelibrary.com].

In general, the histopathological alterations were less frequent in the DR and DIR groups than in the D and DI groups; however, they were more frequent in the DR‐related and DIR than in the SC, R and RV groups. Table 4 shows the histopathological analysis summarized and represented as a score related to the major alterations observed, ranging from 1 to 3.

Table 4.

Percentage of rats presenting specific histopathological alterations and score of the frequencies (1–3) of the tubular alterations found in non‐diabetic and diabetic groups (n = 7) at 75 dpp

Types of histological alterations Groups (%/score)
SC RV R D DR DI DIR
Sloughed germ cells into the lumen 28.6/1 28.6/1 14.2/1 100/3 57.1/2 85.7/3 57.1/2
Cellular depletion 28.6/1 28.6/1 14.2/1 100/3 42.8/2 71.4/3 42.8/2
Intraepithelial vacuolization 14.2/1 14.2/1 0/1 100/4 42.8/2 57.14/2 42.8/2
Epithelial invagination 0/1 0/1 0/1 42.8/2 28.6/1 57.1/2 28.6/1
Multinucleate cells 0/1 0/1 0/1 28.6/1 0/1 28.6/1 0/1
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Discussion

In DM1, the hyperglycaemia‐related increase in oxidative stress is due to the higher levels of reducing sugars that can quickly interact with lipids and proteins, enhancing ROS production (Heidari et al. 2008). Repeated ROS insult can change the permeability of the mitochondrial membrane, a known mechanism of altering sperm motility and morphology (Amaral et al. 2006; Chatterjee et al. 2013). Besides, sperm plasma membrane has a great concentration of polyunsaturated fatty acids (PUFAs), and this makes it highly susceptible to oxidation (Chatterjee et al. 2013; Suresh et al. 2013; Giribabu et al. 2014).

Concerning the experimental model of induction of diabetes mellitus in rats, we have found methodological variations in the database regarding the (i) insulin administration, (ii) hyperglycaemia rate used for the selection of diabetic animals and (iii) duration of the experiment and age of euthanasia. However, the great majority of them used animals at adulthood (Seethalakshmi et al. 1987; Soudamani et al. 2005; Scarano et al. 2006). Anyway, STZ‐induced DM1 in rats causes similar reproductive alterations as reported in humans (Ganda et al. 1976). Thus, the model used here seems to be suitable for this type of study.

As expected from previous studies, (Seethalakshmi et al. 1987; Faid et al. 2015), reduction in body weight was observed in diabetes‐induced rats analysed which was otherwise partially reversed by the treatment with either insulin or RES. However, Faid et al. (2015) did not observe a recovery of body weight loss in diabetes‐induced adult rats treated with RES (5 mg/kg of b.w.) for 21 days (5 days per week/once daily), which suggests that higher doses of RES can be more effective in order to improve this parameter.

In the current study, BGM was also progressively normalized until adulthood when diabetic rats were treated with RES and/or insulin. In contrast, Faid et al. (2015) did not observe amelioration of the glycaemia in RES‐treated diabetic rats; however, they induced diabetes in adult animals and used a different protocol, administering a much lower RES dose (5 mg/kg of body weight) by an intraperitoneal route. In addition, Yonamine et al. (2017) showed that RES improves glycaemic control in diabetic rats treated with insulin; according to this report, such effect was a consequence of an increase in sirtuin 1 nuclear protein content in the liver and changes in the expression downregulation of some specific genes, favouring a reduction in glucose production and efflux. However, they did not compare the glycaemic levels of diabetic RES‐treated and diabetic‐insulin‐treated rats among them. In the current study, there was no improvement of glycaemia in diabetic rats of DIR group in comparison with the DI group; this raises the hypothesis that an enhancement of glycaemic control – through gene expression control – may also have occurred in these treated rats that had diabetes induced from prepuberty.

The scavenger properties of RES could explain such phenomena (Bagul & Banerjee 2015). These results confirm the previously referred antidiabetic properties of RES (Chi et al. 2007; Silan 2008; Chang et al. 2011; Mohamad Shahi et al. 2011; Ku et al. 2012; Jiang et al. 2013) and show, for the first time, a comparison between the effects of RES and insulin on glucose levels. The non‐supplementary effect of RES on glycaemia in short and long term, when administered together with insulin, is also suggested in the present study. In fact, prolonged administration of RES does not affect glucose levels in normoglycaemic animals (Szkudelska & Szkudelski 2010). Likewise, we did not observe alterations of BGM at any age analysed here in non‐diabetic rats that were treated only with RES. However, different anti‐hyperglycaemia mechanisms are involved between insulin and RES, and this subject must be more thoroughly investigated in the future. Impairment of male reproductive function is among the hyperglycaemia‐related complications (Hassan et al. 1993; Soudamani et al. 2005; Rohrer et al. 2007), which includes the delay in pubertal onset (Clarson et al. 1985; Elamin et al. 2006; Rohrer et al. 2007; Vendramini et al. 2014). The establishment of puberty is orchestrated by intense hormonal regulation, especially of testosterone feedback mechanisms (Korenbrot et al. 1977). As presented here, the diabetic rats had a delay in the PS, an androgen‐dependent phenomenon that characterizes pubertal development (Andretta et al. 2014). Although RES and insulin have improved the testosterone plasma level, neither could normalize the time of PS in the diabetic rats. However, we could not find comparable results in the literature relating to the balanopreputial separation and induced diabetes binomial (Qiu et al. 2013).

Besides the increase in testosterone levels, diabetic animals that received insulin and/or RES also showed an improvement in LH plasmatic level, while the E2 level was only improved in RES‐treated diabetic rats. Indeed, RES is known to be a phytoestrogen and stimulates the activity of the pituitary–hypothalamic–testicular axis in healthy rats, which positively affects sperm quality (Juan et al. 2005). Another explanation for the protective effect of RES on steroidogenesis regarding testosterone production might be as a consequence of the direct activity of RES on the Leydig cells, where it contributes to the upregulation of the anti‐oxidant defence mechanisms and of the LH level (Wang et al. 2014). On the other hand, it is important to mention that the anti‐oestrogenic activity of RES is not a consensus finding, and remains to be better understood (Bhat et al. 2001).

Concerning the seminiferous epithelium histopathological alterations, previous evidence indicates that the induction of DM1 by STZ promotes extensive tissue damage, culminating with a series of reproductive disturbances (Rohrer et al. 2007; Chandrashekar 2010) as observed here. In fact, in the current study, diabetic animals demonstrated the presence of cellular debris as well as germ cell detachment into the tubular lumen as previously described by Ricci et al. (2009) and Faid et al. (2015). Diabetic animals also displayed a decrease in androgen‐dependent tubule diameter and a reduction in seminiferous epithelium height, as already observed by previous studies (Rohrer et al. 2007; Gosálvez et al. 2013).

On the other hand, our results show that the atrophy and reduction in seminiferous tubules diameter were more efficiently attenuated by the RES treatment in comparison with the insulin. Thus, we can hypothesize that there was a multiple action on steroidogenesis and spermatogenesis promoted by RES, especially through its extensive free radical scavenger and anti‐apoptotic properties (Aitken & Roman 2008).

The effects of diabetes on sperm quality and function are still poorly understood (Mallidis et al. 2011); however, traditional semen parameters (sperm morphology, motility and concentration) are usually reported as being reduced in patients with diabetes (La Vignera et al. 2012; Alves et al. 2013) as well as in diabetes‐induced rats (Faid et al. 2015; Abdelali et al. 2016). Furthermore, the relationship between diabetes and increase in DNA damage on the spermatozoa of men with diabetes impairs their fertility and reproductive health (Agbaje et al. 2007). Also, the Zucker rats, an experimental model of metabolic syndrome that progressively develop hyperglycemia, show lower sperm DNA integrity (Vendramini et al. 2014).

In the current study, diabetic rats showed altered quantitative spermatic parameters as previously observed by Scarano et al. (2006). In contrast to our results, these authors did not detect alterations in the sperm transit time. This divergent result could be mainly due to the difference in the diabetes protocol of induction and to the time of euthanasia, considering that they used adult rats at the beginning of the experiments. The improvement of sperm number in the RES‐treated diabetic rats was also expected, as RES is known to improve sperm output also in normal rats (Juan et al. 2005). In this matter, using the protocol presented here, insulin seemed to improve sperm counts but not as effectively as RES, which reinforces the theory that the mechanism of action of RES on the germinal epithelium is multifactorial. In a similar way, the series of alterations in sperm morphology in the current analysis, which were already described by Azeez et al. (2010) and Suresh et al. (2013), were also improved by the treatment with RES, but not by the treatment with insulin.

Regarding exogenous insulin therapy as an approach to prevent the damage caused by hyperglycaemia on male reproductive system, there are few experimental studies in vivo. Besides, it seems that even plasma insulin cannot pass through the blood–testis barrier. However, the beneficial action of exogenous insulin on hypothalamic–pituitary–gonadal axis observed in Akita diabetic mice seems to promote an improvement in spermatogenesis (Schoeller et al. 2012).

Increased levels of ROS are known to have a negative impact on the quality of sperm (Aitken & Roman 2008). Herein, we showed that the diabetic rats treated with both RES and insulin had reduced testicular levels of two markers of oxidative stress: MDA and NO. Indeed, a reduction in ROS production promoted by the RES was already detected in a model of experimental testicular ischaemia (Yuluğ et al. 2014), as well as against the oxidative stress caused by doxorubicin treatment, in rats (Türedi et al. 2015). In addition, the competence of RES to reduce lipid peroxidation within the testis has been confirmed in rats with diabetes induced in adulthood (Faid et al. 2015). However, this capability had not yet been well documented at the level of the epididymides.

So far, a reduction in MDA concentration and an improvement in superoxide dismutase activity had only been observed at the corpus cavernosum level in RES‐treated diabetic rats with erectile dysfunction (Yu et al. 2013). Conversely, a recent report suggested that in left‐sided experimental varicocele, there was no significant reduction in testicular concentration of MDA provided by RES against the oxidative stress seen in the varicocelized rats (Mendes et al. 2016).

Considering that there was also an improvement of mitochondrial activity in DM1 animals, promoted by RES, we believe that the decrease in testicular ROS production was effectively promoted by the treatment with RES in DM1 STZ‐induced rats, reinforcing the important biological properties of RES as a scavenging and/or anti‐oxidant substance in this condition (Bhat et al. 2001). As observed in other sperm parameters, RES seems to be more prone to protect the germ cells within the seminiferous epithelium than insulin, and we believe this observation has not yet been reported.

In conclusion, RES has shown antidiabetogenic activity without interfering with the BGM in non‐diabetic rats and was more efficient than insulin to improve most of the reproductive parameters analysed in the model of STZ‐induced diabetes used in the present report. The inclusion of RES in clinical protocols as an adjuvant therapy for patients with diabetes could potentialize the conventional treatments and deserves detailed investigation.

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this article.

Funding source

This work was financially supported by The National Council for the Improvement of Higher Education (CAPES/ Brazil).

Author contributions

JNS performed all the experimental work, contributed to the analysis of the results and writing of this manuscript. TBM contributed to the experimental work. CCP contributed to the writing and revision of the manuscript. VV contributed to the results analysis and revision of the manuscript. SMM executed the experimental design, supervised all the experimental work and analysis of results, writing and reviewing the manuscript. All authors read and approved the final manuscript.

Supporting information

Table S1. Body weight and the reproductive organ weight of non‐diabetic and diabetic rats, at 75 dpp.

Click here for additional data file. (476.4KB, pdf)

Acknowledgements

The authors thank the Laboratory of Urology at UNIFESP for helping with the equipment for the mitochondrial activity analysis; the Institute of Pharmacology (INFAR) – Laboratory of Experimental Endocrinology – UNIFESP for the use of the spectrophotometer in oxidative stress measurements. We would also like to thank Flávia M.O. Neves and Regina E. L. Cabral for the help with oxidative stress analysis protocol at the Laboratory of Developmental Biology – UNIFESP.

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Table S1. Body weight and the reproductive organ weight of non‐diabetic and diabetic rats, at 75 dpp.

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