|Year : 2019 | Volume
| Issue : 3 | Page : 203-209
Can pentoxifylline recover reproductive parameters' damage induced by high-protein diet in male rats?
Mohammad Reza Salahshoor1, Amir Abdolmaleki1, Cyrus Jalili2, Shiva Roshankhah1
1 Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Department of Anatomical Sciences, Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
|Date of Web Publication||6-Sep-2019|
Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Daneshgah Ave., Taghbostan, Kermanshah
Source of Support: None, Conflict of Interest: None
Introduction: Pentoxifylline (PEN) is a xanthine derivative used as a drug to inhibit the inflammatory factors activity, reduce blood viscosity and improve peripheral blood flow. Proteins play the most important role in reproductive parameters. The aim of the present investigation is to evaluate the effects of PEN against high-protein diet (HPD)-induced damage to the reproductive parameter of male rats. Materials and Methods: In this experimental study, 48 male rats were randomly divided to 8 groups: sham (normal protein diet) and HPD (35% protein) groups; PEN groups (25, 50 and 100 mg/kg) and HPD + PEN groups (25, 50 and 100 mg/kg). Animals of the HPD group have fed with high protein daily for 10 months. Daily PEN treatment was injected intraperitoneally. The sperm parameters, spermatogenesis index, total antioxidant capacity, testosterone level and seminiferous tubule diameter were analysed. Results: The values of all reproductive parameters reduced significantly in the HPD group compared to the sham group (P < 0.01). The whole doses of PEN and PEN + HPD groups increased all parameters significantly compared to the HPD group (P < 0.01). Conclusions: No significant modifications were observed in PEN groups in all doses compared to the sham group. PEN relieved the effects of HPD on reproductive parameters.
Keywords: High-protein diet, pentoxifylline, reproductive parameters
|How to cite this article:|
Salahshoor MR, Abdolmaleki A, Jalili C, Roshankhah S. Can pentoxifylline recover reproductive parameters' damage induced by high-protein diet in male rats?. Adv Hum Biol 2019;9:203-9
|How to cite this URL:|
Salahshoor MR, Abdolmaleki A, Jalili C, Roshankhah S. Can pentoxifylline recover reproductive parameters' damage induced by high-protein diet in male rats?. Adv Hum Biol [serial online] 2019 [cited 2020 Mar 31];9:203-9. Available from: http://www.aihbonline.com/text.asp?2019/9/3/203/266226
| Introduction|| |
As an accepted fact, the male reproductive system has a life cycle for physiological, biochemical and anatomical evolutions. It is not surprising to know that the nutrition factors are considered to be effective on function and structure of male reproductive system. Nearly two-third of metabolic items of the body comprised proteins. High-protein diet (HPD) may affect the biochemical structure of male reproductive parameter. There are little published data on how protein diets can affect the reproductive parameters. The relationship between a normal protein diet and appropriate function of reproduction has always been discussed. The development of male reproductive system depends on the amount of protein intake. Biosynthesis of male reproductive system proteins is related to the efficient and consistent normal receipt of amino acids. Furthermore, it seems that long-term consumption of HPD induces the oxidative stress, and consequently, the production of free radicals (such as superoxide and hydroxyl radicals) leads to reproductive system damage. Free radicals' presence in tissue results in membrane lipid peroxidation and changes of enzymatic activity which eventually lead to cellular damage and necrosis. This phenomenon occurs by an attack on unsaturated fatty acids and alkylation of protein groups and other cellular macromolecules. The production of reactive oxygen species (ROS) leads to arrest in the cell cycle and increases the apoptosis process, thereby daily sperm production and total sperm number reduce. However, cellular defense system is not fully defensible to prevent free radical damages, especially under acute conditions. In this regard, it should be mentioned that the application of antioxidant agents will help to reduce the damages and prevent related diseases. Pentoxifylline (PEN) as one of the methylxanthine derivatives and non-competitive inhibitors of phosphodiesterases increases the intracellular cyclic adenosine monophosphate (cAMP) that activates the kinase protein and inhibits the tumour necrosis factor activities, thereby reduces the leukotriene production and inflammation and also enhances the innate immunity. PEN has antioxidant effects too which seem to be triggered by activated neutrophils in response to the superoxide produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In addition, the use of PEN powder can increase the sperms' motility. Regarding the high prevalence of male infertility, the HPD in some people, especially bodybuilders, has destructive effects due to the increased oxidative stress induction. The purpose of this article is to evaluate the effects of PEN on reproductive parameters changes of male rats followed by HPD consumption damage.
| Materials and Methods|| |
Forty-eight male Wistar rats) 220–250 g (were purchased from Pasteur Institute and transferred to the animal house of a medical school. During the study, the animals were kept under standard conditions including special cages, straw bed and free access to food and municipal water. 12-h light/12-h dark and 22°C ± 2°C were provided too. All investigations conformed to the ethical and humane principles of research and were approved by the Ethics Committee of our institute (ethics certificate No. 1396.134).
Study groups and treatment of animals
Forty-eight male rats were randomly divided into eight groups and six rats for each group. The first group as the sham group received a normal protein diet (17.5% protein). Second group as the HPD group received a HPD (35% protein). Third to fifth groups as the PEN administration groups, respectively, received 25, 50 and 100 mg/kg of PEN orally (gavage) and normal protein diet. Sixth to eighth groups as HPD + PEN administration groups in which each animal, respectively, received 25, 50 and 100 mg/kg of PEN orally and HPD., The base energy in normal and HPD was similar, and protein diet for all groups was applied for 10 months.
Animals' dissection and sampling
After 28 days of animals' treatment, the rats were killed by placing in a plastic chamber containing ether-impregnated cotton (Merck). Immediately, the blood sample was collected by subxiphoid aspiration with no excision on the chest wall. The blood was poured into test tubes and was kept in a 37°C incubator for 20 min to be prepared for centrifugation (255 g for 15 min). The blood serum was isolated, part of which was kept at −70°C for measurement of total antioxidant capacity (TAC), nitric oxide and testosterone levels. Then, the thoracotomy and laparotomy were applied, respectively. The epididymis tail was isolated from the testes and placed in DMEMF12/fetal bovine serum 5% culture medium. The testicles were removed from the abdominal cavity and fixed in a 10% formalin solution.
Sperm cells' collection
The epididymides were dissected. The caudal pole was used for valuation of sperm parameters, and the left testis was selected for histological staining. Both cauda epididymides from each animal were crushed and conserved in a warmed petri dish containing 10 ml Hank's balanced salt solution at 37°C. The spermatozoa were allowed to disperse into the buffer. After 15 min, the cauda was removed, and the suspension was slightly shaken to be normalised. Then, it was observed by a light microscope at ×400.
In this section, four types of sperm motility were studied based on the WHO methods, Class A: progressive motility. Progressive motility of the sperms was examined by an optical microscope at ×40 in 10 fields of view. For this purpose, at first, about 50 μl of semen liquid culture medium was placed on a slide culture that was previously cleaned and dried with alcohol. Then, the slide culture was placed on this and examined by a microscope. In each sample, about 100 sperms were counted through a cell count device. In all experimental and control groups, the count was repeated.
Eosin staining was used to identify the living sperms from dead ones. The basis of this method is the transition of stain from the membrane of dead cells and its excretion by the membrane of living cells. At the end of the given time, about 20 μl of medium containing semen fluid was collected from each dish and then mixed with the same volume of eosin staining solution (about 20 μl). 2–5 min later, a part of the mixture was poured onto a Neubauer culture slide. The living sperms lack stain and dead sperms become pink. The prepared slide culture was examined by ×40. At least 100 sperms from each random sample were calculated to get a percentage of living cells according to the 10 fields of imagining.
The normal sperms' morphology was assessed through the examination of sperm smears driven from the right cauda epididymis. To appraise the spermatozoal malformations, an aliquot of the sample was used to make the smears. Eosin/nigrosin staining technique was used to assess the normal spermatozoal morphology. One drop of eosin stain was added to the suspension and mixed slightly. The slides were then observed by a light microscope (×40). A total of 400 spermatozoa were studied on respective slide (4000 cells in each group) for irregularities of the head and tail.
To analyse the number of sperm cells, 400 μL of the sperm suspension was diluted in formaldehyde fixative (Sigma; USA). Approximately, 15 μL was removed from the diluted solution into a haemocytometer by a Pasteur pipette. The haemocytometer was located into a Petri dish with dampened filter paper and allowed to stand for 10 min. The stable sperms were counted and assessed per 250 small squares of the haemocytometer using a 40× objective lens. The number of sperm per mm3 was obtained by the number of sperm counted × the dilution/number counted in mm2 × the depth of the chamber.
Seminiferous tubules' diameter
After testes' fixation, they were dehydrated, cleared and embedded in paraffin. Hematoxylin and eosin staining was applied for 5-μm sections. More than thirty sections were prepared from each block. The mean diameter of seminiferous tubules was measured using a Motic camera and software (Olympus Optical, Tokyo, Japan).
Ferric-reducing ability of plasma method
Ferric-reducing ability of plasma technique is used to measure the TAC exists in blood serum. In this technique, the ability of the plasma to reinstate ferric ions was measured. This process consumed a great quantity of FeIII. By turning the FeIII-TPTZ in acidic pH to FeII, a blue stain was formed and absorption occurred at the maximum wavelength of 600 nm. The factor defining the speed of the FeII-TPTZ formation and the produced blue colour was only the vitalising power of the sample. The values of TAC are strategised by the means of the standard curve of diverse concentrations of iron sulphate.
The collected blood sample was centrifuged at 5000 g in 23°C for 15 min to get blood serum. The serum samples were kept in a deep freezer (−18°C) for analysis of testosterone level through ELISA (Abcam 108666, USA) technique.
Testis tubules were evaluated for their modified spermatogenesis index (SI) through Johnson's score. Based on this score, a grade from 1 to 10 was given to each tubule cross section ranging from no cells to complete spermatogenesis.
After collecting data, the Kolmogorov–Smirnov test was first conducted to confirm data compliance of the normal distribution. One-way analysis of variance and Tukey's post hoc test were used for statistical analysis and determination of differences between the groups, respectively. SPSS 16.0 (New York: IBM, USA) was used for data analysis, and the results were expressed as mean ± standard error, and P < 0.05 was considered as statistically significant.
| Results|| |
Progressive sperm motility and viability
HPD group caused a significant reduction in the sperm progressive motility and viability compared to the sham group (P < 0.01). No significant variations were detected in PEN groups compared to the sham group (P > 0.05). Furthermore, sperm viability and progressive motility in all PEN and HPD + PEN groups increased significantly compared to the HPD group (P < 0.01) [Figure 1] and [Figure 2].
|Figure 1: Correlation analysis between treatment groups for sperm viability. *Significant difference compared to the sham group (P < 0.01).†Significant difference compared to the HPD group (P < 0.01).‡Significant difference compared to the HPD group (P < 0.01). PEN: Pentoxifylline, HPD: High-protein diet.|
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|Figure 2: Comparison of progressive sperm motility between treatment groups. *Significant decrease compared to the sham group (P < 0.01). †Significant difference compared to the HPD group P < 0.01). ‡ Significant difference compared to the HPD group (P < 0.01). PEN: Pentoxifylline, HPD: High-protein diet.|
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Sperm count and normal morphology
The sperm count and morphological normality reduced significantly in the HPD group compared to the sham group (P < 0.01). No significant deviations were detected in the PEN groups in contrast to the sham group (P > 0.05). However, the sperm count and normal morphology were enhanced significantly in all PEN and HPD + PEN groups compared to the HPD group (P < 0.01) [Figure 3] and [Figure 4].
|Figure 3: Effects of HPD, PEN and PEN + HPD on sperm count. *Significant decrease compared to the sham group (P < 0.01).†Significant difference compared to the HPD group (P < 0.01).‡Significant difference compared to the HPD group (P < 0.01). PEN: Pentoxifylline, HPD: High-protein diet.|
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|Figure 4: Comparison of normal sperm morphology in treatment groups. *Significant increase compared to the sham group (P < 0.01).†Significant difference compared to the HPD group (P < 0.01).‡Significant difference compared to the HPD group (P < 0.01). PEN: Pentoxifylline, HPD: High-protein diet.|
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Seminiferous tubules' diameter
HPD group caused a significant reduction in the diameter of the seminiferous tubules compared to the sham group (P < 0.01). No significant alterations were observed in PEN groups compared to the sham group (P > 0.05). The diameter of seminiferous tubule in all PE
N and HPD + PEN groups enhanced significantly compared to the HPD group (P < 0.01)[Figure 5] and [Figure 6].
|Figure 5: Comparison of seminiferous tubule diameter in treatment groups. *Significant difference compared to the sham group (P < 0.01).†Significant difference compared to the HPD group (P < 0.01).‡Significant difference compared to the HPD group (P < 0.01). PEN: Pentoxifylline, HPD: High-protein diet.|
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|Figure 6: Effect of HPD, PEN and PEN + HPD on seminiferous tubules diameter (×40). Normal seminiferous tubule structure was observed in sham group (a) and PEN group (100 mg/kg) (b). A decrease in seminiferous tubules' diameter, germinal layer and sperm was observed in HPD group (c). Normal seminiferous tubule structure was observed in PEN + HPD (100 mg/kg) group (d). Black arrow identifies the germinal layer (reduction in epithelial height and irregularities in the structure of the margin of tubules), yellow arrow identifies the destruction of the membrane seminiferous tubules structure and blue arrows identify sperms. PEN: Pentoxifylline, HPD: High-protein diet.|
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Total antioxidant capacity
The outcomes displayed that the TAC serum level reduced significantly in the HPD group compared to the sham group (P < 0.01). PEN increased TAC levels in the treated rats of whole doses compared to the HPD groups (P < 0.01). The TAC level enhanced significantly in all HPD + PEN groups compared to the HPD group (P < 0.01) [Figure 7].
|Figure 7: Comparison of total antioxidant capacity in treatment groups. *Significant difference compared to the sham group (P < 0.01).†Significant difference compared to the HPD group (P < 0.01).‡Significant difference compared to the HPD group (P < 0.01). PEN: Pentoxifylline, HPD: High-protein diet.|
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HPD group caused a significant decrease in the testosterone hormone level compared to the sham group (P < 0.01). No significant alterations were detected in PEN groups compared to the sham group (P < 0.01). Furthermore, the testosterone hormone level in all PEN and HPD + PEN groups enhanced significantly compared to the HPD group (P < 0.01) [Figure 8].
|Figure 8: Comparison of testosterone hormone level in treatment groups. *Significant difference compared to the sham group (P < 0.01).†Significant difference compared to the HPD group (P < 0.01).‡Significant difference compared to the HPD group (P < 0.01). PEN: Pentoxifylline, HPD: High-protein diet.|
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HPD group caused a significant decrease in the spermatogenic index compared to the sham group (P < 0.01). No significant changes were observed in all PEN groups compared to the sham group (P > 0.05). Moreover, spermatogenic index in all PEN and HPD + PEN groups showed a significant increase compared to the HPD group (P < 0.01) [Figure 9].
|Figure 9: Comparison of spermatogenesis index in treatment groups. *Significant difference compared to the sham group (P < 0.01).†Significant difference compared to the HPD group (P < 0.01).‡Significant difference compared to the HPD group (P < 0.01). PEN: Pentoxifylline, HPD: High-protein diet.|
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| Discussion|| |
Today, many athletes, especially bodybuilders, use HPDs. It seems that this type of diet in the long term owns toxic effects on many organs of the body, especially the genital system. Long-term HPD can induce the oxidative stress and consequently, the production of free radicals. Oxidative stress induced by the presence of free oxygen radicals is considered as one of the most important pathological factors in many diseases. The sperms like other aerobic cell are able to produce ROS in metabolic pathways; thus, they are highly susceptible to oxidative damage and finally lose their physiological function. The results of the present study showed that the survival rate, number and motility of sperm in the HPD group reduced significantly compared to the sham group. In PEN and HPD + PEN groups, a significant increase was observed in the survival rate, number and motility of sperm cells compared to the nitrosamine control group. It seems that ROS affects the synthesis of DNA and RNA in sperms and inhibits the mitochondrial function. It is also possible that oxidative stress conditions in sexual germ cells also act in the same way and disrupt their divisions and differentiation so that the number of affected spermatogonia above the base membrane, primary and secondary spermatocytes, spermatids and adult sperms reduced. Furthermore, the oxidative stress induced by HPD can disrupt the spermatogenesis and result in the formation of defective gametes with remodelled chromatin. These type of gametes with altered chromatin are susceptible to the attack by free radicals which ultimately lead to reducing the number of spermatogonia, spermatocytes, spermatids and spermatozoa. The results of Braden et al.'s survey were consistent with the results of the present study, which showed that HPD induced the oxidative stress in male rats, caused ROS production and a significant reduction in the number of sperms compared to the control group. The first consequence of ROS attack to membrane structures is the appearance of cell peroxidation within the cell membrane and organelles. Since the sperms during the spermatogenesis stage lose a large amount of their cytoplasm (lack of antioxidant systems), it seems to be more sensitive to increased amount of ROS in the environment than somatic cells. It seems that high levels of ROS can reduce the spermatozoa mobility due to the effect on Ca++ channels and reduction of sperm adenosine triphosphate (ATP) reserves. Furthermore, the reduction in glutathione levels can reduce sperm mobility. It seems that plasma membrane of sperms due to the presence of large amounts of unsaturated fatty acids is susceptible to oxidative damage which can result in reduced mobility and viability of sperms. Roshankhah et al. showed that diabetes-induced oxidative stress in male rats induced ROS production, induced sperm deformity, induced DNA degradation, reduced fertility index, reduced motility and reduced the number of sperm and level of testosterone in comparison with control group. The study results of Mukherjee and Mukhopadhyay showed that administration of HPD in male rats caused a significant reduction in the sperm motility and survival rate compared to the control group, which confirms the results of our study. PEN is a methylxanthine derivative which can increase sperm motility by increasing the intracellular calcium concentration and increased membrane penetration to cAMP analogues that inhibit phosphodiesterase. Due to the low amount of cytoplasmic enzymes, no potential is found for the regeneration of oxidative damage, and antioxidants and antioxidant enzymes are highly necessary for the semen fluid to protect against oxidative damage. PEN has anti-inflammatory properties, which seems to reduce the level of lipid peroxidation (LPO) and prevent damage to cells The results of a study by Sancler-Silva et al. confirmed the results of the present study which showed that the testes exposed to PEN produce non-motile sperms. The results of the current study displayed that HPD reduced the TAC serum level, but this value improved significantly in the PEN and PEN + HPD groups compared to the HPD group. The reduction in TAC level in this study showed that the oxidative stress induced by HPD can affect reproductive parameters. HPD induces oxidative stress in testicular tissue. This demonstrates as a growth in the levels of ROS and lipid peroxidation and a reduction in the action of antioxidant enzymes such as TAC. The amount of antioxidants present in testicular tissue can be extremely harmful by the means of the high metabolism, high levels of unsaturated fatty acids in the membrane of related cells and cell proliferation. In the present study, increased levels of TAC in rats treated with PEN highlight the antioxidant and anti-lipid peroxidation effects of PEN. The results of Brighenti et al.'s study showed that the administration of HPD in Italian adults significantly reduces the serum levels of TAC, which confirms the results of the present study. The results of this survey showed that normal morphology of sperms, diameter of seminiferous tubules and testosterone levels in the HPD group reduced significantly compared to the sham group. In the PEN and HPD + PEN groups, a significant increase was observed in the normal morphology of sperms, diameter of the seminiferous tubules and testosterone level compared to the HPD group. HPD can induce the increased oxidative stress, DNA damage, peroxidation lipid as well as formation of additional protein compounds by producing ROS (such as superoxide and hydrogen peroxide). It seems that cells of seminiferous tubules rapidly differentiated in the HPD group and released from tubules, which increased the diameter of the tubules. In addition, the increase in ROS production along with lipid peroxidation leads to induction of tubules' atrophy and apoptosis of the germ cells. It seems that a significant relationship is found between the production of oxygen species in sperms and disturbance in morphology of sperms. The conditions for increasing the amount of free radicals result in the destruction of enveloping tissue cells, damage to Sertoli cells, collapse of cytoplasmic bridges and consequently, a reduction in the number and motility of sperms. HPD can lead to a significant reduction in testosterone levels due to oxidative stress. The findings of Feyli et al. are consistent with the results of our study that the administration of PEN increased the diameter of seminiferous tubules and testosterone level significantly. It seems that, along with antioxidant properties, vasodilatation and increased blood supply of PEN can be a factor in increasing the production of testosterone in the present study. Moreover, the results of this study indicated that HPD decreases the SI. Similarly, a significant increase was detected in SI in all PEN and PEN + HPD groups compared to the HPD group. Therefore, the SI shifts from level 8 (few spermatozoa) to 5.5 (no spermatozoa and many spermatocytes) during the treatment with HPD, and also, the administration of PEN increases the number of spermatozoa.
| Conclusions|| |
The outcomes of this study demonstrate that HPD can develop defects in some of male reproductive parameters, and PEN can stand with mentioned complications by antioxidant property. PEN increases the quality rate of spermatozoa, normal morphology, SI, sperm cell viability, motility and count. PEN can be valuable for infertile men treatment and male fertility enhancement. The antioxidant properties of PEN could be the main reason for their beneficial outcome on reproductive parameters. Supplementary studies are essential to explain their precise mechanism of action.
Financial support and sponsorship
We gratefully acknowledge the Research Council of Kermanshah University of Medical Sciences for their financial support of this study.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]