Abstract: Background and Objective: Obesity exerts negative influences on male reproductive capacity via changing the molecular and physical structure of male germ cells. This study was conducted to evaluate the mitigating effects of raw juice of pineapple on obesity-associated testicular impairment in male Wistar rats. Materials and Methods: Rats included the control group (GI, n = 6) who received a Normal Diet (ND) and the obese group (GII, n = 18) who received a High-Fat Diet (HFD). Obese rats (GII) were subdivided into 3 groups (6 rats each): GII represents the untreated obesity group that continued to receive HFD with plain Drinking Water (DW), GIII received ND along with raw juice (15% v/v) in DW and GIV continued to receive HFD with raw juice (15% v/v) in DW. Rats were sacrificed at the end of the trial and testis was processed for histopathology and immunohistochemistry. Results: Testis from obese rats revealed a significant increment in spermatogenic cell degeneration, pro-inflammatory Nuclear factor of kappa B (NF-κB) and pro-apoptotic Caspase-3 immunoreactivities. Yet, Proliferating Cell Nuclear Antigen (PCNA) displayed poor immunoreactivity in obese rats’ testis relative to controls. Administration of raw juice of pineapple to obese rats significantly reduced degeneration of spermatogenic cells, NF-κB and Caspase-3 immunoreactivities. Additionally, treatment with the juice significantly increased immunoreactivity to PCNA in obese rats. These ameliorating effects were more obvious in rats who received juice along with ND (GIII) than in those who received it along with HFD (GIV). Conclusion: Treatment of obese rats with pineapple juice restored testicular homeostasis, indicating its potential validity to overcome obesity-induced male fertility disorders.
INTRODUCTION
Obesity is a metabolic condition manifested by an excessive aggregation of fat in adipose tissue. This condition usually originates from hereditary, behavioural and environmental stimuli1.
The modern lifestyle is mainly characterized by shifting the nutritional habits towards a selective and desirable consumption of fast processed foods rich in energy. High-energy foods often induce overweight and result in hyperglycaemia, hyperinsulinemia and dyslipidaemia2,3. These conditions lead to a state of the chronic inflammatory process and a high rate of metabolism to maintain the metabolic balance3. However, a high metabolic rate is usually associated with excessive production of the reactive species of oxygen (ROS), leading to deterioration in DNA and proteins and harm the integrity of the cell membranes. Although involving many tissues, these events are principally vital in the microenvironment of the testis where the cells of the sperm are very sensitive to oxidative stress, compromising male fertility2,3.
Growing evidence displays that obesity exerts negative influences on male reproductive capacity via reducing sperm quality and changing the molecular and physical structure of germ cells in male gonads4,5. Hyperglycemia and hyperinsulinemia represent a regular incidence in obese persons2. They are suggested to have a repressive effect on sperm quantity and quality and consequently are associated with reduced fertility in obese males4,6,7. Many findings reported in the clinical surveys and animal trials showed that obesity is associated with decreases in the concentration and motility of sperm, increases in damages of sperm DNA and alterations in the hormones of reproduction1.
Supplementation of several herbal extracts, for instance, Ginger8 and Moringa9 extracts has been proved to provide effective protection against the obesity-associated negative impacts, particularly those related to male fertility. This protection is mainly attributed to their antioxidant, hypoglycemic, insulinotropic, androgenic and hypolipidemic effects8,9.
The fruit of pineapple (Ananas comosus) is rich in essential cations (potassium and calcium) and vitamins (A and C). Additionally, fresh pineapple is a good source of bromelain which may have an anti-inflammatory capacity10. Remarkably, pineapple fruits contain various phenolic compounds11, which can protect them from oxidative stress and inflammations12. The protective effects of raw pineapple juice against obesity and dyslipidemia have been reported. This may be due to its high content of bromelain, which has a significant lipolytic activity13. The obesity-induced by HFD has been repressed by the treatment with the raw juice of pineapple. This repression may result from the reduction of blood serum lipids, weight gain and hepatic lipid accumulation in obese rats13.
The main goal of this study is the recognition of the potential mitigating effect of the raw juice of pineapple on the testicular stress induced by obesity in adult male Wistar rats through studying testicular histopathological and immunohistochemical alterations.
MATERIALS AND METHODS
Study area: This study has been carried out at the laboratories of the Department of Biotechnology, College of Science, Taif University, Saudi Arabia. The Experimental periods extended from January-March, 2020.
Animals and experimental design: Adult male Wistar rats (n = 24) were obtained from the Experimental Animal Center, King Abdul-Aziz University, Saudi Arabia. Rats were held under a controlled environment of temperature (22±3°C), humidity (50±5%) and a 12 hrs light-dark cycle. Before the beginning of the experiment, rats were left one week for acclimatization. Rats were kept in clean standard cages with bedding and fed on standard pellets and water ad libitum. The Animal Care Committee at Taif University, Saudi Arabia has approved all the experimental procedures (# 6145/1440).
Rats were allocated haphazardly into two main groups. Control group (n = 6) that received Normal Diet (ND) and High-Fat Diet (HFD) group (n = 18) that received HFD as presented in Table 1 as described by Woods et al.14. When rats of the HFD group get obese (after 7 weeks), they were subdivided into 3 groups (6 rats each). Untreated obesity group continued to receive HFD with normal drinking water (GII), GIII) received the raw juice of pineapple (15% v/v) in drinking water concurrently with ND, while GIV continued on HFD along with the pineapple raw juice.
| Table 1: Components of the obesity inducing high-fat diet | |
| Ingredients | Values (g kg–1) |
| Casein | 164 |
| Corn starch | 309 |
| Dextrin | 90 |
| Sucrose | 120 |
| Cellulose | 59 |
| Butter oil | 196 |
| Soya bean oil | 10 |
| Cysteine | 2.1 |
| Choline tartrate | 2.9 |
| Mineral mix | 35 |
| Vitamin mix | 12 |
The food consumption and the BW were recorded weekly for each group.
Food intake and body gain: The body weight and the weight gain (g/day/rat) as well as the food intake, were weekly recorded. Food intake was estimated by subtracting the weight of leftovers in the food bin from the weight of food offered.
Sampling: By the end of the trial, rats were decapitated. The testes were obtained directly and immersed in Bouin's solution for the conduction of histological and immunohistochemical procedures.
Histopathological examination: Samples from the testes of different groups were collected, then fixed in Bouin's solution. Paraffin sections of testicular tissues were prepared and stained according to the standard protocols described by Suvarna et al.15.
Immunohistochemical investigation: The immuno-histochemical staining protocols described by Saber et al.16 have been adopted for the detection of PCNA, Caspase-3 and NF-κB P65. Sections were deparaffinized and immersed in 0.05 M citrate buffer, pH 6.8 for the retrieval of antigen. Sections were then treated with 0.3% H2O2 and protein block. Then, they were incubated with rabbit polyclonal anti-PCNA (Abcam, Cat#ab18197, dilution 1/100), rabbit polyclonal anti-Caspase-3 antibodies (Invitrogen, Cat# PA5-77887, dilution 1/100) and anti-NF-κB P65 (Santa cruz, Cat# (F-6): sc-8008, 1:100 dilution). After rinsing with the buffered saline of phosphate, they were incubated with a goat anti-rabbit secondary antibody (Cat# K4003, EnVision+™ System Horseradish Peroxidase Labelled Polymer, Dako) for 30 min at room temperature. Slides were envisioned with a DAB kit and ultimately stained with Mayer's hematoxylin as a counterstain. The immunolabeling index was evaluated and shown as a percentage of positive expression in a total of 1000 cells per 8 High Power Fields (HPF).
Statistical analysis: Data were analyzed using the ANOVA test and Scheffe’s protected least significant difference test by SPSS software (SPSS version 13.0, IBM, Chicago, IL, USA). The difference was considered statistically significant at p<0.05. Data were displayed as Mean±Standard Errors (SEM).
RESULTS
Effect of pineapple juice on body weight: The weight of the body of the untreated obese rats (GII) was increased markedly than those in the GI (controls). This increment was meaningfully reduced by feeding ND along with the raw juice of pineapple (GIII). Also, administration of juice concurrently with HFD (GIV) significantly reduced the body weight relative to untreated obese rats in GII (Table 2).
Effect of pineapple juice on testis weight: The testis weight displayed no significant change in all the experimental groups. Meanwhile, relative testis weight was meaningfully decreased (p<0.05) in untreated obese rats (GII) relative to other groups.
Effect of pineapple juice on epididymal fat weight: The epididymal fat pad weights were increased meaningfully in untreated obese rats (GII) relative to controls. However, the epididymal fat weights were reduced significantly in groups treated with the juice concurrently with either ND (GIII) or HFD (GIV) (Table 2).
Effect of pineapple juice on histopathological findings: The histological sections of the testis from the control group (GI) displayed normal spermatogenic cell layers lining normal seminiferous tubules, which present free sperm in their lumina (Fig. 1a).
| Table 2: Effect of pineapple juice and/or high-fat diet (HFD) on the body weight, food consumption, epididymal fat pad and testicular weights | ||||
| Items | GI |
GII |
GIII |
GIV |
| Initial BW (g) | 278.83±13.93 |
381±13.6 |
369.14±11.0 |
359.33±14.7 |
| Final BW (g) | 331.5±9.31a |
519±18b |
398±2.19c |
425±23.4d |
| BMI | 0.62±0.02a |
0.82±0.03b |
0.69±0.1a |
0.75±0.03b |
| Food consumption (g/day) | 27.78±0.9a |
17.96±0.3b |
25.87±1.1b |
18.49±1.8c |
| Epididymal fat weight (g) | 3.01±0.12a |
4.96±0.19b |
3.58± 0.17c |
3.97±0.11d |
| Testicular weight (g) | 3.17±0.12a |
3.01±0.09a |
3.05±0.13a |
3.02±0.12a |
| Relative testicular weight (%) | 0.96±0.03a |
0.58±0.02b |
0.77±0.03c |
0.71±0.03c |
All data are expressed as Mean±SEM (standard error of the mean). Values carrying different superscript letters in the same row are significantly different at p<0.05. GI: Control group that received, ND: Normal diet, GII: Obese group was received, HFD: High-fat diet, GIII: Obese group was received pineapple juice and ND along, GIV: Obese group was received pineapple juice along with HFD. Different letters mean the presence of significant differences at p<0.05 |
||||
| Fig. 1(a-d): | Photomicrographs of H and E-stained section of rat testis, (a) Testis of control rat showed normal spermatogenic cell layers (S) lining normal seminiferous tubules with free sperm in the lumen (arrow), (b) Testis of untreated obese rat showing marked necrotic and desquamative changes in spermatogenic cell layers (arrows), (c) Testis of obese rats received pineapple juice along with ND displaying normal spermatogenic cell layers (S) lining normal seminiferous tubules with a marked increase in the density of free sperm in the lumen (arrow) and (d) Testis of obese rats received pineapple juice concurrently with HFD showing distinct reduction of the degeneration in the spermatogenic cells (S) with the reappearance of sperms within the lumen (arrow) H and E, Bar = 50 μm, ×200 |
On the other hand, the testis of untreated obese rats (GII) displayed marked necrotic and desquamative changes within the spermatogenic cell layers (Fig. 1b). Additionally, the treatment of obese rats with raw pineapple juice concurrently with replacement of HFD with ND (GIII) restored the histological features to the normal limits (Fig. 1c) or markedly decreased the degeneration when given to obese rats continued receiving HFD (GIV) (Fig. 1d).
Effect of pineapple juice on the immunohistochemical findings: Spermatogenic cells in testis from the controls (GI) displayed marked nuclear expression of PCNA (Fig. 2a). On the contrary, sections of the testis of untreated obese animals (GII) showed a marked reduction in this expression (Fig. 2b). However, the testicular sections of obese rats treated with raw pineapple juice either along with ND (GIII, Fig. 2c) or persisted on HFD (GIV, Fig. 2d) displayed marked PCNA immunostaining within the spermatogenic cells.
Unlike the case of PCNA-immunoreactivity, the testicular sections from controls (GI) showed a mild expression of Caspase-3 within the spermatogenic cells (Fig. 3a). Whereas, the testis of untreated obese rats (GII) exhibited a marked caspase-immunoreactivity (Fig. 3b). Administration of pineapple juice either along with ND (GIII) presented a marked reduction of caspase-immunoreactivity (Fig. 3c) or along with HFD (GIV) displayed a scanty caspase-immunoreactivity within the spermatogenic cells (Fig. 3d).
The pattern of immunoreactivity against NF-κB P65 was quite similar to that of Caspase-3. The testicular sections of controls (GI) displayed a mild NF-κB P65-immunoreactivity (Fig. 4a). Sections from untreated obese rats (GII) showed marked NF-κB P65-immunoreactivity (Fig. 4b). While treatment of obese rats with raw pineapple juice either along with ND (GIII) displayed a mild NF-κB P65-immunoreactivity (Fig. 4c) or along with HFD (GIV) presented a marked reduction in NF-κB P65-immunoreactivity (Fig. 4d).
| Fig. 2(a-d): | Photomicrographs of testicular sections immunostained with PNCA antibody, (a) Testis of a control rat showing distinct nuclear expression of PCNA in spermatogenic cells (arrows), (b) Testis of untreated obese rat presenting obvious reduction in PCNA-immunoreactivity in spermatogenic cells (arrows), (c) Testis of obese rat received pineapple juice along with ND showing marked PCNA immunostaining in spermatogenic cells (arrows) and (d) Testis of obese rat treated with pineapple juice along with HFD showing a noticeable increase of PCNA immunostaining in spermatogenic cells (arrows) PCNA IHC, ×200, Bar = 50 μm |
| Fig. 3(a-d): | Photomicrographs of testicular sections immunostained with Caspase-3 antibody, (a) Testis of control rat displaying a negative to weak immunostaining of Caspase-3 in spermatogenic cells (arrows), (b) Testis of untreated obese rat showing distinct immunoreactivity of Caspase-3 in spermatogenic cells (arrows), (c) Testis of obese rat treated with pineapple juice along with ND exhibiting marked reduction of Caspase-3 expression in spermatogenic cells (arrows) and (d) Testis of obese rats treated with pineapple juice along with HFD showing scanty immunostaining of Caspase-3 in spermatogenic cells (arrows) Caspase-3 IHC, ×200, Bar = 50 μm |
| Fig. 4(a-d): | Photomicrographs of testicular sections immunostained with NF-κβ P65 antibody, (a) Testis of a control rat showing a negative to weak immunoreactivity of NF-κB P65 in spermatogenic cells (arrow), (b) Testis of untreated obese rat displaying marked NF-κB P65-immunostaining in spermatogenic cells (arrow), (c) Testis of obese rat received pineapple juice along with ND exhibiting a negative to weak NF-κB P65-immunostaining in spermatogenic cells (arrow) and (d) Testis of obese rats treated with pineapple juice along with HFD showing marked reduction of NF-κB P65 expression in spermatogenic cells (arrows) NF-κB P65 IHC, ×200, Bar = 50 μm |
| Fig. 5: | A graph scoring the expression level of PNAC, Caspase-3 and NF-κβ Immunolabeling index is shown as a percentage of positive expression in a total of 1000 cells per 8 High Power Fields (HPF). GI: Control group that received ND, GII: Obese group received High-Fat Diet (HFD), GIII: Obese group received pineapple juice along with ND and GIV: Obese group received pineapple juice along with HFD. Different letters mean the presence of significant differences at p<0.05 |
The expression levels of the immunolabeling of the different investigated proteins (PNAC, Caspase-3 and NF-κβ) has been summarized (Fig. 5).
DISCUSSION
Obesity denotes to extreme aggregation of body fats resulting in many negative health impacts. In the present study, feeding of HFD induced increases both in final BW and in BMI. Increased BW and BMI may occur due to the increase of caloric intake resulting in more adipose tissue deposition17. Treatment of obese rats with raw pineapple juice either in combination with ND (GIII) or with HFD (GIV) resulted in a significant decrease in BW and BMI. These findings go in line with our previous study13.
Nutritional obesity induced by HFD can compromise the normal histological configuration of seminiferous tubules without having obvious immediate effects on male fertility but may impair it through the prolonged effects on spermatogenesis18. Seminiferous tubules in testicular sections of obese rats showed vacuolar changes and spermatogenic cell apoptosis8,18. In the present study, the histopathological examination of testis from obese rats (GII) showed marked necrotic changes within the spermatogenic cell layers. Meanwhile, treatment with pineapple juice either with ND (GIII) or with HFD (GIV) restored the normal architecture, especially with ND. Our findings are consistent with previous works on HFD-induced obese rats who applied extracts of ginger8 and moringa9.
The PCNA is an essential protein for multiple cell cycle pathways, including DNA replication and repair19. (The levels of PNCA are significantly reduced in the case of germinal arrest19,20. In the present study, PNCA expression was down regulated due to HFD administration. This effect was restored in obese rats treated with pineapple juice, especially when combined with ND (GIII). Our findings are consistent with previous works that applied ginger extract8 and bee breed19 to ameliorate the obesity-induced testicular impairment in rats.
Testis from the controls (GI) showed mild or even no immunoreactivity for the pro-apoptotic (Caspase-3) and the pro-inflammatory (NF-κB) proteins. On the other hand, strong immunoreactivities for both Caspase-3 and NF-κB were found in the obese rats (GII). These findings go in line with those reported by Suleiman et al.19. It is worth noting that apoptosis of spermatogenic cells is one of the main causes of reduced male fertility21. The NF-κB plays a key role in promoting transcription genes involved in the initiation of caspase and apoptosis in male germ cells22. However, these immunoreactivities were significantly decreased in the pineapple-treated obese rats especially those accompanied with ND (GIII) than that kept on HFD (GIV) indicating that pineapple juice might have an anti-apoptotic effect. Similar findings were reported by Suleiman et al.20, who indicated that bee breed reduced the immunoreactivity of Caspase-3 and NF-κB in rat testis, Alkafafy et al.8 found that moringa leaves extract was able to reduce Caspase-3 immunoexpression in the testis of obese rats and by Al-Yasi et al.23, who mentioned that the rosewater pretreatment has reduced Caspase-3 and NF-κB immunoreactivities induced by lead acetate in the testis of male rats.
Obesity impacts sperm functions and male fertility through different mechanisms. These include extreme conversion of androgens to estrogens within the overgenerous fatty tissues, oxidative stress and severe inflammatory process in the male reproductive tracts induced by adipokines released from adipose tissues1,24 and elevated gonadal heat resulting from the increased scrotal adiposity1. Analysis of the fruits and peels of pineapple (Ananas comosus) showed that it contained vital compounds such as polyphenols25. In addition, the juice of pineapple has suppressed obesity induced by HFD in rats via modulation of genes controlling fat metabolism13.
Additionally, the fresh raw juice of pineapple is rich in bromelain which is one of the most crucial active principles of pineapple and may act as an anti-inflammatory10. Also, pineapple fruits contain various phenolic compounds11, which display antioxidant and anti-inflammatory activities12. Our findings are consistent with those reported by Yeh et al.26, who reported that bromelain suppressed the apoptotic actions, oxidative stress-induced inflammation and cellular ageing induced by tert-butyl hydroperoxide in mice testis.
CONCLUSION
The HFD-induced obesity resulted in reducing fertility in male rats through compromising testicular homeostasis. The testicular impairment in obese untreated rats was evident from deteriorating the normal histological architecture, down regulation of PCNA and upregulation of the pro-inflammatory (NF-κB) and pro-apoptotic (Caspase-3) genes. However, the treatment of obese rats restored these parameters to limits close to the normal control, indicating their potential validity to overcome obesity-induced male fertility disorders.
SIGNIFICANCE STATEMENT
The obtained results indicated that the raw pineapple juice displayed a significant role in improving the testicular impairment associating with obesity. However, the study is still a preliminary step and discloses a prospective solution for obesity-associated subfertility. The current results may motivate researchers to adopt further future studies to determine the active ingredients as well as revealing their specific impacts.
ACKNOWLEDGMENT
The study was financed by the Deanship of Scientific Research, Taif University, Saudi Arabia [Project Number: 1-440-6145].