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Possible protective effect of Moringa oleifera leaf extract on dexamethasone-induced histological changes in adult rat testes

El-wassimy, Mohamed T.a; El Haliem, Nesreen G.b; Hegazy, Mohammed F.c; Younes, Sabry H.a; Al-Badry, Hazem A.a

The Egyptian Journal of Histology: March 2014 - Volume 37 - Issue 1 - p 112–123
doi: 10.1097/01.EHX.0000444076.09624.f8
Original articles

Introduction Dexamethasone (Dex) is a widely used therapeutic agent for its immunosuppressive and anti-inflammatory action. It has adverse effects on many body organs and systems. Moringa oleifera is an antioxidant-rich natural plant. It contains vitamins C and A, and various phenolic compounds.

Aim of work The present study was designed to evaluate the ability of M. oleifera leaf extract to protect rat testis against Dex-induced spermatogenic defects.

Materials and methods Thirty adult male rats were divided equally into three groups (10 animals each): the control group (group I) and two experimental groups (groups II and III). Rats of group II were subjected to intraperitoneal injection of 7mg/kg/day of Dex for 10 days. In group III the rats were treated with M. oleifera leaf extract at 400mg/kg/day, and then after 2h they were administered an intraperitoneal injection of Dex with the same dose as for group II for 10 days. The testes were dissected out and processed for light and electron microscope examination.

Results Microscopic examination revealed that most of the seminiferous tubules of group II were lined with germ cells with dark pyknotic nuclei and vacuolated cytoplasm. The lumen of some tubules was obliterated with exfoliated and sometimes multinucleated giant cells. There was statistically highly significant increase in the percentage of sperm abnormality. Degenerated interstitial Leydig cells were also observed. However, in the moringa-treated group, the histological changes were reduced and the percentage of sperm abnormality was more or less similar to that of the control group.

Conclusion These results demonstrated that M. oleifera leaf extract has a potent protective effect against the testicular toxicity induced by Dex and hence might be clinically useful.

aDepartment of Chemistry, Faculty of Science

bDepartment of Histology, Faculty of Medicine, Sohag University, Sohag

cNational Research Centre, Egypt

Correspondence to Nesreen G. El Haliem, PhD Department of Histology, Faculty of Medicine, Sohag University, Sohag, Egypt Tel: +20 100 656 6743; fax: +0934602963; e-mail: nesreengamal2000@yahoo.com

Received November 1, 2013

Accepted January 27, 2014

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Introduction and aim of the work

Cortisol is the major corticosteroid produced by different tissues under the effect of stress 1. It plays a key role in the restoration of homeostasis during and after stress. It has been frequently known as the major factor mediating the suppressive effect of stress on reproduction 2. Dexamethasone (Dex) is a widely used immunosuppressive and anti-inflammatory agent. Several adverse effects like hypertension, diabetes, and insulin resistance are associated with its use 3. It has been reported that glucocorticoids cause changes in plasma gonadotrophin levels and their pituitary secretion and indirectly contribute to the inhibition of reproductive functions 4. Previous studies have shown that elevation of glucocorticoid levels leads to a decrease in testosterone secretion 5,6. Testosterone is essential for normal function and survival of germ cells in seminiferous tubules 7,8.

Moringa oleifera is commonly known as a drumstick tree and is considered one of the world’s most useful trees. Its leaves can be eaten raw or cooked or they can be stored in dried powder form for several months 9. Its oil and micronutrients have been reported to have antitumor, antiepileptic, antidiuretic, and anti-inflammatory effects. It contains seven times the amount of vitamin A contained in carrots, two times the amount of protein contained in milk, and three times the potassium in bananas 10. M. oleifera is highly valued in tropical and subtropical countries where it is mostly cultivated 11. Its leaves have the highest nutrient content when compared with other parts. They are a source of protein, β-carotene, vitamins A, B, C, and E, riboflavin, nicotinic acid, folic acid, pyridoxine, amino acids, minerals, and various phenolic compounds 11,12. Various parts of the plant have wide medicinal applicability in the treatment of cardiovascular diseases 13.

Its antioxidant activity is due to the presence of various bioactive compounds such as chlorogenic acid, rutin, quercetin glucoside, and kaempferol rhamnoglucoside. It is considered an important nutrient that enforces the body’s defense against free radical damage 14–16.

However, few studies have attempted to describe the protective effect of systemic administration of this agent on the testis. The aim of this study was to investigate the protective role of M. oleifera extract against the toxic effect of Dex on rat testes.

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Materials and methods

Animals

In this study, 30 adult male albino rats, weighing ∼200g each, were used. All animals were kept under the same hygienic conditions and received balanced diet and water.

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Drugs

Dex was obtained as a vial for injection from El-Ameria Pharmaceutical Chemicals Co. Alexandria, Egypt.

The M. oleifera leaves were obtained from the National Research Center of Cairo, Egypt. Powdered leaves are sold commercially in sachets to people for medicinal and nutritional purposes.

A measure of 750g of the sieved powder was weighed accurately and subjected to extraction in a soxhlet apparatus at room temperature using ∼12 L methanol in the Faculty of Science, Sohag University. The extract obtained was filtered, concentrated in a rotary flash evaporator, and maintained at 30°C. The percentage yield of each extract was calculated and the dried extracts were stored.

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Experimental design

The animals were divided into three main groups as follows:

  • Group I: This group consisted of 10 animals that served as controls and received physiological saline at 7mg/kg/day through intraperitoneal injection for 10 days.
  • Group II: This group consisted of 10 animals that received intraperitoneal injection of Dex at 7mg/kg/day for 10 days 17.
  • Group III: This group consisted of 10 animals that received intraperitoneal injection of M. oleifera leaf extract at 400mg/kg/day 2h before the injection of Dex with the same dose and for the same time as group II 18.

All experimental protocols were in compliance with the University of Sohag Ethics Committee on Research in Animals as well as internationally accepted principles for laboratory animal use and care.

Twenty-four hours after the last dose, the rats were anesthetized with ether inhalation and sacrificed; the testes were dissected out and specimens were taken from the right and left testes of each control and treated animal for light and electron microscopic study.

For light microscopic examination the specimens were fixed in 10% formalin and processed to prepare 5 μm-thick paraffin sections for H&E staining. For electron microscopic preparation, 10–12 small pieces from the right and left testes of each group were fixed immediately in 5% gluteraldehyde for 24h. The specimens were processed and ultrathin sections were prepared 19 and examined using an electron microscope ‘JEOL TEM 1010 (Tokyo, Japan) in the electron microscopic unit of the Faculty of Medicine, Sohag University.

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Sperm abnormality test

Sperm abnormality was studied as described by Wyrobek et al. 20 as follows: the caudal part of the epididymis was excised and placed in a beaker containing 2ml of physiological saline, after which each section was quickly incised with a pair of sharp scissors and left for a few minutes to liberate its spermatozoa into the saline solution.

Smears were prepared and stained with 5% toluidine blue. One drop of the prepared seminal fluid and another of the stain were dropped onto the edge of the precleaned slide and mixed using the edge of another clean slide; the smear was spread over the entire surface of the slide. The slides were left to dry and coded for subsequent microscopic examination. Quantitative analysis was carried out whereby the sperm smears were examined with a light microscope to detect sperm abnormalities. Sperms were counted in 20 high-power fields in five smears from each animal using a light microscope (CX21; Leica, Japan) in the Histology Department, Faculty of Medicine, Sohag University.

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Statistical analysis

These data were expressed as mean±SD and analyzed using an independent t-test for comparing the differences in percentages of sperm abnormality between the different groups. The statistical analysis was carried out using the SPSS (version 9)/PC program (Chicago, Illinois, USA). The significance of data was determined by the P value; P values greater than 0.05 were considered nonsignificant; P values less than 0.05 were considered significant; and P values less than 0.001 were considered highly significant.

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Results

Histological results

Control group (GI)

Examination of the H&E-stained sections of the control group showed that the testis was formed of seminiferous tubules and interstitial tissues in-between. Each tubule was surrounded by a thin basement membrane and flat myoid cells. The tubules were lined with spermatogenic cells that constitute the successive stages of spermatogenesis, as well as Sertoli cells. The spermatogenic cells included several distinct types: the spermatogonia, primary spermatocytes, spermatids, and mature spermatozoa (Fig. 1). Spermatogonia appeared as rounded cells with rounded nuclei resting on a thin, straight basement membrane. Primary spermatocytes appeared as rounded cells with large rounded nuclei. Sertoli cells were found between spermatogenic cells with a pale cytoplasm and oval indented nuclei. The interstitial cells of Leydig were observed singly or in clumps and were richly supplied with blood capillaries. They were either rounded or polygonal and could be easily detected by their acidophilic cytoplasm and central vesicular nuclei (Fig. 2).

Figure 1

Figure 1

Figure 2

Figure 2

Electron microscope examination showed spermatogonia and Sertoli cells resting on a thin, straight basement membrane. Spermatogonia exhibited more or less rounded nuclei with prominent nucleoli. Their cytoplasm contained numerous free ribosomes and mitochondria. Primary spermatocytes with large rounded nuclei and evenly distributed coarse chromatin granules were also observed. The Sertoli cells showed large indented nuclei with prominent nucleoli, mitochondria, a few strands of rough endoplasmic reticulum, free ribosomes, some lipid droplets, and lysosomes. Tight junctions were observed between the cell membrane of the adjacent Sertoli cells above the level of the spermatogonia (Fig. 3). Early or round spermatids had acrozomal vesicles over the large rounded nuclei. Their cytoplasm contained small spherical mitochondria with electron-lucent matrix arranged in rows beneath the cell membrane (Fig. 4). Sperm heads appeared hook-shaped with elongated nuclei (Fig. 5). Their tails had a central axonem fibrous and mitochondrial sheath (Figs 4 and 6).

Figure 3

Figure 3

Figure 4

Figure 4

Figure 5

Figure 5

Figure 6

Figure 6

Interstitial cells of Leydig contained oval to rounded euchromatic nuclei, mitochondria, electron-dense bodies, and smooth endoplasmic reticulum. The cytoplasm of macrophages exhibited numerous organelles such as lysosomes, strands of rough endoplasmic reticulum, lipid droplets, and mitochondria (Fig. 7).

Figure 7

Figure 7

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Dexamethasone-treated group (GII)

Examination of the H&E-stained sections of the Dex-treated group revealed various histological changes in both spermatogenic and interstitial tissue. A number of spermatogenic cells with small dark pyknotic nuclei and vacuolated cytoplasm were observed. Some seminiferous tubules were lined with a few layers of spermatogenic cells and revealed widening in the intercellular spaces in between. Many germ cells were detached from the basement membrane and coalesced together, forming abnormal multinucleated spermatogenic cells inside the tubules. The interstitial tissue contained congested blood vessels, interstitial acidophilic exudates, and increased number of interstitial cells (Fig. 8). The basal lamina of the tubules was irregular and separated from the germinal epithelium in some parts. Exfoliated cells were frequently observed inside the lumen of some tubules (Fig. 9).

Figure 8

Figure 8

Figure 9

Figure 9

Electron microscopic examination showed an irregular thick basement membrane. Spermatogonia and primary spermatocytes had irregular heterochromatic nuclei and numerous vacuoles. The cytoplasm of the spermatogonia contained ballooned mitochondria with destructed cristae (Figs 10 and 11). Most of the round spermatids appeared with variable degrees of acrosomal-nuclear dysgenesis. Separation of acrosomal granules was seen in some spermatids (Figs 12 and 13). Multinucleated cells were observed and were formed by the fusion of two or more spermatids. Their cytoplasm contained two or more nuclei with acrosomal vesicles, free ribosomes, and mitochondria, with the same arrangement as in the rounded spermatids (Fig. 14). Numerous spermatozoa appeared with abnormally angled heads and abnormal Y tails. The mid-pieces in some sperms showed vacuolated mitochondria (Figs 15 and 16). Sertoli cells exhibited degenerative changes with separation from the underlying basement membrane. The junctions between them were interrupted and their cytoplasm contained phagocytosed parts of the apoptotic cells, lipid droplets, and large irregular secondary lysosomes (Fig. 17). Degenerative changes were observed in some of the Leydig cells. They contained irregular small-sized nuclei with marginated heterochromatin, large lipid droplets, dilated cisternae of the SER, mitochondria, and vacuoles (Fig. 18). However, the previous described histopathological and ultrastructural alterations were observed in only ∼60% of the treated rat population.

Figure 10

Figure 10

Figure 11

Figure 11

Figure 12

Figure 12

Figure 13

Figure 13

Figure 14

Figure 14

Figure 15

Figure 15

Figure 16

Figure 16

Figure 17

Figure 17

Figure 18

Figure 18

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Moringa oliefera and dexamethasone-treated group (GIII)

Examination of the H&E-stained sections of the combined treatment group showed marked improvement in the testicular structure as compared with the Dex-treated group. Most of the tubules appeared more or less similar to those of the control group (Fig. 19).

Figure 19

Figure 19

Electron microscopic examination of the testis after combined treatment revealed a more or less normal appearance for the spermatogenic epithelium. Sertoli cells contained mitochondria and primary lysosomes and showed intact junctions between each other (Fig. 20). Round spermatids in different developing stages of acrosome formation were observed. Their cytoplasm showed the Golgi zone and mitochondria (Fig. 21). Most of the spermatozoa appeared similar to the control, apart from some with mitochondrial sheath defects (Fig. 22). The interstitial cells of Leydig exhibited numerous dilated SER cisternae and Golgi saccules (Fig. 23).

Figure 20

Figure 20

Figure 21

Figure 21

Figure 22

Figure 22

Figure 23

Figure 23

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Sperm abnormality results

Light microscopic examination of sperm smears of the control group revealed that most of the sperms had normal morphology (a hook-shaped head and a long straight tail) (Fig. 24).

Figure 24

Figure 24

In group II, sperm abnormalities were markedly increased in the form of head anomalies (heads without tails, hook at the wrong angle) and tail abnormalities (coiled tails, tails with absent heads, and fusion of tails at different positions) (Fig. 25).

Figure 25

Figure 25

Animals of group III treated with M. oleifera extracts exhibited a reduction in total sperm abnormalities compared with group II, apart from some sperms with tail abnormalities such as absent tails or coiled ones (Fig. 26).

Figure 26

Figure 26

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Statistical analysis

The statistical study revealed a highly significant increase in the percentage of sperm abnormality in the Dex-treated group compared with the control group. The group treated with combined M. oleifera and Dex showed a nonsignificant change compared with the control. In contrast, there was a highly significant decrease in the combined group compared with the Dex-treated one. All these data are presented in Table 1 and graphically illustrated in Histogram 1.

Table 1

Table 1

Histogram 1

Histogram 1

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Discussion

This study demonstrated that Dex caused marked histological changes in testicular structure. This might lead to a disturbance in the process of spermatogenesis. The surrounding basal lamina of the seminiferous tubule was thickened, irregular, and wavy. Similar changes were found in different types of testicular damage and also after hypophysectomy 21,22. It was suggested that this basal lamina thickening is the result of either the overproduction of collagen fibers by fibroblasts or the decrease in the rate of collagen removal 23. It was found that Dex increased the amount of collagen fibers, especially type IV, through its effect as a metalloproteinase inhibitor that suppresses extracellular matrix degradation 24. It was also proved that Dex promotes connective tissue growth factor production and activation, which stimulates fiber formation by fibroblasts 25,26. Furthermore, some studies found that Dex induced overproduction of reactive oxygen species by different cells such as macrophages and vascular endothelium 27,28. These species could increase the production of collagen fibers and induce fibrosis 29. It is well known that the basal lamina plays an important role in maintaining substance transmission between the interstitial tissue and spermatogenic epithelium and in maintaining the structural and functional integrity of these tissues 30.

In the present work Dex induced degenerative changes in most of the germ cells in the form of increased chromatin texture and cytoplasmic density, ballooned mitochondria with destructed cristae, and dilated SER cisternae. The spermatogonia were the most affected cells; this might have been due to their proliferating character, which makes them the target for the toxic effect of Dex. These results are in agreement with other studies that reported an increase in many apoptotic markers such as Bax and Fas ligand expression and TUNEL reactivity in spermatogonia 31. These degenerative changes in germ cells might result from increased oxidative stress production of malondialdehyde in testicular tissue that was detected by other authors in plasma and aorta after Dex administration 32. There were also structural abnormalities in round and late spermatids in the present work. Some round spermatids exhibited irregular nuclei, abnormal acrosome, and numerous mitochondria. Other late spermatids had abnormally angled heads and abnormal Y tails. However, these changes might be secondary to Sertoli cell injury that affected the synthesis of certain proteins essential for germ cell differentiation. It was found that these proteins are secreted at their highest rate during spermatid elongation and spermiation 33. This could explain the highly significant increase in the percentage of sperm abnormalities and their ultrastructural defects. Furthermore, it was found that degenerative changes in germ cells and the release of reactive oxygen radicals produced varieties of morphologically abnormal sperms and caused DNA damage 34,35. In addition, the presence of numerous mitochondria might be attributed to the high-energy consumption of the detoxification process 36. These results are in agreement with those of previous researchers who found low concentrations of free radical scavenging enzymes in the sperm cytoplasm and hence it was more vulnerable to Dex toxicity 37.

Ultrastructural features of Sertoli cells revealed interrupted junctions, marked nuclear enfolding, vacuoles, irregular secondary lysosomes, and numerous lipid droplets. The desquamated germ cells and the intercellular spacing detected in this work might be secondary to Sertoli cell injury and subsequent retraction of their cytoplasmic processes. This led to disruption of cell arrangement, which became easily sloughed out. Similar findings were reported by other authors who explained the luminal shedding of germinal cells as alteration of intercellular junctions between Sertoli cells 38. Moreover, it was suggested that Sertoli cells secrete Glial cell line-derived neurotrophic factor, which is known to influence the fate of undifferentiated spermatogonia. They reported that Sertoli cell injury was associated with decreased production of this factor, which led to apical sloughing of desquamated spermatocytes or spermatids into the lumen and a massive loss of germ cells 39,40.

Multinucleated giant cells were observed in some of the seminiferous tubules and comprised mainly round spermatids in the Dex-treated group. Similar findings were also seen in the testis of prepubertal normal rats, in old people, and under toxic conditions 41. It was found that these cells formed as a result of the breakdown in the intercellular bridges that connect groups of each specific cell type of the germinal lineage 42. However, it was also reported that such multinucleated cells could be considered a sign of apoptosis 43.

The present results revealed inflammatory signs in the interstitial tissue. There were acidophilic exudates, apparent hyperplasia of interstitial cells, and congested blood vessels. It was found previously that at the site of tissue injury activated innate immune cells release various chemical mediators that are responsible for inflammation 44. Furthermore, the molecules derived from plasma proteins and cells in response to tissue damage could mediate inflammation by stimulating vascular changes and leukocyte migration 45.

Degenerative changes in some Leydig cells were detected in Dex-treated animals in the form of irregular small-sized nuclei with marginated heterochromatin, dilated cisternae of the SER, large lipid droplets, and vacuoles. It was suggested that the degenerative changes in the germ cells may occur secondary to their deprivation of testosterone. This could be a result of excess production of reactive oxygen radicals, which lead to disruption of the mitochondria and inhibition of steroidogenic acute regulatory protein expression 46. In addition, it was stated that corticosteroids decrease the Leydig cell sensitivity to gonadotropins, either by reducing the LH receptor content or by inhibiting the 17α-hydroxylase and/or C17, 20-lyase activity 47–50.

The pretreatment with M. oleifera extract in the present work decreased the toxicity caused by Dex and this was evidenced by an improvement in testicular germ cell morphology. This may result from the antioxidant and phenolic compounds in M. oleifera that help in protecting the testis against the oxidative toxicity caused by Dex treatment 51,52. In agreement with this explanation, many studies showed that M. oleifera could elevate a variety of antioxidant enzymes and testicular biomarkers that improve the reproductive potential 53,54. The protective effect of M. oleifera on degenerative changes induced by Dex in testicular germ cells might have been from inhibition of the lipid peroxidation and DNA damage caused by reactive oxygen radicals 16.

The highly significant decrease in the percentage of sperm abnormalities observed in this work might be secondary to the decrease in the activities of glutathione peroxidase and testicular malondialdehyde, which affects the antioxidant ability of the sperms 55. Commensurate with these findings, it was observed that pretreatment of spermatozoa with free radical scavengers protected the sperm DNA from damage by reactive oxygen species and improved its performance in in-vitro fertilization 56,57.

In the M. oleifera-treated group, Leydig cells exhibited signs of hyperactivity such as irregular euchromatic nuclei, dilated Golgi body, and numerous dilated SER cisternae. Such changes will lead to increased secretion of testosterone by these cells. These findings are in agreement with those of other studies that reported that M. oleifera produced its androgenic effect by increasing serum and testicular testosterone levels and improving blood flow to the male reproductive organs 58. Moreover, it was found that β-carotene in M. oleifera leaves is efficiently converted into vitamin A in the body, which has an essential role in the maintenance of normal reproductive functions 18,59.

In conclusion, the present findings demonstrated that M. oleifera had a protective and modulating effect against Dex-induced spermatogenic defects in rat. Further studies are recommended to clarify the clear-cut mechanism and the actual dosage of M. oleifera leaf extract required for such protection.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

Table

Table

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Keywords:

dexamethasone; Moringa olifera; testis; ultrastructure; sperm

© 2014 The Egyptian Journal of Histology