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The effect of stem cell therapy versus melatonin on the changes induced by busulfan in the testes of adult rat: histological and immunohistochemical studies

Abd El Aziz, Dalia H.a; Metwally, Hala G.b

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The Egyptian Journal of Histology: March 2013 - Volume 36 - Issue 1 - p 175-184
doi: 10.1097/01.EHX.0000425579.77855.ea
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Abstract

Introduction

Treatment with cytotoxic chemotherapy and radiotherapy is associated with significant gonadal damage 1. Successful chemotherapeutic regimens have led to a significant increase in the survival rate of patients with conditions such as testicular cancer, lymphomas, or leukemia. Therefore, chemotherapy-induced impairment in fertility has gained increasing clinical importance. Semen cryopreservation has been the most reliable method for this purpose. However, the quality of cryopreserved sperm after thawing does not always allow fertilization by artificial insemination 2.

Busulfan is an effective antineoplastic and alkylating agent that adversely affects spermatogenesis and it is the drug of choice in the treatment of leukemia 3.

The neurohormone melatonin (N-acetyl-5-methoxytryptamine) is the chief secretory product of the pineal gland 4. Melatonin facilitates various physiologic processes: circadian rhythm functions, such as sleeping and wakefulness, sexual activity and reproductivefunctions, tumor growth, immune response, andaging 5. Melatonin receptors have been detected in the male genitalia system 6. Melatonin also affects cellproliferation and differentiation 7.

Stem cells are self-renewing elements with the capacity to generate multiple distinct cell lineages. They exist in various tissues, even in adults, and have been isolated from a variety of differentiated tissues, including bone marrow, umbilical blood, brain, and fat 8. Transplantation of stem cells for infertility has been a major research topic in recent years, especially cancer patients, because one of the most devastating adverse effects of cancer treatments is damage to the reproductive system.

Human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (HUMSCs) have pluripotent characteristics for indefinite proliferation and can be induced to differentiate into advanced derivatives of all three germ layers, such as bone, cartilage, fat, muscle, heart, and brain cells 9.Unlike the isolation of stem cells derived from other sources, such as embryos and bone marrow, that of HUMSCs is noninvasive and does not have surrounding moral or ethical issues 10. Furthermore, HUMSCs have a relatively high proliferation rate and self-renewal capacity compared with other adult stem cells 11. Clinical application of HUMSCs has been considered promising for regenerative medicine. Because the multipotent properties of HUMSCs are intermediate between those of ESCs and adult stem cells, the induction of germ cells from HUMSCs might be possible 12.

Aim of the work

The present study was carried out to compare the effect of stem cell therapy and melatonin in the amelioration of the harmful changes induced by busulfan in rat testes.

Materials and methods

The study was carried out at the animal house of Faculty of Medicine, Cairo University, according to the guide for the care and use of laboratory animals.

Drugs

  • Myleran (busulfan) (1,4-butanediol dimethanesulfonate), used as tablets (2 mg), was dissolved in dimethyl sulfoxide (GlaxoSmithKline, London, United Kingdom).
  • Melatonin was used as melatonin 5 mg (Natures Bounty Inc., Bohemia, New York) tablets dissolved in 1% ethanol.

Animals

Forty-three adult male albino rats weighing 150–200 g were included in the study. They were housed in a temperature-controlled and light-controlled room (12-h light/dark cycle), with free access to food and water. They were divided into five groups.

Group I (control group)

Group I included eight rats; two in each experimental group. Each rat was administered a single dose of 0.5 ml dimethyl sulfoxide (a solvent of busulfan).

Group II (busulfan group)

Group II included 10 rats; each rat received 20 mg/kg busulfan intraperitoneally in a single dose 13.

  • Three animals from this group were sacrificed to confirm testicular injury.
  • The remaining seven animals received a single dose of 20 mg/kg busulfan intraperitoneally, and did not receive therapy for 35 days 13.

Group III (busulfan+stem cell therapy group)

Group III included 10 rats; each rat received a single dose of 20 mg/kg busulfan intraperitoneally 35 days before stem cell transplantation. The animals were injected with 0.5 ml cultured and labeled stem cells suspended in phosphate buffer saline in the tail vein 14. The rats were sacrificed 15 days following stem cell therapy 15. Stem cells were isolated from cord blood 8.

Group IV (melatonin group)

Group IV included five rats; each rat received 10 mg/kg melatonin intraperitoneally for 5 days 13.

Group V (busulfan group+melatonin)

Group V included 10 rats; each rat received 10 mg/kg melatonin intraperitoneally for 5 days upon an in initial dose of busulfan (20 mg/kg). Thirty-five days after the treatments, all the animals were sacrificed 13.

Cord blood collection was performed at the Gynaecology Department, Faculty of Medicine, Cairo University. Stem cell culture, labeling, and phenotyping were carried out at the Hematology Unit, New Kasr El Aini Hospital, Cairo University.

Methods

Stem cells

Cord blood collection16: The storage and transport temperature was 15–22°C, the transport time was 8–24 h, the sample volume was 65–250 ml, and no sample had signs of coagulation or hemolysis.

Mononuclear cell fraction isolation and propagation16: The mononuclear cell fraction was isolated by loading 30 ml of whole blood onto 10 ml of Ficoll density media (Healthcare Bio-Sciences Piscataway, NJ United States) in 50 ml polypropylene tubes, centrifuged for 30 min at room temperature. The interphase was collected after aspirating and discarding the supernatant. The interphase was washed with 20 ml PBS and centrifuged at room temperature. The supernatant was aspirated and the cells were washed with PBS a second time. The cells were resuspended in the isolation media and transferred to culture dishes. The isolation media were low-glucose Dulbecco’s modified Eagles medium (Cambrex Bio-Science HOPKINTON, MA, United States) supplemented with low dexamethazone(10−7 mol/l) (Sigma-Aldrich Taufkirchen Germany), penicillin (100 IU/ml) (Invitrogen), streptomycin (0.1 mg/ml) (Invitrogen, Van Allen Way Carlsbad, CA. USA), and ultraglutamine (2 mmol/l) (Cambrex Bio-Science). Incubation was carried out at 38.5°C in a humidified atmosphere of 5% CO2.

Culture16: The isolation media were replaced after overnight incubation (12–18 h) in order to remove nonadherent cells. The media were replaced every 3 days until mesenchymal stem cell colonies were noted. The cultures were inspected daily for the formation of adherent spindle-shaped fibroblastoid cell colonies. Subculturing was carried out by chemical detachment using 0.04% trypsin. Later, when cell numbers allowed, expansion was performed in 25or 75 cm2 tissue culture flasks.

Labeling17: Mesenchymal stem cells were labeled by incubation with a ferumoxide injectable solution(25 μg Fe/ml, Feridex; Berlex Laboratories, Montville, New Jersey, USA) in culture medium for 24 h, with 375 ng/ml poly-L-lysine added 1 h before cell incubation. Labeling was assessed histologically using prussian blue. Feridex-labeled HMSCs were washed in PBS, trypsinized, washed, and resuspended in 0.01 mol/l PBS at a concentration of 1×106 cells/ml.

Cell viability analysis: Cell viability was assessed using the trypan blue dye exclusion test. This method is based on the principle that viable cells do not take up certain dyes, whereas dead cells do.

Flow cytometry18: Flow cytometric analyses were carried out on a fluorescence activated cell sorter flow cytometer (Coulter Epics Elite, Miami, Florida, USA). HMSC were trypsinized and washed twice with PBS. A total number of 1×105 HMSC were used for each run. To evaluate the HMSC marker profile, cells were incubated in 100 μl of PBS with 3 μl of CD44-FITC for 20 min at room temperature. The antibody concentration was0.1 mg/ml. Cells were washed twice with PBS and finally diluted in 200 μl of PBS. The expression of the surface marker was assessed by the mean fluorescence.

Histological study

The animals were sacrificed using a lethal dose of ether. Testicular specimens were removed and fixed in Bouin for 24 h. Paraffin blocks were prepared and 5-μm-thick sections were subjected to H&E stain 19.

Histochemical study

Testes sections were stained with prussian blue stain 20 for the observation of iron oxide-labeled mesenchymal therapeutic stem cells in testicular tissue.

Immunohistochemical study

For proliferating cell nuclear antigen (PCNA) and caspase-3 using the avidin–biotin peroxidase complex technique 21, the following were used:

PCNA antibody, which is a mouse monoclonal antibody PC 10 (Novocastra, Newcasle UK.).

Caspase-3 antibody, which is a rabbit polyclonal antibody (CPP32) Ab-4 (Thermo Fisher Scientific, Fermont, California, USA).

Steps of immunostaining: The primary antibody was applied on the sections for 60 min. Then the sections were incubated with the secondary antibody (biotinylated rabbit polyclonal antibody). The site of the reaction was visualized with diaminobenzodene tetrahydrochloride and was indicated by the presence of brown nuclear deposits for PCNA and by brown, predominantly cytoplasmic, with some nuclear deposits of caspase-3.

Sections were counterstained with Meyer’s hematoxylin.

Morphometric study

The software Leica QWin 500 (Cambridge, UK.) for image analysis was used to count the number of positive PCNA cells and active caspase-3-positive cells in semineferous tubules. In each specimen, 10 random nonoverlapping fields were chosen and the number of each type of positive cells was counted using a magnification of ×200. The mean number/low-power field for each specimen and the mean value of each experimental animal group were obtained. The data obtained were subjected to statistical analysis and compared with the control.

Statistical analysis

Quantitative data were summarized as means and SD and compared using one-way analysis of variance. Any significant analysis of variance was followed by a Bonferroni post-hoc test to detect which pairs of groups induced the significant difference. P-values of less than 0.005 were considered statistically significant. Calculations were carried out on SPSS IBM, united state. (version 16 windows) software 22.

Results

H&E stains

Sections in the testes of normal control showed the normal architecture of semineferous tubules that had all types of spermatogenic cells and interstitial cells of Leydig (Figs. 1 and 2).

Figure 1
Figure 1:
Photomicrograph of a section in the testis of a control rat showing many semineferous tubules with interstitial tissue in between.Figure 1. H&E, ×100.
Figure 2
Figure 2:
Photomicrograph of a section in the testis of a control rat showing the normal architecture of semineferous tubules. Note the presence of spermatogonia (Sg). Primary spermatocytes (S1), rounded spermatids (S3), elongated spermatid (S4) in the lumen, and sertoli cells (St). Interstitial cells of Leydig (L) can be seen in the interstitium between tubules.Figure 2. H&E, ×400.

Examination of histological sections in the testes of rats treated with busulfan showed various degrees of affection. Some semineferous tubules appeared atrophied and irregular in shape. Others showed a reduction in germ cell layers (Fig. 3). The tubules showed a marked decrease in germ cell numbers; only spermatogonia appeared. There was exfoliate of apoptotic germ cells into the tubular lumen (Fig. 4a). In addition, multinucleated giant cells were observed (Fig. 4b).

Figure 3
Figure 3:
Photomicrograph of a section in the testis of a busulfan-treated rat showing some atrophied, irregular-shaped semineferous tubules (asterisks). The rest of the tubules showed a reduction in germ cell layers (arrows).Figure 3. H&E, ×100.
Figure 4
Figure 4:
(a) Photomicrograph of a section in the testis of a busulfan-treated rat showing a marked decrease in germ cells number; only spermatogonia (Sg) appear. Note the exfoliate of apoptotic germ cells into the tubular lumen (S). (b) Photomicrograph of a section in the testis of a busulfan-treated rat showing few spermatogonia (Sg). Note the presence of multinucleated giant cells (arrow).Figure 4. H&E, ×400.

Sections of the stem cell-treated group showed individual variations. Some tubules had an irregular shape and appeared atrophied. There was marked depletion of germ cells, whereas other tubules were more or less normal (Figs. 5 and 6). Some tubules contained spermatogonia, primary spermatocytes, sertoli cells, and exfoliated germ cells inside the lumen of tubules (Fig. 7).

Figure 5
Figure 5:
Photomicrograph of a section in the testis of a stem cell-treated rat showing some irregular-shaped and atrophied tubules (asterisks). Some tubules showed marked depletion of germ cells (arrows); others are more or less normal.Figure 5. H&E, ×100.
Figure 6
Figure 6:
Photomicrograph of a section in the testis of a stem cell-treated rat showing the normal architecture of semineferous tubules containing spermatogenic cells and some shedded cells (S). Note the prominence of interstitial cells of Leydig.Figure 6. H&E, ×100.
Figure 7
Figure 7:
Higher magnification of the previous section showing a tubule that contains spermatogonia (Sg), primary spermatocytes (S1), sertoli cells (St), and exfoliated germ cells inside the lumen of tubules (S).Figure 7. H&E, ×400.

Sections of the melatonin-treated group showed more or less normal tubules; one tubule contained only sertoli cells. Interstitial capillaries were congested (Fig. 8). The normal semineferous tubules in this specimen showed spermatogonia, primary spermatocytes, elongated spermatid, and sertoli cells (Fig. 9).

Figure 8
Figure 8:
Photomicrograph of a section in the testis of a melatonin-treated rat showing more or less normal tubules (N); one tubule contains only sertoli cells (St). Note congested interstitial capillaries (c).Figure 8. H&E, ×100.
Figure 9
Figure 9:
Photomicrograph of a section in the testis of a melatonin-treated rat showing more or less normal semineferous tubules, spermatogonia (Sg), primary spermatocytes (S1), elongated spermatids (S3), and sertoli cells (St).Figure 9. H&E, ×400.

The histological structure of the semineferous tubules in the melatonin only-treated group (group IV) was similar to that of the control groups.

Prussian blue-stained sections

Sections in the testes of control rats showed negative staining with prussian blue (Fig. 10). The stem cell therapy group showed spindle-shaped (Fig. 11a), branched, and globular-shaped (Fig. 11b) prussian blue positive-stained cells in the interstitial space. In addition, globular cells were observed in the basal compartment of the semineferous tubule (Fig. 11c).

Figure 10
Figure 10:
Photomicrograph of a section in the testis of a control rat showing the absence of prussian blue-stained cells.Figure 10. Prussian blue staining, ×400.
Figure 11
Figure 11:
(a) Photomicrograph of a section in the testis of a stem cell-treated rat showing a spindle-shaped prussian blue positive-stained cell (arrow) in the interstitial space. (b) Photomicrograph of a section in the testis of a stem cell-treated rat showing branched and globular-shaped prussian blue positive-stained cells in the interstitial space (arrows). (c) Photomicrograph of a section in the testis of a stem cell-treated rat showing a globular-shaped prussian blue positive-stained cell in the basal compartment of the semineferous tubule.Figure 11. Prussian blue staining, ×1000.

Caspase-3-activity immunostaining

A few active caspase-3-activity-positive immunostaining cells were observed in the control group (Fig. 12). There were multiple active caspase-3-activity-positive immunostaining cells in the busulfan-treated group (Fig. 13), moderate in rats treated with stem cells (Fig. 14), and few in rats treated with melatonin (Fig. 15).

Figure 12
Figure 12:
Photomicrograph of a section in the testis of a control rat showing a few active caspase-3 immunostaining cellsFigure 12. Caspase-3 immunostaining, ×400.
Figure 13
Figure 13:
Photomicrograph of a section in the testis of a busulfan-treated rat showing multiple positive active caspase-3 immunostaining cells.Figure 13. Caspase-3 immunostaining, ×400.
Figure 14
Figure 14:
Photomicrograph of a section in the testis of a stem cell-treated rat showing moderate positive active caspase-3 immunostaining cells.Figure 14. Caspase-3 immunostaining, ×400.
Figure 15
Figure 15:
Photomicrograph of a section in the testis of a melatonin-treated rat showing few positive active caspase-3 immunostaining cells.Figure 15. Caspase-3 immunostaining, ×400.

Proliferating cell nuclear antigen immunostaining

Positive-PCNA immunostaining cells were strongly detected in the spermatogenic cells of the control group (Fig. 16). However, there were few positive-PCNA immunostaining cells in the germinal cells of the busulfan-treated group (Fig. 17). The positive-PCNA immunostaining cells were moderate in the stem cell-treated group (Fig. 18). Multiple positive PCNA cells were seen in the germinal cells of melatonin treated groups (Fig. 19).

Figure 16
Figure 16:
Photomicrograph of a section in the testis of a control rat showing PCNA-positive cells in the spermatogenic cells.Figure 16. PCNA immunostaining, ×400.
Figure 17
Figure 17:
Photomicrograph of a section in the testis of a busulfan-treated rat showing a few PCNA-positive cells.Figure 17. PCNA immunostaining, ×400.
Figure 18
Figure 18:
Photomicrograph of a section in the testis of a stem cell-treated rat showing moderate PCNA-positive cells in the germinal cells.Figure 18. PCNA immunostaining, ×400.
Figure 19
Figure 19:
Photomicrograph of a section in the testis of a melatonin-treated rat showing multiple PCNA-positive cells in the germinal cells.Figure 19. PCNA immunostaining, ×400.

Morphometric results

The mean number of active caspase-3-positive cells was significantly increased in the busulfan group compared with the control group, whereas the mean number of active caspase-3-positive cells was significantly decreased in both group III and group V compared with group II (Table 1). The mean number of PCNA-positive cells was significantly decreased in the busulfan group compared with the control, whereas the mean number of PCNA-positive cells in group V was significantly increased comparable to group II (Table 2).

Table 1
Table 1:
Mean number of active caspase 3-positive cells in the different groups studied
Table 2
Table 2:
Mean values of the number of proliferating cell nuclear antigen-positive cells in different groups studied

Discussion

The current study was carried out to compare the effect of cord blood stem cell therapy and melatonin administration on busulfan-induced testes injury in albino rats.

In the present study, in group II, semineferous tubules appeared atrophied and irregular in shape. Some investigators have confirmed these findings 23.

Moreover, some tubules showed a reduction in germ cell layers; only spermatogonia appeared. In addition, exfoliation of apoptotic germ cells into the tubular lumen was detected. These findings have been confirmed by other workers 24. Some authors have reported that spermatogenic epithelium was the target of cytotoxic drugs because of their high mitotic activity 25. Other investigators have attributed these changes to the direct toxic effect of busulfan on spermatogenic series cells and to a secondary effect of busulfan on the hypothalamo–pituitary–gonadal 26. Morphometrically, there was a significant increase in the mean number of caspase-3-positive cells compared with the control group. Some workers have reported that busulfan exerts an apoptotic effect on spermatogenic cells, mostly on spermatogonia and primary spermatocytes 27.

The mean number of PCNA-positive cells was significantly decreased in the busulfan-treated group in the present study compared with the control group. Other authors, declared that there was a suppression of spermatogonial cell division and the arrest of spermatogenesis at the Zygotene stage due to the maturation blockages at this stage. Also, They added that busulfan induced acute testicular regression associated with a severe depletion of sperm count and resulted in sterility for a specific period. Such a suppressive effect of busulfan on testicular spermatogenesis suggests that the drug acted directly on the semineferous tubules 28.

In the stem cell-treated group in the present study, a few tubules appeared more or less normal. In addition, the mean number of active caspase-3-positive cells was significantly decreased compared with group II. Some investigators have reported that HUMSCs could differentiate into germ cells in vitro12. Some researchers have reported that stem cells can regenerate various cell lineages by transdifferentiation or cell fusion mechanisms 29.

Other sections of the stem cell-treated group showed no improvement, with some irregular-shaped and atrophied tubules; there was depletion of germ cells. Some tubules contained spermatogonia, primary spermatocytes, sertoli cells, and exfoliated germ cells inside the lumen. Some authors have suggested that stem cells might have been recognized as foreign cells and may have been eliminated by sertoli cells. They added that being an antimitotic agent, busulfan could affect many other organs. In this way, different inflammatory signals could guide stem cell migration to other organs, where the specific cytokines were secreted, and promoted homing 15. However, in the present work, using prussian blue stain, sections in animals treated with stem cells showed spindle-shaped, branched, and globular cells in the interstitial spaces and in the basal compartment of the semineferous tubules, indicating migration of injected stem cells to the injured testes.

In the present work, we injected undifferentiated stem cells into the tail vein of busulfan-treated rats; we suggested that for better results we might transplant spermatogonial stem cells (SSCs). This was confirmed by investigators who transplanted SSCs into busulfan-treated mice. The results showed complete spermatogenesis in recipient mouse testes; the transplantation of SSCs stimulated endogenous spermatogenesis in the recipient mice 30.

In the present study, in the stem cell-treated group, we analyzed recipient rat testes 15 days after transplantation. However, some investigators have reported that stem cells could regenerate testes epithelium, but they waited for ∼10 weeks after transplantation 31. Therefore, the longer duration might be responsible for better improvement.

In the present study, it was found that animals treated with melatonin showed normal semineferous tubules, the mean number of active caspase-3-positive cells was significantly decreased in the melatonin-treated group compared with the busulfan group, and the mean number of PCNA-positive cells in the melatonin-treated group was significantly increased comparable to that in the busulfan-treated group. Some investigators have concluded that melatonin exerted a possible protective effect against busulfan-induced testicular damage 13.

Some authors have reported that melatonin is the most powerful antioxidant in the attenuation of testicular injury 32. Other investigators added that the protective effects of melatonin might be because of both the inhibition of lipid peroxidation and increased antioxidant activity 33. Other investigators have shown the indirect antioxidant role of melatonin in stimulating antioxidant enzymes, its ability to augment the activities of other antioxidants, its protective role of antioxidant enzymes against oxidative damage, and its ability to increase the efficiency of the mitochondrial electron transport chain, thereby reducing electron leakage and free radical generation. They added that the ability of melatonin to function as a direct free radical scavenger may be related to its electron-donating ability 34.

Some authors have reported that melatonin exerts an antiapoptotic effect. They added that melatonin alleviated testicular germ cell apoptosis through inhibition of endoplasmic reticulum stress and the unfolded protein response in testes 35. Other investigators have confirmed that melatonin improved histopathological changes in the testes and reduced germ cell apoptosis 36. Some investigators have reported that the increase in PCNA in testicular germ cells indicated high proliferative activity and stimulation of spermatogenesis 37.

Conclusion

We concluded that melatonin exerts a possible protective effect against busulfan-induced testicular damage. Taken together, our study shows that HUMSCs transplantation led to no or minimal improvement in the testes.

Table
Table:
No title available.

Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

busulfan; melatonin; stem cells; testes

© 2013 The Egyptian Journal of Histology