الفهرس 
Pyrethroids (cyhalothrin)Only 14 pages are availabe for public view
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Abstract
Pesticides, especially synthetic pyrethroids have been extensively used in daily life and industry, and documented to be associated with increased incidences of infertility and reproductive dysfunction. ʎ-cyhalothrin (LCT), one of the newer synthetic pyrethroid insecticides, was initially recommended as a low-toxicity agent, while recent scientific evidence suggests that accumulation of LCT is extremely detrimental to humans and animals, specifically to the reproductive system, where was associated with poor sperm quality and sexual functions which finally lead to infertility.Cleome droserifolia, a natural antioxidant, plant of the Cleomaceae family, grows in Egypt, Libya, Palestine, and Syria. This plant has a long history of medicinal use. Extract of leaves and stems for Cleome droserifolia (CDE) is rich in bioactive compounds such as flavonoids which have long been recognized to possess anti-inflammatory, antioxidant, hepatoprotective, antithrombotic and anticarcinogenic activities.
Stem cells are undifferentiated biological cells that can differentiate into specialized cells and can divide through mitosis to produce more stem cells. Mesenchymal stem cells (MSCs) from bone marrow have great potential for tissue repair, and it has the ability to form bone, fat, cartilage, muscle, neurons, hepatocytes, skin and as well germ cells in the appropriate conditions in vivo.
The present study aimed to elucidate the protective and therapeutic role of a natural antioxidant, Cleome droserifolia extract (CDE), and mesenchymal stem cells (MSCs) derived from the bone marrow (BM) in the restoration of fertility and reproductive functions after testicular disfunctions induced by ʎ-cyhalothrin (LCT) exposure in male rats.
Seventy adult male Wister albino rats weighing 110-130g were used, and divided into seven groups each containing 10 rats as follows:
G I (control): Rats received orally olive oil+distilled water, parallel to the drug treated groups.
G II (LCT group): Rats received LCT only (6.23 mg/kg b.w.,i.p.) dissolved in olive oil three times a week for two weeks.
G III (CDE group): Rats received CDE only (100 mg/kg b.w., p.o) dissolved in distilled water daily for eight weeks.
G IV (LCT+CDE-protective group): Rats given CDE (100 mg/kg b.w., p.o.) daily for eight weeks. On the 7th week just after CDE treatment they received doses of LCT (6.23 mg/kg b.w., i.p.) three times a week for two weeks with the continuation of the extract.
G V (LCT+CDE-therapeutic group): Rats receiving dose of LCT (6.23 mg/kg b.w.,i.p.) three times a week on the 1st and 2nd weeks, then administered doses of CDE (100 mg/kg b.w., p.o.) daily for 8 weeks.
G VI (LCT+MSCs-protective group): Rats injected (i.v.) with MSCs through the tail vein with a single dose (1x106 cells/ ml PBS), then received doses of LCT (6.23 mg/kg b.w., i.p.) three times a week for two weeks, and left until the 8th week.
G VII (LCT+MSCs-therapeutic group): Rats received doses of LCT (6.23 mg/kg b.w.,i.p.) three times a week on the 1st and 2nd weeks, then injected (i.v.) with MSCs through the tail vein with a single dose (1x106 cells/ ml PBS) and left for 8 weeks.
Animals were sacrificed at the end of the experiment. The morphological investigation included detecting of mesenchymal stem cells (MSCs), total body and testis weights and the morphological study of sperms (count, viability, motility and abnormality).
The biochemical investigation included serum testosterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), total protein and albumin level, total cholesterol, triglyceride (TG), lipoproteins (HDL, LDL and VLDL). It was also measured tumor necrosis factor (TNF-α), interleukin-10 (IL-10) and interleukin-12 (IL-12). In addition, testicular reduced glutathione (GSH), superoxide dismutase (SOD) and lipid peroxidation malondialdehyde (MDA) were measured in tissue. The histological and ultrastructural alterations in testis tissue were examined.
The results obtained from the present investigation can be summarized as follows:
Morphological studies:
Detecting of mesenchymal stem cells:
In vitro, the cell culture showed mesenchymal stem cells (MSCs) as spindle and fibroblastic in morphology which later became the rounded cells.
MSCs showed negative immune reaction to CD34 and CD45 but showed positive to CD44, and CD105. Also it showed its ability to differentiate into mesodermal cell lineages (adipocytes, chondrocytes and osteocytes) in vitro.
MSCs groups showed spindle-shaped, branched, and globular-shaped prussian blue positive-stained cells in various areas of testicular tissue.
Total body and testis weights:
The study showed a very highly significant decrease in body and testis weight in LCT-treated rats (GII) compared to control group (GI), whereas all groups (LCT+CDE-treated groups and LCT+MSCs treated groups) showed significant increase in total body weight compared to GII.
A very highly significant increase occurred in testis weight in both, LCT+CDE-protective group (GIV) and LCT+MSCs-therapeutic group (GVII). In addition, the average of testis weights of LCT+CDE-therapeutic group (GV) recorded highly significant increase, while a non-significant increase occurred in the mean testis weight in LCT+MSCs-protective group (GVI).
Sperm indices:
This study revealed that rats treated with LCT (GII) had markedly impaired sperm quality. LCT significantly lowered sperm count, viability and motility percent and significantly increased sperm abnormalities as compared to control group.
The administration of CDE (GIV and GV) in the present investigation indicated significant improvement as regards sperm count, viability, motility and abnormality especially in LCT+CDE-protective group (GIV) where the value was nearly returned to the control.
The LCT+MSCs-protective group (GVI) showed partial improvement in sperm parameters. But, significant improvement was observed in LCT+MSCs-therapeutic group (GVII) as compared to control group.
Biochemical studies:
Sexual hormones:
The present study showed a marked decrease in the levels of serum testosterone and a significant increase in the levels of serum FSH and LH in rats exposed to LCT (GII) as compared to control group. However, protection was shown greatly in LCT+CDE-protective group (GIV) and to a lesser in LCT+CDE-therapeutic group (GV).
The LCT+MSCs-protective group (GVI) revealed minimal improvement in sex hormones, while, LCT+MSCs-therapeutic group (GVII) showed significant improvement in the levels of these hormones.
Protein profile:
The administration of LCT (GII) induced an obvious depletion in both total protein and albumin levels in the serum as compared to control.
LCT+CDE-protective group (GIV) showed very highly significant improvement in the level of both albumin and total protein, while CDE in the LCT+CDE-therapeutic group administered LCT followed by CDE (GV) revealed partial recovery in total protein.
LCT+MSCs-therapeutic group (GVII) showed significant increase in the levels of both total protein and albumin, whereas, minimal improvement occurred in the LCT+MSCs-protective group (GVI).
Lipid profile:
In LCT rats (GII) a significant elevation of the levels of cholesterol, TG, LDL and VLDL and significant reduction of HDL were recorded as compared to control groups. However, in the present investigation, a protection occurred in lipid profile levels in LCT+CDE-protective group (GIV) while, a partial improvement was recorded in the LCT+CDE-therapeutic group (GV) in serum cholesterol, TG, HDL, LDL and VLDL.
Also, a partial improvement occurred in lipid profile levels in the LCT+MSCs-protective group (VI), while a marked improvement was recorded in LCT+MSCs-therapeutic group (VII) in serum cholesterol, TG, HDL, LDL and VLDL.
Cytokines:
Administration of LCT (GII) resulted in disruption of some immune biomarkers reflected by the significant increase in serum TNF-α and IL-12 levels, and significant decrease in IL-10 level.
Significant improvement was observed in LCT+CDE-protective group (GIV) in the same parameters, while a partial improvement in LCT+CDE-therapeutic group (GV).
The intravenous injection of MSCs into male rats in GVI and GVII showed marked restoration of cytokines (TNF-α, IL-12 and IL-10) levels as compared to control group especially in LCT+MSCs-therapeutic group (GVII).
Antoxidants and oxidative stress:
A significant depletion in the testicular GSH and SOD was designated, while a significant elevation was realized in MDA in LCT-treated rats (GII) as compared with the control group (GI).
Protection was shown in LCT+CDE-protective group (GIV) more than the LCT+CDE-therapeutic group (GV) in testicular GSH, SOD and MDA.
The results revealed minimal protection in tissue GSH, SOD and MDA of LCT+MSCs-protective group (GVI), while significant improvement was shown in LCT+MSCs-therapeutic group (GVII).
Microscopic studies:
- Light microscopic study:
The histopathological examination of testis sections from the rats administered LCT revealed severe damage of the seminiferous tubules with separating of cells from underlying layer of the tubules, exfoliation and germ cell depletion and hyalinization of the luminal contents. Edema and widening of the intertubular spaces together with diminution of the stromal interstitial tissue were also illustrated.
In rats treated with CDE as a protective agent then followed by the administration of LCT (LCT+CDE-protective group, GIV), the histopathological changes were greatly reduced. This group showed recovery and preservation of seminiferous epithelium except separating of few cells from underlying layer and appearance of large intraepithelial vacuoles.
The group exposed to LCT then treated with the CDE (LCT+ CDE-therapeutic group, GV) showed partial recovery of some seminiferous tubules represented in the moderate restoration of spermatogenesis. Also, incomplete recovery of germinal epithelium was illustrated. However, a relatively large number of spermatozoa in the tubular luminae were noticed.
Testis sections of rats that received MSCs then exposed to LCT (LCT+MSCs-protective group, GVI) revealed poor protection with abnormal morphology of many seminiferous epithelium. Shrunken seminiferous tubules, widened intertubular spaces, disruption of the boundary tissue and diminution of the interstitial cells were still evident.
Light microscopic preparation of testis sections of rats that received LCT then followed by MSCs (LCT+MSCs-therapeutic group, GVII) revealed improvement in the seminiferous tubule structure with an increase in the number of spermatogenic cells compared to the LCT group, also, showed nearly restoration of spermatogenesis to its normal situations. Spermatogenic layers were comparatively well organized and developed.
- Electron microscopic study:
Electron microscopic examination of ultrathin sections of the rat testis of LCT-treated group (GII) revealed that Sertoli cell does not rest on the basement membrane. The cytoplasm of Sertoli cell, spermatogonia and primary spermatocytes is highly degenerated and contained smooth endoplasmic reticulum-derived vacuoles along with disintegration or overall decreases of cytoplasmic organelles. Irregular boundary tissue of seminiferous tubules, deformed spermatids, with distorted acrosome and degenerated mitochondria were also evident.
The ultrastructure of the seminiferous tubules of the rat testis in LCT+CDE-protective group (GIV) showed nearly normal structure of boundary tissue and relatively unaffected spermatogonia, spermatocytes and spermatids. Almost all the mitochondria appear normal, except fusion of few ones.
At the ultrastructural level, partial improvement was observed in the structure of the seminiferous tubules of LCT+CDE-therapeutic group (GV). However, it showed signs of testicular damage included small-sized spermatogonia with flattened nnucleus and abnormal chromatin, as well Sertoli cell with compressed crescent-shaped nucleus, dilated smooth endoplasmic reticulum and lipid droplets with variable sizes were also noticed, whilst, many little affected mitochondria and slightly irregular basement membrane appeared.
Electron microscopical examination of ultrathin sections of LCT+MSCs-protective group (GVI) revealed poor improvement in the structure of the seminiferous tubules. This group showed spermatogonia with very dense nuclear chromatin and altered cytoplasm, primary spermatocyte with abnormal elongated nucleus. Sertoli cell appeared abnormal with degenerated mitochondria and dilated smooth endoplasmic reticulum, in addition to the intercellular vacuoles.
The ultrastructure of the seminiferous tubules of a testis of rats of LCT+MSCs-therapeutic group (GVII) showed marked restoration of the general ultrastructure of testicular tissues. Spermatogonia, primary spermatocytes and Sertoli cells appeared with normal fine structure. In addition, Sertoli cell with large elongated nucleus and its cytoplasmic processes contain intact mitochondria; however, large lipid droplets were illustrated.
It is important mentioning that, no remarkable changes were reported after rats treated with CDE only (GIII) through the experimental duration in all parameters.