Asker, Samar A.
The oviduct is a dynamic organ and an important source of factors that play key roles in reproductive and developmental events in the female genital system . The reproductive cycle in the rat includes proestrous, estrous, metestrous, and diestrous phases . The diestrous phase correlates to the luteal phase in human . The oviduct is divided into four anatomical segments: preampulla (consisting of fimbriae and infundibulum), ampulla, isthmus, and uterotubal junction . In the ampullary region, significant variations in the morphology and in localization of leptin correlated with the stages of the reproductive cycle .
Leptin is a hormone originally identified in adipocytes that plays a critical role not only in the control of energy balance and metabolism but also in diverse functions such as reproduction . Leptin regulates the ovarian steroid synthesis , which in turn controls the different oviductal functions  from transport and maturation of the oocytes, spermatozoa, and embryos to fertilization .
Female infertility is a serious medical problem. The female reproductive system is sensitive to different harmful environmental chemicals, industrial pollutants, and food additives [2,7]. Monosodium glutamate (MSG) is the salt of nonessential glutamic acid. It is widely used as a food additive. It is present in flavored chips and snacks, canned soups or sauces, prepared meals, frozen foods and meals, fresh sausages, marinated meats, bottled soy or oriental sauces, manufactured meats, luncheon chicken and turkey, flavored tuna, and vegetarian burgers. This popular taste enhancer is widely used not only in the food industry but also in homes and restaurants . There are conflicting reports concerning the safety of MSG; many studies reported its effect on liver, kidney, brain , and ovary  but the reports concerned with its effect on the oviduct are deficient.
Coenzyme Q10 (CoQ10) is a naturally occurring compound with properties similar to those of vitamins. It is a cofactor in the mitochondria respiratory chain and in the production of ATP. Furthermore, it is a potent lipophilic antioxidant and is capable of recycling and regenerating other antioxidants such as tocopherol and ascorbate . Dietary CoQ10 is present in fish, meat, chicken heart, liver, dairy products, vegetable oils, parsley, broccoli, grape, avocado, and cauliflower . It was demonstrated that CoQ10 can protect against the changes induced by other toxicants in the female genital system .
Therefore, this study was conducted to investigate the effect of MSG on the histological structure and the immunohistochemical distribution of leptin in the ampulla of adult female albino rat oviduct during the diestrous phase, and the effect of CoQ10 supplementation.
Materials and methods
All procedures in this study were performed in accordance with the Medical Research Ethics Committee of Mansoura University. Adult (120 days old) virgin (isolated since weaning) female albino rats (150–200 gm) were examined for the regularity of their estrous cycle. Vaginal smears were collected daily (between 9:00 a.m. and 10:00 a.m.) for three consecutive cycles, and only rats with consistent 4-day cycles were included in the study . A total of 50 adult female rats were used. They were housed in a room temperature of 22±2°C, exposed to a 12-h dark/light cycle and had access to normal rat chow and water ad libitum. Rats were randomized into four groups of control and experimental animals. Group I (n=20) included control rats that were equally subdivided into subgroup (a) including negative control rats not receiving any substance, and subgroup (b) including rats intraperitoneally injected daily with 25 ml of corn oil (C8267, Sigma, St. Louis, Missouri, USA)  (the solvent media of CoQ10). Group II (n=10) included rats that received MSG (G 1626, Sigma) dissolved in distilled water at a daily dose of 100 mg /kg body weight/ rat by gastric tube . Group III (n=10) included rats that received intraperitoneal injections of CoQ10 (C 9538, Sigma) at a daily dose of 4 mg/kg body weight/rat  dissolved in corn oil . Group IV (n=10) included rats that received the similar doses of MSG and CoQ10.
Overnight fasting rats from all groups were weighed and then anesthetized by an intraperitoneal injection of pentobarbital (40 mg/kg)  after 2 months from the beginning of the experiment  during the diestrous phase. Venous blood was obtained from the heart of the animals of all groups, centrifuged, and the sera were stored at −70°C for biochemical study. The rats were then perfused through the left ventricle with 30 ml of isotonic saline solution containing procaine hydrochloride to prevent vasoconstriction, followed by a solution of 0.66% paraformaldehyde. The right atrium was incised to allow exsanguination and fluid drainage . The oviducts were excised.
Tissue preparation for histological study
The ampullary region of the right oviduct was taken and fixed in 10% neutral buffered formalin, dehydrated in alcohols, cleared in xylol, and embedded in paraplast. Sections of 5 μm thickness were stained with hematoxylin and eosin and Masson trichrome stains  for the light microscopic examination and for morphometric analysis.
The height of epithelial cells that extended as far as the luminal surface and the section that clearly passed through the nucleus, parallel to the longitudinal axis of the cell were selected and counted with an ocular micrometer and those in very obliquely cut areas were excluded. The number of epithelial cells was counted by light microscopy per ten high-power fields (×400) in the mucosa of each specimen by means of a micrometer .
Immunohistochemical technique for localization of leptin
Sections of 4 μm thickness were used for the immunohistochemical (IHC) localization of leptin [1,12]. Sections from the ovaries of control rats were used as positive control slides for leptin [5,12].
Ready-to-use target retrieval solution (S1700, Dakocytomation, Glostrup, Denmark), primary antibody polyclonal antibody against leptin (Y-20 sc-843; Santa Cruz Biotechnology, California, USA), and ready-to-use antibody diluent with background reducing components (S3022, Dakocytomation) were used. Universal detection kits (K 0673, Dakocytomation) were based on a modified avidin–biotin (laboratory) technique in which a biotinylated secondary antibody forms a complex with peroxidase-conjugated streptavidin molecule .
Sections were dewaxed in xylol and hydrated in descending grades of alcohol down to distilled water. They were immersed into a preheated target retrieval solution at 95–99°C (without boiling) in a water bath for 40 min and then allowed to cool for 20 min at room temperature. Sections were rinsed three times with phosphate-buffered saline (PBS). Excess liquid was tapped off the slides. Enough hydrogen peroxide was applied to cover the specimen for 5 min, then the slides were rinsed gently with PBS, and excess liquid was tapped off. Primary antibody (diluted 1 : 200)  was applied on specimens, and was incubated for 2 h in a humidity chamber at room temperature. Slides were rinsed in PBS. Biotinylated link was applied on specimens for 10 min and sections were rinsed in PBS. Streptavidin (horseradish peroxidase) reagent was applied on specimens for 10 min and sections were then rinsed in PBS. Freshly prepared 3,3-diaminobenzidine tetrahydrachloride substrate chromogen solution was removed from 2–8°C storage and applied on specimens for 10 min. Slides were rinsed gently in distilled water, immersed in hematoxylin for ½ min, and were rinsed in tap water until blue. Slides were dehydrated in ascending grades of alcohol, cleared in xylol, mounted by Canada balsam, and covered with a cover slip. Negative control slides were prepared by the same steps, except they were incubated with the antibody diluent instead of primary antibody. Positive reaction appeared brown in color .
Tissue preparation for the ultrastructural study
Small pieces (1 mm3) of the ampullary region of the left oviduct were fixed in a mixture of 2.5% gluteraldehyde and 2.5% paraformaldehyde (pH 7.3) overnight at 4°C. Specimens were postfixed in 1% osmium tetroxide. The specimens were dehydrated in ascending grades of alcohol and then passed through two changes of propylene oxide and embedded in epon. Ultrathin sections (60 nm thick) were cut, mounted on copper grids, and stained with uranyl acetate and lead citrate . The grids were then examined with the transmission electron microscope (Seo-Russia, 2000, Soma, Ukraine) in Military Veterinary Medicine Hospital, Cairo, Egypt.
Total serum cholesterol was detected using an enzymatic method .
The statistical data included rat weight, total serum cholesterol level, height of epithelial cell, and number of epithelial cells. Data were expressed as arithmetic mean±standard deviation. The Student t-test was used to test the significant changes of the parameters in groups II, III, and IV compared with negative control rats in group I. Statistical significance was realized at probability of P value of less than 0.05. Statistical analysis of the data was performed by MedCalc software (Mariakerke, Belgium) for medical statistics .
Control group I
Light microscopic structure of oviducts: vaginal smear of female albino rat during the diestrous phase showed leukocytes and some epithelial cells (Fig. 1). Cross-sections stained with hematoxylin and eosin revealed no apparent differences in the structure of subgroups (a) and (b). The ampulla of the oviduct was formed of mucosa, musculosa, and serosa. The mucosa was longitudinally folded. It consisted of epithelium and an underlying corium. The epithelium was formed of simple columnar cells with oval basal nuclei, whereas the corium was formed of a loose connective tissue containing blood vessels, nerves, and lymphatics. Occasional intraepithelial lymphocytes with dense rounded nuclei surrounded by a thin zone of clear basophilic cytoplasm were detected. The musculosa was formed of smooth muscle fibers arranged in two layers: inner circular and outer longitudinal one. The serosa was formed of simple squamous mesothelial cells and submesothelial layer of loose connective tissue containing blood vessels, nerves, and lymphatics. The lumen appeared empty (Figs 2 and 3). In sections stained with Masson trichrome, few blue collagen fibers in the connective tissue between the smooth muscle fibers of musculosa and serosa were detected (Fig. 4).
Ultrastructure of oviducts: the epithelium was formed of two types of columnar cells having euchromatic nuclei; the predominant type was the secretory cells, whereas ciliated cells were few (Fig. 5). Ciliated cells showed mitochondria and minimal cilia (mainly solitary cilium) projecting from the apical surface of the cells into the lumen. The secretory cells revealed apical microvilli and many secretory blebs arising into the lumen (Figs 5–7). The cytoplasm of secretory cells was moderately electron dense and contained secretory vesicle variable in size and shape; some of them had electron-dense center (Fig. 6), mitochondria with closely packed transverse cristae, well-developed Golgi apparatus, and rough endoplasmic reticulum (Figs 6–8).
IHC detection of leptin: positive control slides showed positive IHC reaction for leptin in the form of fine brown granules in the oocyte of primary follicles, oolema, and granulose cells of growing follicles as well as in the endothelium of blood vessels (Fig. 9). In control group I, the immune reaction for leptin was positive in the cytoplasm of epithelial cells and in endothelium lining blood vessels (Fig. 10).
Light microscopic structure of oviducts: the epithelium of the mucosa appeared overcrowded and showed some mitotic figures. Intraepithelial lymphocytes were frequently encountered. The corium and musculosa appeared thick. The fibroblasts in the corium also revealed some mitotic figures. Many inflammatory cells were seen in the lumen (Figs 11 and 12). Excess collagen fibers in the thickened corium and musculosa were detected in sections stained with Masson trichrome (Fig. 13).
Ultrastructure of oviducts: the epithelial cells showed short rarely present cilia and microvilli on the surface, but occasional blebs were still seen (Figs 14–16). Some cells appeared vacuolated. Some polymorph nuclear leukocytes and intraepithelial lymphocytes were detected between the epithelial cells (Fig. 14). The nuclei were corrugated (Figs 14 and 15). The cytoplasm revealed dilated Golgi apparatus and rough endoplasmic reticulum (Fig. 15). Many mitochondria showed irregular cristae and failed to fuse together. Many secondary lysosomes were seen (Fig. 16).
IHC detection of leptin: the IHC reaction for leptin was negative in the epithelium of the oviducts and in the endothelium lining blood vessels (Fig. 17).
No changes in the histological structure or IHC reaction for leptin were detected in this group compared with the control group I.
Light microscopic structure of oviducts: longitudinally folded mucosa lined with columnar epithelium with some intraepithelial lymphocytes was observed. The vascular corium was relatively thin. The muscle fibers in musculosa were arranged in the form of inner circular and outer longitudinal smooth muscle fibers. The lumen appeared empty (Fig. 18). Some collagen fibers were seen among the muscle fibers (Fig. 19).
Ultrastructure of oviducts: numerous microvilli and rare cilia were evident on the surface of the columnar secretory cells (Fig. 20).
IHC detection of leptin: positive immune reaction for leptin was detected in the cytoplasm of the epithelial cells and in the endothelium lining blood vessels (Fig. 21).
Compared with negative control rats in group I, rats receiving MSG in group II revealed a significant (P<0.05) increase in body weight, total serum cholesterol level, and height of epithelial cells. However, there was an insignificant increase (P>0.05) in the number of epithelial cells. Rats receiving CoQ10 together with MSG in group IV revealed an insignificant increase in the body weight, total serum cholesterol level, height of epithelial cells, and number of epithelial cells (P>0.05) (Table 1).
The ampullary region of oviduct is a strategic site in which fertilization and early embryonic development occur. Its fluid composition is involved in the optimization of the microenvironment for fertilization and early cleavage stage in embryonic development .
In this study, the histological structures of the ampulla of control group at diestrous phase showed predominant secretory cells and few ciliated cells. Different studies reported that, during the diestrous phase, the secretory cells are the dominating cell type and the ciliated cells are reduced in number [3,20,21]. The secretory cells have apical microvilli and many secretory vesicles variable in size and shape . The secretory cells secrete glycoproteins, which reduce the incidence of polyspermy and increase postcleavage development to blastocyst .
Increased use of chemicals due to advanced technology can seriously harm female fertility. A great danger is hidden in the increased use of different food additives such as MSG . It is generally believed that more than 70 million couples suffer from infertility worldwide .
In this study, group II receiving MSG showed a significant increase in the total serum cholesterol level and epithelial cell height. However, the increase in the epithelial cell number was not significant. Moreover, different structural changes in the oviduct in the form of overcrowded epithelial cells and thickening in the corium and musculosa associated with mitotic figures in fibroblasts of the corium were encountered. Similar findings were reported in other studies [24,25]. The exact mechanism of how MSG induced cellular overcrowding is not clear. However, some investigators suggested that increase in the levels of total protein, cholesterol, and estrogen by MSG may lead to an increase in cell proliferation . Furthermore, other studies reported that hypercholesterolemia can promote the proliferation of fibroblasts .
In oviducts of rats that received MSG, many inflammatory cells were seen in the lumen and between epithelial cells. This was reported before by another study . MSG cause oxidative stress, leading to an increase in the inflammatory markers . The accumulation of inflammatory cells in the female genital organs may lead to infertility .
Dilated Golgi apparatus and rough endoplasmic reticulum were detected in group II. It was reported that glutamate metabolism occurs through transport from the endoplasmic reticulum to the Golgi apparatus ; therefore, MSG can alternate protein synthesis .
Some epithelial cells showed many vacuoles and lysosomes in their cytoplasm as a result of MSG intake. In agreement with this finding, a previous study reported cell death due to degeneration induced by MSG in the form of apoptosis and necrosis . MSG is a cytotoxic substance that may enhance autophagy of the cells resulting in apoptosis .
Disrupted mitochondria with failure of their division and fusion were detected in group II. The mitochondrial division and fusion play a major role in controlling the morphology of the organelle and maintain the function of healthy cells. Loss of fusion and division have been linked to loss of mitochondrial membrane potential in addition to reduced respiratory activity and apoptosis . It was mentioned that glutamate induces Ca2+ influx and disruption of the inner transmembrane potential of the mitochondria, resulting in opening of the mitochondrial permeability transition pore . This will lead to release of essential factors of apoptosis, which have the ability to activate caspases into the cytosol resulting in apoptosis .
Leptin is a hormone originally identified in adipocytes. It is involved in the regulation of fat deposition and energy expenditure and in other functions, such as reproduction .
In this study, control group showed a positive IHC reaction for leptin in the cytoplasm of the ampullary epithelium of the oviduct and in the endothelium lining blood vessels. Different studies proved a strong positive reaction for leptin in the cytoplasm of secretory cells of the ampulla than in other parts of the oviduct  and in endothelial cells [5,12].
Overweight animals showed negative immune reaction for leptin in this study. Studies have indicated for decades that MSG intake causes overweight and obesity as a result of hypothalamic and arcuate nuclei damage, resulting in the lack of control between absorption and energy expenditure [34,35]. Many studies reported a link between the decrease in leptin production and obesity as leptin modulates food intake and energy homeostasis [36,37]. In contrast, some investigators postulated that mice with diet-induced obesity such as MSG exhibit circulating hyperleptinemia. However, there is a resistance to the metabolic actions of leptin and absence of its expression in the cells [38,39]. This negative immune reaction for leptin may cause infertility. It was reported that a negative IHC reaction for leptin in an ovulatory cycle may contribute to subfertility .
CoQ10 is an endogenously synthesized compound that acts as an electron carrier in the mitochondrial respiratory chain. It is involved in the reactive oxygen species removal and prevention of oxidative stress-induced apoptosis. In addition, it has potential benefits to decrease inflammation [41,42] and to enhance neuroprotection . It was reported that CoQ10 improves fertility in aged female mice through an increase in the quantity and quality of ovulated eggs .
In this study, CoQ10 was given alone in group III rats in a dose that did not alter the structure, ultrastructure, or the IHC reaction. When a similar dose of CoQ10 was given with MSG, the structural and ultrastructural degenerative changes induced by MSG were prevented and the positive immune reaction for leptin in the epithelium of the oviduct was preserved. Different studies reported that CoQ10 can inhibit fibrosis  and dramatically reduced apoptotic cell death [46,47]. Moreover, there was no significant increase in the body weight and total serum cholesterol in this group compared with the control group. It was proved that CoQ10 can strongly inhibit adipogenesis  and can decrease the body weight and serum cholesterol , reflecting a partial protective role of CoQ10.
Administration of MSG resulted in changes in the histology and in the IHC expression of leptin in the ampulla of the oviducts in adult female albino rats during the diestrous phase. The administration of CoQ10 with MSG partially prevented the occurrence of such changes and regulated leptin expression, suggesting a possibility to use CoQ10 in concomitant with MSG to decrease its toxic effect.
The safety use of MSG should be reconsidered with a potential to withdraw MSG from the food chain. Furthermore, the possible role of CoQ10 in ameliorating the toxic effect of MSG on the oviducts and on other female genital organs should be examined to raise a possibility to use CoQ10 in cases of unexplained infertility, which may be due to nutritive habits.
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