Ahmed, Fayza E.
Monosodium glutamate (MSG) is a sodium salt of the naturally occurring (nonessential) L-form of glutamic acid. It is one of the main flavor enhancers used in various food products. Glutamate is also produced in the body and plays an essential role in human metabolism. Glutamate and its receptors are found in a variety of hypothalamic nuclei critical for the regulation of various reproductive functions such as puberty, LH pulsatility, the midcycle surge of gonadotropin, and reproductive behavior [1,2].
MSG enhances taste perception. This popular taste enhancer is widely used not only in the food industry but also in homes and restaurants. It is present in flavored chips and snacks, soups or sauces (canned, packed), prepared meals, frozen foods and meals, manufactured meats, luncheon chicken and turkey, flavored tuna, vegetarian burgers, and sausages .
MSG improves the palatability of meals and thus influences the appetite center positively, with a resultant increase in body weight. Although MSG improves taste stimulation and enhances appetite, reports have indicated that it is toxic to human and experimental animals [4,5]. It has a toxic effect on the testis by inducing significant oligozoospermia and an increase in abnormal sperm morphology in a dose-dependent manner in male Wistar rats . It has been reported that MSG has neurotoxic effects resulting in brain cell damage, retinal degeneration, endocrine disorders, and some pathological conditions such as addiction, epilepsy, neuropathic pain, schizophrenia, anxiety, depression, and Alzheimer's disease. It cannot be stated with certainty that MSG is the cause of such varied conditions as epilepsy and Alzheimer's disease, although there may be concerns of its involvement in their etiology .
Parenteral administration of high doses of MSG to animals during the neonatal period may cause lesions of the preoptic nuclei, arcuate nuclei, the circumventricular organs, and the retina [8,9]. It was also found that animals treated with injections of MSG during the first 10 neonatal days exhibited a series of neuroendocrine disorders during their adult lives. MSG can be harmful when given orally too . Negative effects on the uterine tube of female albino rats caused by MSG have also been reported .
The aim of this study was to investigate the histological and immunohistochemical effects of MSG on the ovary and to study the possible role of diltiazem (L-calcium channel blocker) in the prevention of these effects in adult female rats.
Materials and methods
This study was carried out on 30 adult female albino rats (180–200 g. average weight) that had a regular 4-day estrous cycle. Before the start of the experiment, all the female rats were sexually cocycled in a lab lacking male rats; cocycling was brought about by preparing vaginal smears. These vaginal smears were collected daily from all of the animals. The smears were stained by hematoxylin and eosin (H&E) and examined microscopically. Three types of cells could be recognized: epithelial cells, cornified cells, and leukocytes. The proportions of these cells were used for determination of the estrous cycle phase . The animals were in the estrous phase at the start of the experiment and were classified randomly into three equal groups: I, II, and III (10 animals each). The control group (group I) received fixed amounts of grower's marsh (550 g) without addition of MSG for 14 days. The treated group (group II) was given 6 g of MSG (obtained from El-Kahera Company for Chemical and Medical Trading, Zagazig, Egypt) thoroughly mixed with an equal amount of feeds (grower's marsh) daily for 14 days . In addition to MSG, group III (prophylactic group) was given diltiazem (its trade name is delay-tiazem SR capsules, obtained from GlaxoSmithKline, Egypt) dissolved in water daily by an oro-gastric tube (5 mg/g body weight) for 14 days. The rats were sacrificed on day 15 of the experiment. The ovaries were quickly dissected and prepared for light microscope evaluation .
Specimens were fixed in 10% formal saline and processed to prepare 5-μm-thick serial sections. They were stained with H&E  and immunohistochemically stained for detection of inducible nitric oxide synthase (iNOS) .
An immunohistochemical reaction was carried using a streptavidin system with antibody against the iNOS marker for oxidative stress. The primary antibody was a mouse monoclonal antibody; the kits were obtained from Dako Life Trade, (Egypt; clone 608, code no. M 616 for iNOS). The universal kit used was biotinylated secondary antibodies. The immune reaction was visualized with 0.05% diaminobenzidine and the slides were counter stained with Mayer's hematoxylin before mounting. The positive results for the iNOS immune reaction were indicated by brown coloration of the cytoplasm .
Quantitative morphometric measurements
Sections stained with H&E and immunohistochemical reactions were analyzed morphometrically using an image analyzer computer system. The data were obtained using Leica Q 500 DMLB (Cambridge, England) at the image analysis unit in the National Research Centre. This image analyzer computer system was used to evaluate the number of ovarian follicles (primordial, secondary, and mature Graafian follicles) using the interactive measure menu. The procedure was performed on H&E-stained sections at a magnification of ×100. Various fields were chosen and 10 readings were obtained from each group. The optical density (OD) of the iNOS immune reaction was measured in the cytoplasm of granulosa and stromal cells, using the gray measure menu in 10 measuring frames in each specimen using an objective lens of magnification ×40.
The data obtained from the image analyzer were analyzed using SPSS version 11 (Chicago, USA). A comparison between groups was made using ANOVA and least significant difference. Data were expressed as mean±SD. Results were considered significant when P value is less than 0.05.
Group I (control group)
Examination of H&E-stained sections of the ovary of the control group showed a wide cortex with numerous primordial follicles under the tunica albuginea consisting of an oocyte surrounded by a single layer of squamous follicular cells. Clusters of stromal cells were seen (Figs 1 and 2). The ovary also contained secondary follicles with a cavity surrounded by follicular granulosa cells and peripheral fusiform theca folliculi cells (Fig. 3). Graafian follicles were noticed consisting of an oocyte with a well-defined zona pellucida, corona radiata, many layers of granulosa cells, and fusiform theca cells (Fig. 4). A weak immune reaction to iNOS was seen in the granulosa cells and in the stromal cells (Fig. 5).
Group II (monosodium glutamate-treated group)
The ovary of MSG-treated rats showed that the cortex was occupied by many interstitial stromal cells arranged in clusters (Fig. 6). The ovary also contained some atretic follicles, secondary follicles, Graafian follicles, and corpus luteum. These structures are separated from each other by abundant interstitium and blood vessels (Fig. 7). Secondary follicles consisted of a degenerated oocyte, a small cavity, disorganized follicular granulosa cells with darkly stained nuclei, and fusiform theca folliculi cells (Fig. 8). Also, the Graafian follicle consisted of a degenerated oocyte surrounded by disorganized cells of the corona radiata, antrum, follicular granulosa cells with darkly stained nuclei, and vacuolated theca folliculi cells (Fig. 9). The blood vessels contained hyaline eosinophilic material in their lumina and their walls were thickened and contained hypertrophied smooth muscle cells (Fig. 10). There was an increase in the cellularity of the tunica albuginea under the surface epithelium. The stromal cells appeared vacuolated and arranged in clusters (Fig. 11). Some ovarian follicles had markedly degenerated granulosa cells and multiple vacuoles (Fig. 12). The immune reaction to iNOS was strongly positive in the cytoplasm of most of the granulosa cells and in the stromal cells (Fig. 13).
Group III (prophylactic group)
The ovaries, in this group, had some primary follicles under the surface, corpus luteum and many dilated congested blood vessels. These follicles were composed of oocytes surrounded by cuboidal granulosa cells (Figs 14 and 15). The ovary also contained many apparently healthy follicles at different stages of development (Fig. 16). The follicles were almost identical to those of the control group. The secondary follicles were composed of an oocyte with a well-defined zona pellucida, a fluid-containing cavity, many layers of granulosa cells, and flattened theca cells (Fig. 17). The Graafian follicles consisted of an oocyte with a well-defined zona pellucida, corona radiata, many layers of granulosa cells, and fusiform theca cells (Fig. 18). Weak immunopositivity for iNOS was observed in the granulosa cells and in the stromal cells (Fig. 19).
Morphometric and statistical analysis
Morphometrical study and statistical analysis of the data obtained confirmed the histological results and showed a statistically significant decrease in the number of ovarian follicles in the treated group compared with the control or the prophylactic group. In contrast, there was a nonsignificant difference in the number of these follicles in the prophylactic group compared with the control (Table 1 and Histogram 1). There was a statistically significant increase in the OD of iNOS in the treated group compared with the control or the prophylactic group. The OD of iNOS in the prophylactic group showed a statistically significant increase compared with the control group (Table 2 and Histogram 2).
Female infertility is a very real medical problem in developing countries. The female reproductive system is very sensitive to different harmful environmental factors. A major advancement in technology was followed by an increased use of chemicals that could seriously impair female fertility. Some chemicals are counteractive with estrogen . There is a potential danger in the increased use of different food additives like MSG .
The results of this study revealed observable alterations in the structure of the ovary of MSG-treated rats. The ovary showed an increase in the cellularity of tunica albuginea. The stromal cells appeared vacuolated and arranged in clusters. Some atretic follicles were present whereas other types of follicles were distorted, having a degenerated oocyte surrounded by disorganized cells of follicular granulosa cells with darkly stained nuclei and vacuolated theca folliculi cells. Some follicles had markedly degenerated granulosa cells and multiple vacuoles. The blood vessels contained hyaline eosinophilic material in their lumina and their walls were thickened and contained hypertrophied smooth muscle cells. These results were in agreement with those of other investigators who reported that MSG treatment in the neonatal female rats resulted in cystic degeneration of the ovaries, with the appearance of many atretic follicles, abundant interstitium, and hyalinosis of arteriola . Prolonged administration of high doses of MSG induced degenerative and atrophic changes in rat ovaries as MSG had a dose-dependent toxic effect on the oocyte and follicular cells .
Other authors attributed the toxic effect of MSG on the female reproductive system to its direct effect on nuclei of the hypothalamus. Rats treated with MSG showed a decrease in cytosol estrogen receptors in the arcuate median eminence region . It was found that reduction of endogenous production of ovarian estrogen by the follicular zona granulosa initiated widespread atrophic reproductive tract changes. Ovarian atrophy was associated with inactive interstitial glands, reduction in the numbers of follicles and interstitial stromal cell hypertrophy or hyperplasia. These stromal cells may be collections of theca cells remaining after atresia . Anovulatory follicular cysts may develop, forming an immature ovum that may lead to infertility . Similarly, other investigators have reported that nicotine inhibits the release of gonadotrophins, FSH, and LH from the pituitary, acting through the hypothalamus, blocking the neural stimulus to GnRH . In females, FSH stimulates the growth of Graafian follicles and LH is required for its maturation and ovulation. Thus, a decrease in gondatrophins is reflected in atrophy of both gonads in structure and function .
Glutamate receptors are present in different tissues including the hypothalamus, endocrine system, ovaries, and uterus . Glutamate receptors play a very important role in the pathogenesis of disorders induced by MSG. Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system [25–27]. There are two basic types of glutamate receptors: ionotropic and metabotropic [28,29]. The neurotoxicity of MSG is related to glutamate receptor activation . Sustained high-concentration MSG could alter the ionic permeability of neural membranes for calcium and induce persistent depolarization. This excessive activation of glutamate receptors and overloading with intracellular calcium could induce neural death, including nuclei of the hypothalamus .
In this study, the immune reaction to iNOS was highly positive in the granulosa cells and in the stromal cells of the treated group. Nitric oxide is an important biological messenger in animals. It is synthesized from l-arginine by enzyme nitric oxide synthase (NOS), which exists in multiple isoforms in a wide range of mammalian cells. Nitric oxide is considered a vital molecule controlling the hypothalamic–pituitary–gonadal axis . The NOS had been classified depending on the tissue of origin, the functional and structural properties in neuronal constitutive NOS, endothelial constitutive NOS (eNOS), and iNOS. iNOS is expressed in response to inflammatory cytokines and lipopolysaccarides .
Results from different studies have indicated that rat granulosa cells from primary, secondary, and small antral follicles and rat stroma, thecal, and luteal cells express eNOS. In contrast, iNOS was barely detectable . eNOS and iNOS from aortic endothelial cells showed an increased expression in ovariectomized compared with intact pigs . It has been proposed that locally produced NO may help modulate follicle development and possibly prevent apoptosis, at least at low concentrations, whereas high levels might promote cell death by means of peroxynitrite formation .
It has been reported that a reduction in the level of gonadotrophins, known as inhibiting factors of apoptosis in granulosa cells, results in apoptosis and shrinkage of granulosa cells. Apoptosis of granulosa cells could be considered another reason for the reduction of the FSH level [37,38]. In addition, MSG induces oxidative stress in different organs including the thymus. MSG increased rat thymocytes apoptosis by decreasing BCL-2 expression in rat thymocytes , caused a significant increase in lipid peroxidation, glucose, and ascorbic acid and a decrease in the glutathione content in the hepatocytes. Oxidative stress and hyperglycemia observed after the administration of MSG seem to be because of the metabolic products of MSG .
In this study, a concomitant administration of diltiazem and MSG in the prophylactic group resulted in improved histological and immunohistochemical changes instead of pictures. Many apparently healthy follicles at different stages of development were seen and separated by dilated congested blood vessels. This was in accordance with the observations of other authors who reported that the pretreatment with diltiazem prevented the development of morphological and functional disorders of ovaries following MSG toxicity because calcium overload played an important role in the mechanisms of this toxicity [14,18]. Diltiazem has been shown to be a potent vasodilator by inducing relaxation of smooth muscles and the resultant decrease in peripheral vascular resistance. Diltiazem is used in the treatment of the vascular smooth muscle dysfunction because of calcium overload .
Calcium channel antagonists have found widespread use in the treatment of cardiovascular abnormalities. They have been used experimentally in a range of disorders besides the cardiovascular system; for example, it has been reported that diltiazem attenuates acute hepatocellular damage in the perfused rat liver induced by D-galactosamine and CCl4. Also, calcium channel antagonists prolong the survival time in various animal models of endotoxic shock . The addition of a calcium chelator maintained the DNA integrity of cooled pig oocytes .
Thus, in this study, administration of MSG to adult female rats resulted in histological and immunohistochemical alterations, suggesting harmful effects on the function of the ovary. Pretreatment with diltiazem prevented the development of these alterations caused by MSG. These results suggest that the safety profile of MSG should be reexamined and that calcium overload plays an important role in the mechanisms of MSG toxicity.
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