The Effects of Sub-lethal Concentrations of Sodium Fluoride on the Number and Diameters of Ovarian Follicles

Fluoride was the first compound utilized in the fluoridation of drinking water, and still used today to prevent dental caries. Sodium fluoride exerts it toxic effects on different organs. Ovarian follicle considered as the fundamental unit of ovary. The current work aimed to study the toxic effects of sodium fluoride (NaF) on the number and diameters of ovarian follicles. Twenty seven adults, non-pregnant female albino rats were used in this study. Rats were randomly divided into three equal groups. The 1 st group was untreated group, 2 nd group was treated with 150ppm of sodium fluoride by gavage daily for 90 days and the third group was treated with sodium fluoride (300ppm) by gavage daily for 90 days. The sodium fluoride had adverse effects on the number and diameters of ovarian follicle in both treated groups in contrast to the control group. Also in addition, inhibition of the maturation of ovarian follicles. These results suggest that sodium fluoride damage the structures of ovary, and has adverse effects on the coefficient of the female reproductive system. ـــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
Excessive exposure to pollutant and toxic materials in the environment is a major cause of reproductive health problems. Metal toxicity like aluminum, mercury, fluoride and lead effects on the coefficient of organs and inducing damage even at low level ( AL Taee et al., 2020). Fluoride is a common pollutant found in nature ( Piyal and Alok, 2018 ). Chronic fluoride exposure causes osteon and dental abnormalities (Choubisa, 2013); neurological disorders; disturbances of gastrointestinal tract, and disorders of the reproductive system (Choubisa, 2012). The association between long-term fluoride exposure and impairment of fertility has piqued peoples interest ((Choubisa ,2012).
Clinical research and trials on experimental animals suggested that, sodium fluoride has adverse effects on the male reproductive system ( AL-Sabaawy and AL-Kaisie, 2020). Hormones play important role in the singling pathway of endocrine system (Abdel Dayem et al., 2020). Thyroid gland hormones stimulate the consumption of O2 cell in the body, that help in regulate the metabolism of carbohydrate and lipid which are necessary for the maturation of tissue like uterus endometrium and ovary ( Krassas et al., 2010 andBakeer et al., 2021). Thyroid function is necessary for normal health and reproduction (Abdelsamad et al., 2018) as hormones of the thyroid gland effects on the reproductive system at all levels. Thyroid hormone receptors are expressed by stromal cell and granulose cell of the ovary; thus, thyroid hormones play an important role in the ovarian physiology.
Administration of big with 100,250, and 400mg/kg of NaF for 50 days showed marked decreased in T4, and increase in TSH; these related to decrease of iodine level in the thyroid gland due to fluoride toxicity (Zhan et al., 2006). Female reproductive system is inhibited by hypothyroidism and hyperthyroidism (Kang et al., 2013). Estrus cycle is characterized by morphological changes in the uterus and ovary (Arbind, 2015). Decreased estrus cycle and disturbances of ovulation was observed in mice according to the previously mentioned facts. Due to lack of data on the effects of sodium fluoride on the female reproductive system, the current study was conducted to evaluate the toxic effects of sub-lethal doses of sodium fluoride on the number and diameters of the ovarian follicles.

Animals:
Twenty seven adults, non-pregnant female albino rats were used in this study .The weight ranged from 200-250 grams. The age ranged from 90 to 100 days. The animals were held in cages and kept at room temperature in the animal house at College of Veterinary Medicine University of Mosul. The rations and water were continuously served freely distributed.

Experiment design:
Animals were divided into three equal group, the first group was untreated (control group) which received tap water for 90 days and standard rat pellet ; while the 2 nd group received 150 ppm of sodium fluoride (NaF) by gavage daily for 90 days and the 3 rd group received 300ppm of sodium fluoride by gavage, daily for 90 days. All animals were adapted for at least one week before the day of dosing. Following scarifying of the empirical animals at 90 days the ovaries were extracted and fixed in 10% formaldehyde. The normal histological procedures were used to dehydrate the samples and embedded in paraffin wax. Each section was cut into 4-5mm thickness, stained with Hematoxylin and Eosin and finally examined under the light microscope (Survarn et al., 2018).

Evaluation of the ovarian follicle:
All micro morphometric measurements of ovary were done using the colour USB 2.0 digital image camera (Novel-H1C) provided with image processing software (Scope Image 9.0-China). The camera's software was calibrated to all objective lenses of Microscope (Olympus CX31) with a 0.01 mm stage micrometer (ESM-11/Japan). The ovary's micro morphometric parameters include: (1) number of primordial, primary, secondary, mature and atretic follicles per section / µm, and (2) diameter of follicles/µm.

Effects of sub lethal concentration of sodium fluoride on the number of ovarian follicle:
Sodium fluoride toxicity on ovarian follicle number was showed in table (1) and Plate (1). Results of the both treated groups showed a significant decrease at (p≤0.05) in the number of primordial, primary, and mature follicle when compared with the control group; while the number of secondary follicle showed significant reduction in the 3 rd group in contrast to the 1 st and 2 nd groups. The number of atretic and luteal follicle showed a significant decrease at (p≤0.05) in both treated groups but the luteal and atretic follicle was the highest in the 3 rd groups when compared with the 1 st &2 nd groups.  Table (2) and Plate (2) revealed a significant changes at (p≤0.05) in the diameters of secondary and mature ovarian follicle in the both dose group in contrast to the control group while the corpus luteal diameters of the 3 rd group showed significant changes when compared with the 1 st and 2 nd group .  The similar letters in columns means non-significant differences (p<0.05) among age's. The different letters in columns means there is a significant difference (p<0.05) among age's.

Control groups
The histological examination of the ovary control group showed normal composition of ovarian tissue, primordial follicles, corpus lutes, mature follicles and secondary follicles (Fig.1).

DISCUSSION
The microscopic examination of the ovary in the both dosed adults albino rats showed decreased in the number and diameters of ovarian follicle most of them degenerated leading to increase number of the atretic follicle especially in the 3 rd group these result agree with Varsha et al., (2017) who attributed the decreased in the number and diameters of ovarian follicle in the both dosed group to decreased availability of the proteins necessary for cell growth, fluoride toxicity effects on the secretion of protein that synthesized in the endoplasmic reticulum and transported to plasma membrane and Golgi apertures, as well the biological consequences of fluoride toxicity including its effects on differentiation of germ cell during the production or development of an ovum, and cell division, or due to inhibition of hypothalamus by fluoride toxicity (Kinawy, 2019).
The toxicity with sodium fluoride damage the structures of thyroid gland, disturbed the secretion of hormone (Bouaziz et al., 2005) inducing hypothyroidism, thus in hypothyroid animals the ovarian follicle are not fully develop and these concerning to that thyroid hormone may have a direct impacts on the growth and development of ovarian follicles (Armada et al ., 2001).Thyroid gland function is depended on the appropriate amount of iodine.
Fluoride and iodine are similarly structure, since both belong to the halogen group, but F is more active chemically than iodine thus it binds with iodine receptor and inhibit the activity of Na/K-ATPase and reduce iodine level in the thyroid gland (Zhan et al., 2006) leading to distributed enzyme activity that stimulate the conversion of thyroxin (T4) into triiodothyronine (T3) and interrupted the physiology of thyroid gland (Xiang et al., 2009). These findings confirm that ingestion of sodium fluoride can result in hypothyroidism these results are in accordance with