Archives of Anatomy and Physiology

The present study investigated the effect of oral administration of sodium fl uoride on testicular histopathology of rats and evaluated ameliorative effect of Boerhaavia diffusa L. Adult male rats weighing 100-150g were administered with sodium fl uoride (NaF) at three different doses 100, 200 and 300 ppm/ kg body weight, orally, daily for 40 days. At the end of the experimental period, half of the animals were sacrifi ced and their testis was removed. Remaining half rats were post treated with two doses (250 mg and 500 mg/kg body weight) of leaf extract of Boerhaavia diffusa L. for 20 days. Both light and scanning electron microscopy revealed dose dependent increase in testicular histopathological anomalies in NaF treated groups as compared to control. The normal histoarchitecture of seminiferous tubules was distorted. There was severe atrophy of seminiferous tubules as they were devoid of epithelium, with only sertoli cells and spermatogonia present within the depleted tubules. The spermatogenic cells showed degeneration and necrosis. There was severe tissue disruption, with absence of epithelial layers and complete cessation of spermatogenesis. The spermatozoa were absent in the tubules, only spermatocytes and spermatogonia were visible. The Leydig cell had scanty cytoplasm. The interstitial tissues were wide and congested. There was congestion and dilation of blood vessels. However, NaF groups post treated with leaf extract of Boerhaavia diffusa L. showed normal architecture of seminiferous tubules. The present study suggests that fl uoride exerted disruptions to testicular histology was ameliorated by the administration of Boerhaavia diffusa L. extract. Research Article Boerhaavia diff usa L. Supplementation Attenuates Fluoride Induced Testicular Impairments in rats Shashi A* and Imtiaza Khan Department of Zoology and Environmental Sciences, Punjabi University Patiala147002, Punjab, India Dates: Received: 28 October, 2017; Accepted: 06 November, 2017; Published: 07 November, 2017 *Corresponding author: Shashi A, Department of Zoology and Environmental Sciences, Punjabi University Patiala147002, Punjab, India, Tel: 08054280827; E-mail:


Introduction
A large part of population in the industrialized world is exposed to variety of pollutants on daily basis, fl uorides being one of them [1]. Besides immunotoxic, neurotoxic and cytotoxic effects induced by fl uoride, the adverse infl uence of fl uoride on the reproductive system has become a major concern in many countries. The importance of reproductive health to offspring developments has prompted epidemiological investigations of the apparent connection between excessive fl uoride exposure to male fertility and low birth rates [2].
Involvement of the reproductive organs due to fl uorosis in animals had also been studied extensively. Fluoride consumption for a longer period of time has many pathological effects as a result of increased oxidative stress on soft tissues like muscle, liver, gastrointestinal tract in addition to the reproductive and endocrine organs by the property of simple diffusion. Several clinical investigations and animal experiments suggested that fl uoride has adverse impacts on male reproductive function [3,4], such as spermatogenesis and steriodogenesis [5], alterations in the epididymis and accessory reproductive glands and testicular cell cycle [6]. Moreover, high doses of fl uoride could result in apoptosis of leydig cells [7], and vascular dystrophy in seminal cells and necrosis in mice [8].
Due to exposure to environmental pollutants and physical stress, the rate of male infertility has been increasing, which has become a very serious social problem. Boerhaavia diff usa L. is an important medicinal plant much used in Ayurveda and Unani medicines and in other traditional medicines in many parts of the world. Each part of this plant has a different therapeutic value. This plant rejuvenates liver, male reproductive system and other organ system, aphrodisiac, increases libido, erection and quality and quantity of semen [9]. Therefore, the present study was conducted to assess the toxic effects of fl uoride on testicular histology and its alleviation by B. diff usa.

Materials and Methods
Young male Wistar rats weighing between 100-150 g were housed in polypropylene cages with stainless steel grill tops and fed with standard rat pellet diet (Hindustan Lever Limited, India) and maintained in an air conditioned animal house facility

Preparation of plant extract
The leaf extract of B. diff usa was prepared by the method of Narendhirakannan, et al. [10].

Experimental design
After acclimatization to environment for one week, the animals were randomly divided into six groups with twelve animals in each group. The fi rst group served as normal control administered with 1 ppm deionized water/kg b.w./day. The remaining three experimental groups received 100, 200 and 300 ppm sodium fl uoride (NaF)/kg b.w./day. All the treatments were given daily by oral gavage for 40 days. Remaining half rats were post treated with B. diff usa (250 and 500 mg/kg b.w.) for next 20 days. Last two groups were kept as positive control I and positive control II administered with 250 and 500 mg/kg b.w./ day B. diff usa leaf extract, respectively. At the end of the experimental period, rats were fasted overnight and sacrifi ced and testis was removed, blotted free of blood, and processed for light and scanning electron microscopy.

Histopathological study
Light microscopy: After the completion of experimental regimen, the rats were fasted overnight and were excised under ether anesthesia and the reproductive organ (testis) were removed, blotted free of blood, fi xed in Bouin's fl uid for 24 hours. The tissue was dehydrated in graded series of alcohol followed by tertiary butyl alcohol, cleared in amyl acetate, processed for paraffi n embedding and 7μ thin paraffi n sections were stained with haematoxylin and eosin [11]. Histopathological changes were studied under research binocular microscope and subsequently micro photographed.
Electron microscopy: After fi xation in Karnovsky's fi xative, the specimens were washed in 0.1 M phosphate buffer and then fi xed for 1 hr in 0.5% osmium tetraoxide. After few washes in 0.1 M phosphate buffer, the specimens were dehydrated through ascending grades of alcohol. After critical point drying followed by coating with gold pellets, the tissue were examined under a scanning electron microscope-JSM(6100) JEOL [12].

Results
In control rat, the seminiferous tubules were surrounded by                         spermatocytes, early and late spermatids were nearly more or less similar to the control group ( Figure 6).
The presence of degenerating spermatogenic cells, pyknotic nuclei and vacuolation were less in B. diff usa leaf extract treated groups.

Scanning electron microscopic observations
Scanning electron micrograph of control rat showed that the outer surface of the seminiferous tubule was covered with single layer of fl at myoid cells, the nuclei of which appeared as small bulges. The myoid cells were arranged in a continuous monolayer (Figure 7a). Germ cells layers were visible. The tubular lumen fi lled was fi lled with fl agella of spermatozoa (Figure 7b).
Scanning electron micrograph of testicular sections of rats treated with 100 and 200 ppm NaF showed narrow seminiferous tubule having irregular outlines (Figure 8a), with exfoliated germ cells in the lumen (Figure 8b). The number of spermatozoa was reduced (Figure 8c).
In rats treated with 300 ppm NaF scanning electron micrograph of seminiferous tubule showed many cavities (Figure 9a), and depletion of the germinal epithelial layers (Figure 9b), with decreased number of spermatozoa in the lumen (Figure 9c). Empty seminiferous tubules were also visible ( Figure 9d).         The fl uorotic rats post treated with 500 mg/kg bw of B.
diff usa leaf showed a signifi cant improvement of testicular structure. The seminiferous tubules were well defi ned and lined by several layers of spermatogenic cells. The tubular lumen was fi lled with spermatozoa ( Figure 11).

Discussion
In the present study, the microscopic examination of the testes of the control rats showed normal structure and normal spermatogenesis with different stages of differentiation and maturation. By contrast, the NaF treated group animals showed many pathological alterations in testis including necrosis and a prominent decline in the number of spermatogonial cells resulting in epithelial disorganization [13], and absence of all internal layers except scattered cells of spermatogonia layer along the basement membrane [14], leading to complete cessation of spermatogenesis and the tubular lumen showed a few scattered clusters of spermatozoa with luminal  cellular debris [15], alongwith vaccular degeneration of the spermatogenic and sertoli cells as well as leydig cells necrosis [16]. Moreover, there was thick and irregular basement membrane [8]. In addition to, marked infl ammatory infi ltration in the interstitial tissue of the seminiferous tubules was observed. Congestion of interstitial blood vessel and thickening of tunica albuginia was also observed [17].
The fi ndings on the altered histoarchitecture of testis are in accordance with the study of Sakr and Azab [18], and Manna et al. [19].
Wan et al. [20]. Also recorded that NaF (150mg/L of drinking water) caused disorganization, denudation and reduction in germinal epithelial cells of the seminiferous tubules and an accompanying absence of sperm in the lumina in histological sections of rats. Sekhar et al. [21]. Found that cypermethrin and NaF -treated mice testes exhibited clumped spermatozoa, vacuolation, severe necrosis and degenerative changes with increased lumen of seminiferous tubules. Beside these degenerative changes in spermatids, atrophied seminiferous tubules, necrotic changes in theca albuginea and scattered spermatids were also reported. Feng et al. [22], observed that on administration of 2.5 g/L NaF resulted in degeneration of seminiferous epithelium and depletion of sperm cells.
On the other side, the microscopic examination of the rats post treated with B. diff usa leaf extract revealed an improvement in histological picture of fl uoride toxicity of testis sections. The later showed normal seminiferous tubules epithelium with Citation: Shashi  Thus, histopathological results of the present study confi rmed the observations of previous authors in mice and rabbits [23,24]. Moreover, the NaF exposure showed severe loss of Leydig cells as a result of the oxidative stress that affected on steroidogenesis and testicular apoptosis [25].
The necrosis of seminiferous tubules was in consonance with the study of Yang et al. [26]. Who explained that fl uoride stimulated free radicals which increased lipid peroxide levels, and decreased the activities of glutathione peroxidase and ATPase in testis and epididymis that picked up by mitochondria producing swelling and distortion of mitochondrial cristae, uncoupled energy metabolism, inhibited cellular respiration, and altered calcium kinetics, Moreover, Agha et al. [27], suggested that the oxidative stress results from the loss of equilibrium between oxidative and antioxidative mechanisms that can produce DNA fragmentation, resulting in apoptosis.
The same fi nding was attributed by Shashi et al. [28], who explained that fl uoride toxicity leads to loss of selective permeability of the cell membrane, resulting in dilatation of cytoplasmic component secondary to intracellular fl uid and electrolyte redistribution.
Sertoli cell degeneration related to decreased sperm quality and abnormal spermatozoa has also been reported [29], which supported our observations, in addition to the apoptotic appearance of sertoli cells which are essential for nourishment. Yang et al. [26], documented that the severe irritation of fl uoride ions on the tissue parenchyma may lead to the infi ltration of infl ammatory cells in testis and epididymis that causes apoptosis and necrosis of germinal cells.
The present study demonstrated that exposure to NaF caused histopathological changes in the testes that are indicative of changes in spermatogenesis function, especially in the seminiferous tubule. Furthermore, normal testicular structure and the maintenance of its internal microenvironment are important for sperm production and for maintaining good quality of sperms. By post treatment with B. diff usa leaf extract the testicular histology was resumed to normal architecture.