Histopathological alterations of male and female reproductive systems induced by alloxan in rats

Article history: Received September 1, 2019 Accepted October 21, 2019 Available online September 7, 2020 The objective of this study is to determine the histological effects experimentally induced by injection of alloxan 100 mg/kg B.w. on the histopathological structure of reproductive organs of male and female albino rats. The results showed that treatment with alloxan cause alteration in testis include irregular shape and size of seminiferous tubules, irregular division of spermatid cells, degeneration and necrosis of Sertoli cells and paucity of sperms in the lumen of tubules. While histological examination of epididymis showed the lumen of it free from sperms, thickening of muscular layer and interstitial tissue between the epididymis canal. The histological alteration of female reproductive organs includes disturbances in development of primary follicles of ovaries, hemorrhage in the interstitial tissue as well as atrophy in the uterine glands with hyperplasia of the epithelial cells of uterus. The conclusion of this study showed that alloxan cause histological alteration in reproductive organs of male and female rats.


Introduction
Oxidative stress is a status results from the imbalance between the oxidative agents and the antioxidant system leading to production of free radicals, which have a role inducing harmful effects on living tissues (1). Alloxan used to cause type I diabetes mellitus (2) it considers as a cause of free radicals' production especially reactive oxygen species (ROS) like superoxide onion radicals which transformed into hydrogen peroxide (3). Numerous studies have demonstrated the role of oxidative stress in many pathological conditions (4) including male and female reproductive abnormalities which characterized by infertility, in ability to fertilize and effects the functions of sperms which decrease in sexual behavior (5). Also, female reproductive system affected by oxidative stress via genital mutability during and after reproductive period, as well as it affects the functional process starting from the maturity of oocyte to the ovulation, implantation of blastocytes and lysis of corpus luteum (5). Alloxan cause increase the blood glucose, this leads to oxidative stress which cause structural and functional reproductive impairment, finally contribute infertilities (6).

Animals
Twenty adult male and female (females separated from) albino rats 200-250 g. The animals obtained from house of laboratory animal in Veterinary Medicine College, University of Mosul. The animals were kept in plastic cages and maintained under laboratory controlled. The rats had free water and food.

Grouping of experiments
Rats were divided into four groups included diabetic and normal (control group). The first and second groups 5 male and 5 females considered as control. The third and fourth groups 5 male and 5 females are diabetic groups.

Induction of diabetic mellitus
Diabetes was induced in the male and female albino rats by single subcutaneous injection with the dose 100mg /kg BW of alloxan according to previous study (7). The rat fasted 12 hours before and after alloxan injection.
Rats were allowed to drink 5% glucose solution over night to prevent hypoglycemia (8,9).
The alloxan treated rats were confirmed the accuracy of diabetes by examining the urine using tap detector once every three days for month for glucose urea to ensure that the rats do not return to normal state. Following a month of treatment with alloxan, all animals were euthanized with diethyl ether.
Removing of testes and epididymis of male rats, ovaries and uterus of female rats was done immediately then fixed in 10% neutral buffer formalin for 72 hours before starting the process of histological slides.
After fixation, dehydration in a series of increasing alcohol concentration and embedded in paraffin wax was used. The section of 5 microns thickness was stained by hematoxylin and eosin and examined by light microscope (10).

Result
Histopathological findings induced diabetes by alloxan after month cause histological changes in the testes characterized by irregular shape and size of seminiferous tubules, irregular division of germ cells, with degeneration and necrosis of Sertoli cells, paucity of sperms into the lumen of tubules comparing with control group (Figure 1).
The histopathological examination of epididymis of diabetic rats showed thickening of muscular layer and interstitial tissue between the epididymis canal, the lumen of epididymis free from sperms, degeneration and necrosis of epithelial cells lining the epididymis canal also observed, while the control group showed the normal architecture of epididymis ( Figure 2).
Moreover, different histological section of ovaries from the female control group indicate well development of ovarian follicles, normal blood vessels and normal stromal cells, while the ovarian section of female rats treated with alloxan showed histopathological changes include disturbance in the development of ovarian follicles, reduction in ovarian follicle number with congestion of blood vessels and vacuolar degeneration of granulosa cell layer, also numerous empty cystic follicles, with decrease in oogenesis and depletion of corpus luteum and atreric follicles also observed. The uterine tissue sections from female control group showed common uterine histology, while uterine sections of female rats treated with alloxan showed atrophy of uterine glands, thickening of muscular layer, myometrium, congestion of blood vessels with infiltration of inflammatory cells (Figure 3).

Discussion
Diabetes mellitus is a metabolic disease which characterized by hyperglycemia which induced oxidative stress (11). This will cause alteration of sperm function and sperm damage then decrease fertility (5). This occur due to stimulation detriment blood testes barrier (BTB) changes which induce alteration of testis that cause disrupting the metabolic action between cellular content of BTB with the consequences on sperm quality and fertility (12).
Hyperglycemia induce oxidative stress this associated with failure of testis function to dysfunction because the testicular tissue and spermatocytes are susceptible to free radical's damage due to high level of poly unsaturated fatty acid, with low oxygen tension and with lack of antioxidant defense mechanism (13,14). This will cause alteration in the histological structure of both seminiferous tubules and epididymis in diabetic animals, this result agrees with the results reported by (15). Hyperglycemia has adverse effect on the density and motility of the sperm as a result of alteration in the production of energy and free radical's management (15). Diabetes also cause reduce the concentration of testosterone, androgen binding protein, sialic acid in the epididymis tissue, this will have effect on the secretory activity and the capacity of epididymal epithelium which lead to defective sperm maturation (16). These results of our study agree with results of researchers (17). Also, hyperglycemia has harmful effects on female reproductive function (18,19) and affect the development of blastocytes (20).
Hyperglycemia induced histopathological and morphological alteration of ovaries and uterus (21). These resulting in reproductive sterility and gonad atrophy and cause disturbances in primary follicles development, in agreement with results (22) that found hyperglycemia cause disturbances in the ovarian follicles development, also agreement with researchers (23,24). The vascular changes in the uterus are due to the level of glucose, the hyperglycemia which occur due to hypoinsulinemia, these results reported by (25). Hyperglycemia cause endocrine disorder that leads to multi system dysfunction (26). The main reproductive problems include disturbances in foliculogenesis and ovulation which result in low fertility.

Conclusion
This study concluded that the induction of diabetes mellitus by using of alloxan cause severe effect in the male and female reproductive organs.